U.S. patent application number 16/279798 was filed with the patent office on 2019-08-15 for multiple virus resistance in plants.
The applicant listed for this patent is MONSANTO TECHNOLOGY LLC. Invention is credited to Stanislaw Flasinski, Alessandra Frizzi, Brad Gabor, Charles Hagen, Shihshieh Huang, John Kao, Raquel Salati.
Application Number | 20190249189 16/279798 |
Document ID | / |
Family ID | 42272567 |
Filed Date | 2019-08-15 |
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United States Patent
Application |
20190249189 |
Kind Code |
A1 |
Huang; Shihshieh ; et
al. |
August 15, 2019 |
MULTIPLE VIRUS RESISTANCE IN PLANTS
Abstract
The present invention provides gene targets, constructs and
methods for the genetic control of plant disease caused by multiple
plant viruses. The present invention relates to achieving a plant
protective effect through the identification of target coding
sequences and the use of recombinant DNA technologies for
post-transcriptionally repressing or inhibiting expression of the
target coding sequences of plant-parasitic viruses.
Protein-expression based approaches may also be utilized to augment
phenotype resistance. Thus, transcription of a single transgenic
event comprising one or more plant expression cassettes can allow
for broad spectrum resistance of a plant to multiple plant viral
strains and species among the geminiviruses, tospoviruses, and
potexviruses.
Inventors: |
Huang; Shihshieh; (Woodland,
CA) ; Flasinski; Stanislaw; (Ballwin, MO) ;
Frizzi; Alessandra; (Davis, CA) ; Gabor; Brad;
(Woodland, CA) ; Hagen; Charles; (Davis, CA)
; Kao; John; (Davis, CA) ; Salati; Raquel;
(Hollister, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MONSANTO TECHNOLOGY LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
42272567 |
Appl. No.: |
16/279798 |
Filed: |
February 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15598095 |
May 17, 2017 |
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16279798 |
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13750971 |
Jan 25, 2013 |
9670502 |
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15598095 |
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12763790 |
Apr 20, 2010 |
8455716 |
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13750971 |
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61171021 |
Apr 20, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8218 20130101;
C12N 15/8283 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A tomato plant comprising resistance to a plurality of plant
virus species, wherein the resistance is provided by at least two
different modes of action selected from the group consisting of
dsRNA, miRNA, and inhibition of virion assembly.
2. The plant of claim 1, wherein: (a) the resistance is provided by
at least three different modes of action; (b) wherein the
resistance comprises resistance against a begomovirus, tospovirus
or potexvirus; (c) wherein the resistance provided to at least one
of the plant virus species is provided by expression of a nucleic
acid construct that produces dsRNA; (d) wherein resistance provided
to at least one of the plant virus species is provided by
expression of a dsRNA fusion construct or wherein resistance
provided to at least one of the plant virus species is provided by
expression of a nucleic acid construct that produces dsRNA; or (e)
wherein resistance is provided against a begomovirus or tospovirus
by a sequence encoded by a stacked miRNA expression cassette.
3-5. (canceled)
6. The plant of claim 2, wherein the dsRNA interferes with
expression of a virus coat protein gene, a virus movement protein
gene or a virus replication gene; or wherein the miRNA interferes
with expression of a virus coat protein gene, a virus movement
protein gene or a virus replication gene.
7. The plant of claim 2, wherein the nucleic acid construct which
produces dsRNA comprises a sequence selected from the group
consisting of SEQ ID NOs:379-455.
8-10. (canceled)
11. The plant of claim 2, wherein the miRNA comprises a sequence
selected from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25,
31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and
141.
12. The plant of claim 1, wherein: (a) resistance against a
begomovirus is provided by expression of dsRNA which interferes
with expression of a begomovirus replication gene; (b) resistance
against a tospovirus or potexvirus is provided by expression of a
dsRNA which interferes with expression of a virus coat protein gene
or virus movement protein gene; (c) resistance against a potexvirus
is provided by expression of a nucleic acid construct which
produces miRNA; or (d) resistance against a begomovirus or
tospovirus is provided by a sequence encoded by a stacked miRNA
expression cassette.
13. The plant of claim 1, wherein resistance provided to at least
one of the plant virus species is provided by expression of a
tospovirus genome segment terminal sequence that inhibits virion
assembly.
14. The plant of claim 1, wherein resistance provided to at least
one of the plant virus species is provided by inhibiting virion
assembly, wherein virion assembly is inhibited by a sequence
comprised within a nucleic acid construct comprising a first
nucleic acid segment and a second nucleic acid segment, wherein the
first and second segments are substantially inverted repeats of
each other and are linked together by a third nucleic acid segment,
and wherein the third segment comprises at least one terminal
sequence of a tospovirus genome segment that inhibits virion
assembly.
15. The plant of claim 14, wherein the third nucleic acid comprises
a tospovirus genome terminal sequence selected from the group
consisting of: a terminal sequence of a CaCV or GBNV L genome
segment, a terminal sequence of a CaCV or GBNV M genome segment, a
terminal sequence of a CaCV or GBNV S genome segment, a tospovirus
genome terminal repeat sequence, a nucleic acid sequence comprising
SEQ ID NO: 167, a nucleic acid sequence comprising SEQ ID NO:168, a
nucleic acid sequence comprising SEQ ID NO:376, a nucleic acid
sequence comprising SEQ ID NO:377, a nucleic acid sequence
comprising SEQ ID NO:378, and a nucleic acid sequence comprising
SEQ ID NO: 455.
16. The plant of claim 15, wherein the tospovirus genome segment
terminal repeat sequence comprises SEQ ID NO:167 or SEQ ID
NO:168.
17. The plant of claim 1, wherein the plant comprises resistance to
viruses of at least two of the Geminiviridae, Bunyaviridae and
Flexiviridae families; or wherein the viruses are selected from the
genera Potexvirus, Tospovirus, and Begomovirus.
18. (canceled)
19. The plant of claim 18, wherein the viruses are selected from
the group consisting of: a) at least one of TYLCV, ToSLCV, ToLCNDV,
PHYVV, PepGMV; b) one or more of TSWV, GBNV, CaCV; and c)
PepMV.
20. The plant of claim 17, wherein the Potexvirus is Pepino mosaic
virus; the begomovirus is TYLCV, ToLCNDV, PHYVV, ToSLCV, or PepGMV;
or the tospovirus is CaCV, GBNV, or TSWV.
21. The plant of claim 18, wherein the begomovirus is TYLCV,
ToLCNDV, PHYVV, ToSLCV, or PepGMV.
22. The plant of claim 18, wherein the topovirus is CaCV, GBNV, or
TSWV.
23. The plant of claim 18, wherein the begomovirus is TYLCV and the
Potexvirus is Pepino mosaic virus; or the tospovirus is TSWV and
the potexvirus is Pepino mosaic virus; or the wherein the
begomovirus is TYLCV, the potexvirus is Pepino mosaic virus, and
the tospovirus is TSWV.
24. The plant of claim 1, further comprising a sequence selected
from the group consisting of SEQ ID NOs:156, 158, 160, 162, 164,
166, and 363-375; or further comprising a sequence selected from
the group consisting of: SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43,
47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, 141, and 379-454.
25. (canceled)
26. The plant of claim 1, comprising (a) at least one sequence
selected from the group consisting of SEQ ID NOs:379-454 and at
least one sequence selected from the group consisting of SEQ ID
NOs:167, 168, 376, 377, 378, and 455; (b) at least one sequence
selected from the group consisting of SEQ ID NOs:379-454 and at
least one sequence selected from the group consisting of SEQ ID
NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85,
99, 113, 127, and 141; or (c) at least one sequence selected from
the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43,
47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141 and at least
one sequence selected from the group consisting of SEQ ID NOs:167,
168, 376, 377, 378, and 455.
27. The plant of claim 1, wherein the plant comprises at least one
heterologous nucleic acid sequence that confers viral resistance
selected from the group consisting of a) a nucleic acid sequence
that encodes an RNA sequence that is complementary to all or a part
of a first target gene; b) a nucleic acid sequence that comprises
multiple copies of at least one anti-sense DNA segment that is
anti-sense to at least one segment of said at least one first
target gene; c) a nucleic acid sequence that comprises a sense DNA
segment from at least one target gene; d) a nucleic acid sequence
that comprises multiple copies of at least one sense DNA segment of
a target gene; e) a nucleic acid sequence that transcribes to RNA
for suppressing a target gene by forming double-stranded RNA and
that comprises at least one segment that is anti-sense to all or a
portion of the target gene and at least one sense DNA segment that
comprises a segment of said target gene; f) a nucleic acid sequence
that transcribes to RNA for suppressing a target gene by forming a
single double-stranded RNA that comprises multiple serial
anti-sense DNA segments that are anti-sense to at least one segment
of the target gene and multiple serial sense DNA segments that
comprise at least one segment of said target gene; g) a nucleic
acid sequence that transcribes to RNA for suppressing a target gene
by forming multiple double strands of RNA and comprises multiple
segments that are anti-sense to at least one segment of said target
gene and multiple sense DNA segments of the target gene, and
wherein said multiple anti-sense DNA segments and said multiple
sense DNA segments are arranged in a series of inverted repeats; h)
a nucleic acid sequence that comprises nucleotides derived from a
plant miRNA; and i) a nucleic acid sequence encoding at least one
tospovirus terminal sequence that interferes with virion
assembly.
28. The plant of claim 27, further comprising a non-transgenic
plant virus resistance trait.
29. The plant of claim 27, wherein expression of the at least one
heterologous nucleic acid sequence results in resistance to two or
more viruses selected from the group consisting of: tospoviruses,
begomoviruses, and potexviruses.
30. A transgenic seed or cell of the plant of claim 1.
31. A method for conferring resistance in a tomato plant to a
plurality of plant virus species, the method comprising expressing
in the plant at least two nucleic acid sequences that collectively
provide resistance to said plurality of plant virus species,
wherein at least 2 different modes of action are utilized to
provide such resistance, comprising expression of at least two
sequences selected from the group consisting of: dsRNA, miRNA, and
a sequence which interferes with virion assembly.
32. The method of claim 31, wherein: (a) the resistance comprises
resistance against a begomovirus, tospovirus or potexvirus; (b) the
resistance provided to at least one of the plant virus species is
provided by expression of a nucleic acid construct that produces
dsRNA; (c) the resistance provided to at least one of the plant
virus species is provided by expression of a dsRNA fusion
construct; (d) the resistance provided to at least one of the plant
virus species is provided by expression of a nucleic acid construct
that produces miRNA; (e) the resistance against a begomovirus or
tospovirus is provided by a sequence encoded by a stacked miRNA
expression cassette; or (f) the resistance provided to at least one
of the plant virus species is provided by expression of a
tospovirus genome segment terminal sequence that inhibits virion
assembly.
33-34. (canceled)
35. The method of claim 32, wherein the dsRNA interferes with
expression of a virus coat protein gene, a virus movement protein
gene or a virus replication gene; or wherein the nucleic acid
construct comprises a sequence selected from the group consisting
of SEQ ID NOs:379-455.
36.-38. (canceled)
39. The method of claim 32, wherein the miRNA interferes with
expression of a virus coat protein gene, a virus movement protein
gene or a virus replication gene; or wherein resistance provided to
at least one of the plant virus species is provided by expression
of a nucleic acid construct that produces miRNA.
40. (canceled)
41. The method of claim 31, wherein: (a) resistance against a
begomovirus is provided by expression of dsRNA which interferes
with expression of a begomovirus replication gene; (b) resistance
against a tospovirus or potexvirus is provided by expression of a
dsRNA which interferes with expression of a virus coat protein gene
or virus movement protein gene; (c) resistance against a potexvirus
is provided by expression of a nucleic acid construct which
produces miRNA; or (d) resistance against a begomovirus or
tospovirus is provided by a sequence encoded by a stacked miRNA
expression cassette.
42. (canceled)
43. The method of claim 31, wherein resistance provided to at least
one of said plant virus species is provided by inhibiting virion
assembly, wherein virion assembly is inhibited by a sequence
comprised within a nucleic acid construct comprising a first
nucleic acid segment and a second nucleic acid segment, wherein the
first and second segments are substantially inverted repeats of
each other and are linked together by a third nucleic acid segment,
and wherein the third segment comprises at least one terminal
sequence of a tospovirus genome segment, expression of which
inhibits virion assembly.
44. The method of claim 43, wherein the third nucleic acid
comprises a tospovirus genome terminal sequence selected from the
group consisting of: a terminal sequence of a CaCV or GBNV L genome
segment, a terminal sequence of a CaCV or GBNV M genome segment, a
terminal sequence of a CaCV or GBNV S genome segment, and a
tospovirus genome terminal repeat sequence.
45. The method of claim 44, wherein the terminal sequence or
terminal repeat sequence comprises SEQ ID NO:167, SEQ ID NO:168,
SEQ ID NO:376, SEQ ID NO:377, or SEQ ID NO: 378.
46. The method of claim 31, wherein the plurality of plant virus
species are selected from at least two of the Geminiviridae,
Bunyaviridae and Flexiviridae families; or wherein the viruses are
selected from the genera Potexvirus, Tospovirus, and
Begomovirus.
47. (canceled)
48. The method of claim 47, wherein the viruses are selected from
the group consisting of: a) one or more of TYLCV, ToSLCV, ToLCNDV,
PHYVV, PepGMV; b) one or more of TSWV, GBNV, CaCV; and c)
PepMV.
49. The method of claim 31, wherein the nucleic acid sequence
comprises at least one gene suppression element for suppressing at
least one first target gene.
50. The method of claim 31, comprising expressing in the plant: a)
at least one sequence selected from the group consisting of SEQ ID
NOs:379-454 and at least one sequence selected from the group
consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455; (b) at
least one sequence selected from the group consisting of SEQ ID
NOs:379-454 and at least one sequence selected from the group
consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55,
59, 63, 67, 71, 85, 99, 113, 127, and 141; or (c) at least one
sequence selected from the group consisting of SEQ ID NOs:1, 7, 13,
19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127,
and 141 and at least one sequence selected from the group
consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455.
51. (canceled)
Description
[0001] This application claims the priority of U.S. Provisional
Appl. Ser. No. 61/171,021, filed Apr. 20, 2009, the entire
disclosure of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to methods and
compositions for enhancing resistance to multiple plant
viruses.
2. Description of Related Art
[0003] Solanaceous plants are subject to multiple potential disease
causing agents, including virus-induced diseases that are
responsible for major crop losses worldwide. For many RNA viruses,
expression of transgenic coat protein (CP) or replicase blocks the
progression of the virus infectious process. RNA-based resistance
makes use of the plant post-transcriptional gene silencing (PTGS)
mechanism to degrade viral RNAs. However, such approaches may yield
resistance that is narrowly based and/or not durable, especially
with rapidly spreading/evolving new viral species or isolates. In
some instances, classically-defined (non-transgenic) resistance
traits are available to aid in development of virus resistant
plants. Additionally, control of plant pests, such as insects that
serve to transmit plant viruses, may help to limit losses due to
viral infection of plants.
BRIEF DESCRIPTION OF THE FIGURES
[0004] FIG. 1: Schematic diagram of genome organization of viruses
of interest.
[0005] FIG. 2A: Schematic diagram illustrating approach for
identifying sequence efficacious for plant virus control.
[0006] FIG. 2B: Schematic diagram of a typical begomovirus DNA-A
genome showing location of regions screened for effectiveness for
viral control when expressed as inverted repeats. Numbered gray
arrows represent portions of genome that were tested.
[0007] FIG. 2C: Schematic diagram of a typical potexvirus (Pepino
mosaic virus; PepMV) genome showing location of regions screened
for effectiveness for viral control when expressed as inverted
repeats. Numbered gray arrows represent portions of genome that
were tested.
[0008] FIG. 2D: Schematic diagram of a tospovirus (e.g. Tomato
spotted wilt virus (TSWV) genome showing location of regions
screened for effectiveness for viral control when expressed as
inverted repeats. Numbered gray arrows represent portions of genome
that were tested.
[0009] FIG. 3: Virus resistance correlating with siRNA production
in transformed tomato plants.
[0010] FIG. 4: Exemplary artificial dsRNA fusion constructs for
conferring multiple virus resistance ("MVR").
[0011] FIG. 5: Suitable 21nt sequences (among SEQ ID NOs:1-42) that
were analyzed against five targeted Geminiviruses (perfect match:
double underline; G:U mis-match: single underline; other mis-match
or not utilized: not underlined).
[0012] FIG. 6: Suitable 21nt sequences (among SEQ ID NOs:43-70)
that were analyzed against tospoviruses (perfect match: double
underline; G:U mis-match: single underline; other mis-match or not
utilized: not underlined).
[0013] FIGS. 7A, 7B: Suitable 21nt sequences (among SEQ ID
NOs:71-154) that were analyzed against targeted potexviruses
(perfect match: double underline; G:U mis-match: single underline;
other mis-match or not utilized: not underlined).
[0014] FIG. 8: Schematic of exemplary construct for deploying
multiple engineered miRNAs in one transgenic cassette, such as with
phased siRNAs.
[0015] FIG. 9: Schematic diagram illustrating expression cassette
for deploying multiple modes of action for virus resistance.
[0016] FIG. 10: Additional exemplary constructs for deploying
multiple engineered miRNAs in a transgenic cassette, as well as for
expressing miRNA along with dsRNA.
[0017] FIGS. 11A, 11B: Scanning of regions of the tospovirus genome
to define segments which may be expressed as dsRNA with anti-viral
efficacy. X axis represents individual events and target regions
(CP: coat protein; GP: envelope glycoprotein; and RdRP:
RNA-dependent RNA polymerase). Y axis represents % of transgenic
R.sub.1 plants displaying virus resistance. FIG. 11A: TSWV results;
FIG. 11B: CaCV and GBNV results. "CP4+" refers to presence of the
selectable marker gene linked to the dsRNA-encoding sequence, in
R.sub.1 plants.
[0018] FIG. 12: Regions of the PepMV genome tested for
effectiveness in generating dsRNA-mediated resistance against this
potexvirus (CP: coat protein; Mov: movement protein; RdRP, or RdR:
RNA-dependent RNA polymerase; TGB: Triple gene block protein).
[0019] FIGS. 13A, 13B: Regions of geminivirus genome assayed for
effectiveness in generating dsRNA-mediated resistance against this
virus group. (CP: coat protein; Rep: replication protein). Other
transgenic plants contain a glyphosate resistance gene. "0%-100%"
denotes the percentage of R.sub.1 plants that are selectable-marker
positive and virus resistant. FIG. 13A: representative results for
TYLCV and ToSLCV; FIG. 13B: representative results for PepGMV,
PHYVV, and ToLCNDV. For PHYVV and ToLCNDV events, "Spc" refers to
the presence of a selectable marker gene conferring spectinomycin
resistance. Other events were transformed with a construct
comprising a selectable marker gene conferring glyphosate
resistance ("CP4 positive").
[0020] FIG. 14: Schematic diagram illustrating representative
expression cassettes for targeting of multiple viruses in multiple
virus families.
[0021] FIG. 15: Results of using an artificial dsRNA fusion
construct targeting CP expression of tospoviruses and PepMV
(potexvirus), for multiple virus resistance as discussed in Example
3. Construct used is schematically shown in FIG. 4B, bottom
construct: TSWV (160 bp), TSWV (296 bp), PepMV (231 bp), CaCV/GBNV
(232 bp).
[0022] FIG. 16: Depicts resistance observed against inoculated CaCV
in inoculated CP4 positive R.sub.1 plants transformed with a
construct comprising tospovirus terminal repeat sequences (SEQ ID
NOs:167, 168, 376, 377, 378, as found in SEQ ID NO:455).
SUMMARY OF THE INVENTION
[0023] The present invention provides methods and compositions for
obtaining plants resistant to multiple plant viruses. In one
aspect, the present invention provides a tomato plant comprising
resistance to a plurality of plant virus species. In certain
embodiments, the resistance is provided by at least two different
modes of action selected from the group consisting of dsRNA, miRNA,
and inhibition of virion assembly. In other embodiments, the
resistance is provided by at least three different modes of action.
The resistance of the tomato plant may comprise resistance against
begomovirus, tospovirus or potexvirus.
[0024] In certain embodiments, the resistance provided to at least
one of the plant virus species is provided by expression of a
nucleic acid construct that produces dsRNA. In some embodiments the
resistance provided to at least one of the plant virus species is
provided by expression of a dsRNA fusion construct. In some
embodiments of the invention, the dsRNA interferes with expression
of a virus coat protein gene, a virus movement protein gene or a
virus replication gene. In particular embodiments, the nucleic acid
construct which produces dsRNA comprises a sequence selected from
the group consisting of SEQ ID NOs:379-455.
[0025] In other embodiments, the resistance provided to at least
one of the plant virus species is provided by expression of a
nucleic acid construct that produces miRNA. Thus, in certain
embodiments, the resistance against a begomovirus or tospovirus is
provided by a sequence encoded by a stacked miRNA expression
cassette. In yet other embodiments, the miRNA interferes with
expression of a virus coat protein gene, a virus movement protein
gene or a virus replication gene. In particular embodiments, the
miRNA comprises a sequence selected from the group consisting of
SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67,
71, 85, 99, 113, 127, and 141.
[0026] In certain embodiments, the tomato plant comprises (a)
resistance against a begomovirus which is provided by expression of
dsRNA which interferes with expression of a begomovirus replication
gene; (b) resistance against a tospovirus or potexvirus which is
provided by expression of a dsRNA which interferes with expression
of a virus coat protein gene or virus movement protein gene; (c)
resistance against a potexvirus which is provided by expression of
a nucleic acid construct which produces miRNA; or (d) resistance
against a begomovirus or tospovirus which is provided by a sequence
encoded by a stacked miRNA expression cassette.
[0027] In other embodiments, a tomato plant is provided wherein
resistance provided to at least one of the plant virus species is
provided by expression of a tospovirus genome segment terminal
sequence that inhibits virion assembly. In certain embodiments,
resistance provided to at least one of the plant virus species is
provided by inhibiting virion assembly, wherein virion assembly is
inhibited by a sequence comprised within a nucleic acid construct
comprising a first nucleic acid segment and a second nucleic acid
segment, wherein the first and second segments are substantially
inverted repeats of each other and are linked together by a third
nucleic acid segment, and wherein the third segment comprises at
least one terminal sequence of a tospovirus genome segment that
inhibits virion assembly. In particular embodiments, the third
nucleic acid comprises a tospovirus genome terminal sequence
selected from the group consisting of: a terminal sequence of a
CaCV or GBNV L genome segment, a terminal sequence of a CaCV or
GBNV M genome segment, a terminal sequence of a CaCV or GBNV S
genome segment, a tospovirus genome terminal repeat sequence, a
nucleic acid sequence comprising SEQ ID NO: 167, a nucleic acid
sequence comprising SEQ ID NO:168, a nucleic acid sequence
comprising SEQ ID NO:376, a nucleic acid sequence comprising SEQ ID
NO:377, a nucleic acid sequence comprising SEQ ID NO:378, and a
nucleic acid sequence comprising SEQ ID NO: 455.
[0028] In certain embodiments, the tomato plant comprises
resistance to viruses of at least two of the Geminiviridae,
Bunyaviridae and Flexiviridae families. Thus, in some embodiments
the viruses are selected from the genera Potexvirus, Tospovirus,
and Begomovirus. In particular embodiments, the viruses are
selected from the group consisting of: a) at least one of TYLCV,
ToSLCV, ToLCNDV, PHYVV, PepGMV; b) one or more of TSWV, GBNV, CaCV;
and c) PepMV. In a more particular embodiment, the potexvirus is
Pepino mosaic virus. In certain embodiments, the begomovirus is
TYLCV, ToLCNDV, PHYVV, ToSLCV, or PepGMV. In some embodiments, the
tospovirus is CaCV, GBNV, or TSWV. In particular embodiments, the
begomovirus is TYLCV and the potexvirus is Pepino mosaic virus; or
the tospovirus is TSWV and the potexvirus is Pepino mosaic virus;
or the wherein the begomovirus is TYLCV, the potexvirus is Pepino
mosaic virus, and the tospovirus is TSWV.
[0029] In some embodiments, the tomato plant may comprise a
sequence selected from the group consisting of SEQ ID NOs:156, 158,
160, 162, 164, 166, and 363-375. In those or other embodiments, the
tomato plant comprises, or further comprises, a sequence selected
from the group consisting of: SEQ ID NOs:1, 7, 13, 19, 25, 31, 37,
43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, 141, and 379-454.
Thus, a tomato plant of the invention may comprise: (a) at least
one sequence selected from the group consisting of SEQ ID
NOs:379-454 and at least one sequence selected from the group
consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455; (b) at
least one sequence selected from the group consisting of SEQ ID
NOs:379-454 and at least one sequence selected from the group
consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55,
59, 63, 67, 71, 85, 99, 113, 127, and 141; or (c) at least one
sequence selected from the group consisting of SEQ ID NOs:1, 7, 13,
19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127,
and 141 and at least one sequence selected from the group
consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455.
[0030] In yet other embodiments, the tomato plant comprises at
least one heterologous nucleic acid sequence that confers viral
resistance selected from the group consisting of a) a nucleic acid
sequence that encodes an RNA sequence that is complementary to all
or a part of a first target gene; b) a nucleic acid sequence that
comprises multiple copies of at least one anti-sense DNA segment
that is anti-sense to at least one segment of said at least one
first target gene; c) a nucleic acid sequence that comprises a
sense DNA segment from at least one target gene; d) a nucleic acid
sequence that comprises multiple copies of at least one sense DNA
segment of a target gene; e) a nucleic acid sequence that
transcribes to RNA for suppressing a target gene by forming
double-stranded RNA and that comprises at least one segment that is
anti-sense to all or a portion of the target gene and at least one
sense DNA segment that comprises a segment of said target gene; f)
a nucleic acid sequence that transcribes to RNA for suppressing a
target gene by forming a single double-stranded RNA that comprises
multiple serial anti-sense DNA segments that are anti-sense to at
least one segment of the target gene and multiple serial sense DNA
segments that comprise at least one segment of said target gene; g)
a nucleic acid sequence that transcribes to RNA for suppressing a
target gene by forming multiple double strands of RNA and comprises
multiple segments that are anti-sense to at least one segment of
said target gene and multiple sense DNA segments of the target
gene, and wherein said multiple anti-sense DNA segments and said
multiple sense DNA segments are arranged in a series of inverted
repeats; h) a nucleic acid sequence that comprises nucleotides
derived from a plant miRNA; and i) a nucleic acid sequence encoding
at least one tospovirus terminal sequence that interferes with
virion assembly. The invention also provides a plant wherein
expression of the at least one heterologous nucleic acid sequence
results in resistance to two or more viruses selected from the
group consisting of: tospoviruses, begomoviruses, and potexviruses.
The plant may also further comprise a non-transgenic plant virus
resistance trait.
[0031] In another aspect of the invention, a transgenic seed is
provided, of any generation of the tomato plant comprising
resistance to a plurality of plant virus species, wherein the
resistance is provided by at least two different modes of action
selected from the group consisting of dsRNA, miRNA, and inhibition
of virion assembly.
[0032] In yet another aspect, the invention provides a method for
conferring resistance in a tomato plant to a plurality of plant
virus species, the method comprising expressing in the plant at
least two nucleic acid sequences that collectively provide
resistance to said plurality of plant virus species, wherein at
least 2 different modes of action are utilized to provide such
resistance, comprising expression of at least two sequences
selected from the group consisting of: dsRNA, miRNA, and a sequence
which interferes with virion assembly. In certain embodiments, the
resistance comprises resistance against a begomovirus, tospovirus
or potexvirus.
[0033] The resistance may be provided to at least one of the plant
virus species by expression of a nucleic acid construct that
produces dsRNA. In particular embodiments, resistance provided to
at least one of the plant virus species is provided by expression
of a dsRNA fusion construct. In more particular embodiments, the
dsRNA interferes with expression of a virus coat protein gene, a
virus movement protein gene or a virus replication gene. In yet
more particular embodiments, the nucleic acid construct comprises a
sequence selected from the group consisting of SEQ ID
NOs:379-455.
[0034] In other embodiments, resistance provided to at least one of
the plant virus species is provided by expression of a nucleic acid
construct that produces miRNA. In one embodiment, it is
contemplated that resistance against a begomovirus or tospovirus is
provided by a sequence encoded by a stacked miRNA expression
cassette. The produced miRNA may further interfere with expression
of a virus coat protein gene, a virus movement protein gene or a
virus replication gene. In particular embodiments, the miRNA
comprises a sequence selected from the group consisting of SEQ ID
NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85,
99, 113, 127, and 141.
[0035] Thus, in certain embodiments, (a) resistance against a
begomovirus is provided by expression of dsRNA which interferes
with expression of a begomovirus replication gene; (b) resistance
against a tospovirus or potexvirus is provided by expression of a
dsRNA which interferes with expression of a virus coat protein gene
or virus movement protein gene; (c) resistance against a potexvirus
is provided by expression of a nucleic acid construct which
produces miRNA; or (d) resistance against a begomovirus or
tospovirus is provided by a sequence encoded by a stacked miRNA
expression cassette.
[0036] In some embodiments resistance provided to at least one of
the plant virus species is provided by expression of a tospovirus
genome segment terminal sequence that inhibits virion assembly. In
certain embodiments, resistance is provided to at least one of said
plant virus species by inhibiting virion assembly, wherein virion
assembly is inhibited by a sequence comprised within a nucleic acid
construct comprising a first nucleic acid segment and a second
nucleic acid segment, wherein the first and second segments are
substantially inverted repeats of each other and are linked
together by a third nucleic acid segment, and wherein the third
segment comprises at least one terminal sequence of a tospovirus
genome segment, expression of which inhibits virion assembly.
Further, in particular embodiments, the third nucleic acid may
comprise a tospovirus genome terminal sequence selected from the
group consisting of: a terminal sequence of a CaCV or GBNV L genome
segment, a terminal sequence of a CaCV or GBNV M genome segment, a
terminal sequence of a CaCV or GBNV S genome segment, and a
tospovirus genome terminal repeat sequence. In more particular
embodiments, the terminal sequence or terminal repeat sequence
comprises SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:376, SEQ ID
NO:377, or SEQ ID NO: 378.
[0037] In other embodiments of the invention, the plurality of
plant virus species are selected from at least two of the
Geminiviridae, Bunyaviridae and Flexiviridae families. Thus, the
viruses may be selected from the genera Potexvirus, Tospovirus, and
Begomovirus. In certain embodiments, the viruses are selected from
the group consisting of: a) one or more of TYLCV, ToSLCV, ToLCNDV,
PHYVV, PepGMV; b) one or more of TSWV, GBNV, CaCV; and c)
PepMV.
[0038] In other embodiments, the nucleic acid sequence comprises at
least one gene suppression element for suppressing at least one
first target gene. For instance, the method may comprise expressing
in the plant: a) at least one sequence selected from the group
consisting of SEQ ID NOs:379-454 and at least one sequence selected
from the group consisting of SEQ ID NOs:167, 168, 376, 377, 378,
and 455; (b) at least one sequence selected from the group
consisting of SEQ ID NOs:379-454 and at least one sequence selected
from the group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37,
43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141; or (c)at
least one sequence selected from the group consisting of SEQ ID
NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85,
99, 113, 127, and 141 and at least one sequence selected from the
group consisting of SEQ ID NOs:167, 168, 376, 377, 378, and
455.
[0039] Another aspect of the invention provides a transgenic cell
of the tomato plant comprising resistance to a plurality of plant
virus species, wherein the resistance is provided by at least two
different modes of action selected from the group consisting of
dsRNA, miRNA, and inhibition of virion assembly.
[0040] The term "about" is used to indicate that a value includes
the standard deviation of error for the device or method being
employed to determine the value. The use of the term "or" in the
claims is used to mean "and/or" unless explicitly indicated to
refer to alternatives only or the alternatives are mutually
exclusive, although the disclosure supports a definition that
refers to only alternatives and to "and/or." When not used in
conjunction closed wording in the claims or specifically noted
otherwise, the words "a" and "an" denote "one or more." The term
"conferred by a transgene," for example, thus encompasses one ore
more transgene(s).
[0041] The terms "comprise," "have" and "include" are open-ended
linking verbs. Any forms or tenses of one or more of these verbs,
such as "comprises," "comprising," "has," "having," "includes" and
"including," are also open-ended. For example, any method that
"comprises," "has" or "includes" one or more steps is not limited
to possessing only those one or more steps and also covers other
unlisted steps. Similarly, any plant that "comprises," "has" or
"includes" one or more traits is not limited to possessing only
those one or more traits and covers other unlisted traits.
[0042] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and any specific examples provided, while indicating
specific embodiments of the invention, are given by way of
illustration only, since various changes and modifications within
the spirit and scope of the invention will become apparent to those
skilled in the art from this detailed description.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The following is a detailed description of the invention
provided to aid those skilled in the art in practicing the present
invention. Those of ordinary skill in the art may make
modifications and variations in the embodiments described herein
without departing from the spirit or scope of the present
invention.
[0044] The present invention provides methods and compositions for
genetic control of virus diseases in plants, including Solanaceous
plants such as tomato (i.e. Lycopersicon or Solanum sp.), pepper
(i.e. Capsicum sp.), petunia (i.e. Petunia sp.), and potato and
eggplant (i.e. Solanum sp). In one embodiment RNA-mediated gene
suppression can be conferred by the expression of an
inverted-repeat transgene cassette that generates a population of
small interfering RNAs (siRNAs) derived from the dsRNA region of a
transgene transcript. Another RNA-mediated approach for gene
suppression is by expression of one or more miRNA segments that
"target" specific transcripts and lead to their degradation. Thus
approaches including engineering dsRNA, miRNA, ta-siRNA and/or
phased siRNA may be utilized in accordance with the invention. For
instance, begomovirus-derived, or other virus-derived sequences
targeting replication, coat protein, and C2 and/or C3 proteins may
also be utilized. Likewise, for control of potexviruses such as
Pepino mosaic virus, sequences targeting portions of coat (capsid)
protein ("CP"), replication protein such as RNA-dependent RNA
polymerase ("RdRP"), and/or one or more movement protein(s) ("MP")
which may include a triple gene block ("TGB") or a "30K" MP may be
used. For control of tospoviruses, sequences targeting, for
instance, the coat protein ("CP", also termed the nucleocapsid, "N"
protein), RdRP, movement protein ("NsM"), and/or non-structural
glyocoprotein(s) (encoded by "G1" or G2" genes) may similarly be
utilized. Such sequences may correspond exactly to sequences from
one or more viral isolates, or may be variants, for instance
designed to increase their antiviral efficacy, or avoid non-target
effects.
[0045] In certain embodiments, multiple virus resistance ("MVR") is
achieved by utilizing dsRNA and/or miRNA expressed from a single
transformed construct, or more than one construct. Further, a
single construct may comprise one or more expression cassettes that
produce dsRNA and/or miRNA that targets one or more functions
necessary for plant viral infection, multiplication, and/or
transmission, as well as, in certain embodiments, one or more
expression cassettes that produce at least one mRNA that encodes a
protein, or portion of a protein, being targeted. Thus, resistance
to multiple plant viruses may be achieved in a single transgenic
"event." RNA mediated resistance may further be enhanced by protein
based approaches utilizing aptamer(s) that inhibit replication,
expression or mutation in replicase or replication associated
proteins, ssDNA binding proteins such as m13-G5 (e.g. U.S. Pat. No.
6,852,907; Padidam et al., 1999), for geminivirus resistance, or a
peptide aptamer that interferes with geminivirus replication may
also be employed (e.g. Lopez-Ochoa et al., 2006).
[0046] It is also contemplated that inhibition of virion assembly,
for instance by a nucleic-acid based approach, may be utilized as a
mode of action in providing virus resistance to a tomato plant.
This inhibition of virion assembly may be provided, for instance,
by use of a tospovirus terminal sequence, such as a terminal repeat
sequence. By "inhibition of virion assembly" is meant interference
with the interaction between viral capsid proteins and nucleic
acid(s) which together may form a viral particle ("virion"). Such
interference may occur, for instance, by expression of a sequence
that can serve as an artificial substrate competing for reverse
transcriptase, and/or may occur by interference with proper
circularization of replicating viral genome components.
[0047] Additionally, classical genetic resistance loci for
tolerance in tomato, peppers, and other Solanaceous plants may be
utilized, for instance through classical breeding approaches. In
certain embodiments, protein-based approaches using tospovirus "N"
gene (nucleocapsid; coat protein) plus/minus inverted repeats and a
potexvirus (e.g. Pepino mosaic virus) coat protein (CP) and
replicase for resistance, are also provided.
[0048] Methods of gene suppression may include use of anti-sense,
co-suppression, and RNA interference. Anti-sense gene suppression
in plants is described by Shewmaker et al. in U.S. Pat. Nos.
5,107,065, 5,453,566, and 5,759,829. Gene suppression in bacteria
using DNA which is complementary to mRNA encoding the gene to be
suppressed is disclosed by Inouye et al. in U.S. Pat. Nos.
5,190,931, 5,208,149, and 5,272,065. RNA interference or
RNA-mediated gene suppression has been described by, e.g.,
Redenbaugh et al., 1992; Chuang et al., 2000; and Wesley et al.,
2001.
[0049] Several cellular pathways involved in RNA-mediated gene
suppression have been described, each distinguished by a
characteristic pathway and specific components. See, for example,
reviews by Brodersen and Voinnet (2006), and Tomari and Zamore
(2005). The siRNA pathway involves the non-phased cleavage of a
double-stranded RNA to small interfering RNAs ("siRNAs"). The
microRNA pathway involves microRNAs ("miRNAs"), non-protein coding
RNAs generally of between about 19 to about 25 nucleotides
(commonly about 20-24 nucleotides in plants) that guide cleavage in
trans of target transcripts, negatively regulating the expression
of genes involved in various regulation and development pathways;
see Ambros et al. (2003). Plant miRNAs have been defined by a set
of characteristics including a paired stem-loop precursor that is
processed by DCL1 to a single specific .about.21-nucleotide miRNA,
expression of a single pair of miRNA and miRNA* species from the
double-stranded RNA precursor with two-nucleotide 3' overhangs, and
silencing of specific targets in trans (see Bartel (2004); Kim
(2005); Jones-Rhoades et al. (2006); Ambros et al. (2003)). In the
trans-acting siRNA ("ta-siRNA") pathway, miRNAs serve to guide
in-phase processing of siRNA primary transcripts in a process that
requires an RNA-dependent RNA polymerase for production of a
double-stranded RNA precursor; trans-acting siRNAs are defined by
lack of secondary structure, a miRNA target site that initiates
production of double-stranded RNA, requirements of DCL4 and an
RNA-dependent RNA polymerase (RDR6), and production of multiple
perfectly phased .about.21-nt small RNAs with perfectly matched
duplexes with 2-nucleotide 3' overhangs (see Allen et al.,
2005).
[0050] Many microRNA genes (MIR genes) have been identified and
made publicly available in a database ("miRBase", available online
at www.microrna.sanger.ac.uk/sequences; also see Griffiths-Jones et
al. (2003)). Additional MIR genes and mature miRNAs are also
described in U.S. Patent Application Publications 2005/0120415 and
2005/144669, which are incorporated by reference herein. MIR gene
families appear to be substantial, estimated to account for 1% of
at least some genomes and capable of influencing or regulating
expression of about a third of all genes (see, for example, Tomari
et al. (2005); Tang (2005); and Kim (2005)). MIR genes have been
reported to occur in intergenic regions, both isolated and in
clusters in the genome, but can also be located entirely or
partially within introns of other genes (both protein-coding and
non-protein-coding). For a recent review of miRNA biogenesis, see
Kim (2005). Transcription of MIR genes can be, at least in some
cases, under control of a MIR gene's own promoter. The primary
transcript, termed a "pri-miRNA", can be quite large (several
kilobases) and can be polycistronic, containing one or more
pre-miRNAs (fold-back structures containing a stem-loop arrangement
that is processed to the mature miRNA) as well as the usual 5'
"cap" and polyadenylated tail of an mRNA. See, for example, FIG. 1
in Kim (2005).
[0051] A "phased small RNA locus," which transcribes to an RNA
transcript forming a single foldback structure that is cleaved in
phase in vivo into multiple small double-stranded RNAs (termed
"phased small RNAs") capable of suppressing a target gene may also
be employed (e.g. U.S. Patent Application Publication 20080066206).
In contrast to siRNAs, a phased small RNA transcript is cleaved in
phase. In contrast to miRNAs, a phased small RNA transcript is
cleaved by DCL4 or a DCL4-like orthologous ribonuclease (not DCL1)
to produce multiple abundant small RNAs capable of silencing a
target gene. In contrast to the ta-siRNA pathway, the phased small
RNA locus transcribes to an RNA transcript that forms hybridized
RNA independently of an RNA-dependent RNA polymerase and without a
miRNA target site that initiates production of double-stranded RNA.
Novel recombinant DNA constructs that are designed based on a
phased small RNA locus are useful for suppression of one or
multiple target genes, without the use of miRNAs, ta-siRNAs, or
expression vectors designed to form a hairpin structure for
processing to siRNAs. Furthermore, the recognition sites
corresponding to a phased small RNA are useful for suppression of a
target sequence in a cell or tissue where the appropriate phased
small RNA is expressed endogenously or as a transgene.
A. Virus Targets
[0052] In accordance with the invention, methods and compositions
are provided for conferring resistance to multiple viruses to
plants, including Solanaceous plants such as tomatoes. Viruses to
which resistance may be targeted in the present invention include,
without limitation, two or more viruses from among the
geminiviruses, tospoviruses, and potexviruses. FIG. 1 illustrates
the genome organization of representatives of these viral
genera.
[0053] 1. Begomoviruses
[0054] The Geminiviridae are a large, diverse family of plant
viruses that infect a broad variety of plants and cause significant
crop losses worldwide. They are characterized by twin icosahedral
capsids and circular ssDNA genomes that replicate through dsDNA
intermediates. Geminiviruses ("begomovirus" and "geminivirus" are
used interchangeably herein) depend on the plant nuclear DNA and
RNA polymerases for replication and transcription. These viruses
contribute only a few factors for their replication and
transcription. The family Geminiviridae contains three main genera
(formerly termed "subgroups") that differ with respect to insect
vector, host range, and genome structure.
[0055] Geminiviridae Subgroup I (genus Mastrevirus) includes
leafhopper-transmitted viruses that generally infect monocot plants
and have single-component genomes.
[0056] Geminiviridae Subgroup III (genus Begomovirus) includes
whitefly-transmitted viruses that infect dicot plants and most
commonly have bipartite genomes.
[0057] Geminiviridae Subgroup II (genus Curtovirus) viruses are
transmitted by leafhoppers and have single-component genomes like
Subgroup I, but infect dicot plants like subgroup III.
[0058] 2. Tospoviruses
[0059] Viruses in the genus Tospovirus cause significant worldwide
crop losses. The genus name is derived from Tomato spotted wilt
virus ("TSWV"). The Spotted Wilt Disease of tomato was first
observed in Australia in 1915 and was later shown to be of viral
origin. Until the early 1990s TSWV was considered to be the sole
member of the tomato spotted wilt group of plant viruses. The
identification and characterization of several similar viruses,
including Impatiens necrotic spot virus (INSV), Capsicum chlorosis
virus ("CaCV"), Peanut bud necrosis virus (also known as Groundnut
bud necrosis virus, "GBNV"), and Tomato chlorotic spot virus led to
the creation of the plant-infecting Tospovirus genus within the
Bunyaviridae family. This family includes a large group of
predominantly animal-infecting viruses. More than twenty
tospoviruses have since been identified and characterized and
previously unknown species of the genus continue to be described on
a regular basis.
[0060] Tospoviruses have a tripartite RNA genome of ambisense
polarity. The three portions of the genome are termed the "L"
segment, the "M" segment, and the "S" segment. A consensus terminal
sequence of each portion of the RNA genome is found, defined by
segments UCUCGUUAGC (SEQ ID NO:167) at the 3'end and AGAGCAAUCG
(SEQ ID NO:168) at the 5'end. The largest RNA, the "L segment,"
encodes replicase. The medium size RNA, "M segment," encodes
glycoproteins G1 and G2 in the complementary-sense RNA and a
nonstructural protein, NSm, in the genome-sense RNA. The smallest
segment, "S segment," encodes the nucleocapsid protein (N) in the
complementary-sense RNA and a cell-to-cell movement, NSs, in the
genome-sense RNA. The virus is transmitted by thrips in the genera
Frankliniella (five species) and Thrips (three species). Mechanical
transmission of the virus is also possible. TSWV can infect more
than 925 plant species belonging to 70 botanical families, whereas
the other tospovirus species have much narrower host ranges.
[0061] 3. Potexviruses
[0062] The Pepino mosaic virus (PepMV) is a representative
potexvirus from among the Flexiviridae, and is highly contagious
with a significant potential to cause damage in protected tomato
production. Significant crop losses are possible if action is not
taken to eliminate infection. The virus is readily spread via
contaminated tools, human hands or clothing, and by direct
plant-to-plant contact. It can also be transmitted by grafting or
when taking cuttings from infected mother plants. The use of coat
protein-mediated resistance may provide good resistance. However,
including inverted repeats to the CP may enhance resistant line
production.
B. Nucleic Acid Compositions and Constructs
[0063] The invention provides recombinant DNA constructs and
methods for use in achieving resistance to multiple (i.e. more than
one) viral species and strains from among the begomoviruses,
tospoviruses, and potexviruses in transgenic plants. In certain
embodiments, resistance is conferred to 2, 3, 4, 5, 6, 7, 8, or
more viral species selected from at least two of the following
groups: begomoviruses, tospoviruses, and potexviruses. The
resistance may be directed by production of siRNA or miRNA, and may
also be complemented by protein based approaches such as resistance
mediated by expressed coat protein or replicase, mutated forms of
replicases, and production of aptamers. Genetically based tolerance
(i.e. as identified in a classical breeding approach) may also be
utilized.
[0064] As used herein, the term "nucleic acid" refers to a single
or double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. The "nucleic acid" may also
optionally contain non-naturally occurring or altered nucleotide
bases that permit correct read through by a polymerase and do not
reduce expression of a polypeptide encoded by that nucleic acid.
The term "nucleotide sequence" or "nucleic acid sequence" refers to
both the sense and antisense strands of a nucleic acid as either
individual single strands or in the duplex. The term "ribonucleic
acid" (RNA) is inclusive of dsRNA (double stranded RNA), siRNA
(small interfering RNA), shRNA (small hairpin RNA), mRNA (messenger
RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged or
discharged with a corresponding acylated amino acid), and cRNA
(complementary RNA) and the term "deoxyribonucleic acid" (DNA) is
inclusive of cDNA and genomic DNA and DNA-RNA hybrids. The words
"nucleic acid segment," "nucleotide sequence segment," or more
generally "segment" will be understood by those in the art as a
functional term that includes both genomic sequences, ribosomal RNA
sequences, transfer RNA sequences, messenger RNA sequences, operon
sequences and smaller engineered nucleotide sequences that express
or may be adapted to express, proteins, polypeptides or
peptides.
[0065] As used herein, the term "substantially homologous" or
"substantial homology," with reference to a nucleic acid sequence,
includes a nucleotide sequence that hybridizes under stringent
conditions to any of SEQ ID NOs:169-455, or a portion or complement
thereof, are those that allow an antiparallel alignment to take
place between the two sequences, and the two sequences are then
able, under stringent conditions, to form hydrogen bonds with
corresponding bases on the opposite strand to form a duplex
molecule that is sufficiently stable under conditions of
appropriate stringency, including high stringency, to be detectable
using methods well known in the art. Substantially homologous
sequences may have from about 70% to about 80% sequence identity,
or more preferably from about 80% to about 85% sequence identity,
or most preferable from about 90% to about 95% sequence identity,
to about 99% sequence identity, to the referent nucleotide
sequences as set forth the sequence listing, or the complements
thereof.
[0066] As used herein, the term "ortholog" refers to a gene in two
or more species that has evolved from a common ancestral nucleotide
sequence, and may retain the same function in the two or more
species.
[0067] As used herein, the term "sequence identity," "sequence
similarity" or "homology" is used to describe sequence
relationships between two or more nucleotide sequences. The
percentage of "sequence identity" between two sequences is
determined by comparing two optimally aligned sequences over a
comparison window such as the full length of a referenced SEQ ID
NO, wherein the portion of the sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison, and multiplying the result by 100 to yield
the percentage of sequence identity. A sequence that is identical
at every position in comparison to a reference sequence is said to
be identical to the reference sequence and vice-versa. A first
nucleotide sequence when observed in the 5' to 3' direction is said
to be a "complement" of, or complementary to, a second or reference
nucleotide sequence observed in the 3' to 5' direction if the first
nucleotide sequence exhibits complete complementarity with the
second or reference sequence. As used herein, nucleic acid sequence
molecules are said to exhibit "complete complementarity" when every
nucleotide of one of the sequences read 5' to 3' is complementary
to every nucleotide of the other sequence when read 3' to 5'. A
nucleotide sequence that is complementary to a reference nucleotide
sequence will exhibit a sequence identical to the reverse
complement sequence of the reference nucleotide sequence. These
terms and descriptions are well defined in the art and are easily
understood by those of ordinary skill in the art.
[0068] As used herein, a "comparison window" refers to a conceptual
segment of at least 6 contiguous positions, usually about 50 to
about 100, more usually about 100 to about 150, in which a sequence
is compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
The comparison window may comprise additions or deletions (i.e.
gaps) of about 20% or less as compared to the reference sequence
(which does not comprise additions or deletions) for optimal
alignment of the two sequences Those skilled in the art should
refer to the detailed methods used for sequence alignment, such as
in the Wisconsin Genetics Software Package Release 7.0 (Genetics
Computer Group, 575 Science Drive Madison, Wis., USA).
[0069] The present invention provides one or more DNA sequences
capable of being expressed as an RNA transcript in a cell or
microorganism to inhibit target gene expression of at least one
plant virus. The sequences comprise a DNA molecule coding for one
or more different nucleotide sequences, wherein each of the
different nucleotide sequences comprises a sense nucleotide
sequence and an antisense nucleotide sequence. The sequences may be
connected by a spacer sequence. The spacer sequence can constitute
part of the sense nucleotide sequence or the antisense nucleotide
sequence or an unrelated nucleotide sequence and forms within the
dsRNA molecule between the sense and antisense sequences. The
spacer sequence may comprise, for example, a sequence of
nucleotides of at least about 10-100 nucleotides in length, or
alternatively at least about 100-200 nucleotides in length, at
least 200-400 about nucleotides in length, or at least about
400-500 nucleotides in length. The sense nucleotide sequence or the
antisense nucleotide sequence may be substantially identical to the
nucleotide sequence of the target gene or a derivative thereof or a
complementary sequence thereto. The dsDNA molecule may be placed
operably under the control of one or more promoter sequences that
function in the cell, tissue or organ of the host expressing the
dsDNA to produce RNA molecules. As used herein, "expressing" or
"expression" and the like refer to transcription of a RNA molecule
from a transcribed polynucleotide. The RNA molecule may or may not
be translated into a polypeptide sequence.
[0070] The invention also provides a DNA sequence for expression in
a cell of a plant that, upon expression of the DNA to RNA and
contact with a plant virus achieves suppression of a target viral
gene or viral replication or symptomatology (i.e. expression of
symptoms). Methods to express a gene suppression molecule in plants
are known (e.g. US Publication 2006/0200878 A1; US Publication
2006/0174380; US Publication 2008/0066206; Niu et al., 2006), and
may be used to express a nucleotide sequence of the present
invention.
[0071] Non-constitutive promoters suitable for use with recombinant
DNA constructs of the invention include spatially specific
promoters, developmentally 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 suppressing expression of the first
target RNA in plastids, roots, pollen, or seeds, respectively). In
many cases a seed-specific, embryo-specific, aleurone-specific, or
endosperm-specific promoter is especially useful. Developmentally
specific promoters can include promoters that tend to promote
expression during certain developmental stages in a plant's growth
cycle, 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). Also of interest are microRNA promoters, especially
those having a developmentally specific, spatially specific, or
inducible expression pattern. 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."
[0072] Thus, a gene sequence or fragment for plant virus control
according to the invention may be cloned downstream of a promoter
or promoters which are operable in a transgenic plant cell and
therein expressed to produce mRNA in the transgenic plant cell that
form dsRNA molecules. Numerous examples of plant expressible
promoters are known in the art (e.g. CaMV 35S; FMV 35S; PC1SV (e.g.
U.S. Pat. No. 5,850,019); ScBV; AtAct7, among others). Promoters
useful for expression of polypeptides in plants include those that
are inducible, viral, synthetic, or constitutive as described in
Odell et al. (1985), and/or promoters that are temporally
regulated, spatially regulated, and spatio-temporally regulated. A
number of organ-specific promoters have been identified and are
known in the art (e.g. U.S. Pat. Nos. 5,110,732; 5,837,848;
5,459,252; 6,229,067; Hirel et al. 1992). The dsRNA molecules
contained in plant tissues are expressed in a plant so that the
intended suppression of the targeted virus gene expression is
achieved. The cauliflower mosaic virus 35S promoter, an archetypal
strong promoter common in transgenic plant applications, or a
related promoter such as the E35S or the FMV promoter, may be
employed for driving virus resistance genes. Promoters have also
been identified that direct tissue specific gene expression.
[0073] A transgene transcription unit includes DNA sequence
encoding a gene of interest. A gene of interest can include any
coding or non-coding sequence from a virus species. Non-limiting
examples of a non-coding sequence to be expressed by a transgene
transcription unit include, but not limited to, 5' untranslated
regions, promoters, enhancers, or other non-coding transcriptional
regions, 3' untranslated regions, terminators, intron, microRNAs,
microRNA precursor DNA sequences, small interfering RNAs, RNA
components of ribosomes or ribozymes, small nucleolar RNAs, RNA
aptamers capable of binding to a ligand, and other non-coding
RNAs.
[0074] Non-limiting examples of a gene of interest further include,
but are not limited to, translatable (coding) sequence, such as
genes encoding transcription factors and genes encoding enzymes
involved in the biosynthesis or catabolism of molecules of interest
(such as amino acids, fatty acids and other lipids, sugars and
other carbohydrates, biological polymers, and secondary metabolites
including alkaloids, terpenoids, polyketides, non-ribosomal
peptides, and secondary metabolites of mixed biosynthetic origin).
A gene of interest can be a gene native to the cell (e. g., a plant
cell) in which the recombinant DNA construct of the invention is to
be transcribed, or can be a non-native gene. A gene of interest can
be a marker gene, for example, a selectable marker gene encoding
antibiotic, antifungal, or herbicide resistance, or a marker gene
encoding an easily detectable trait (e.g., in a plant cell,
phytoene synthase or other genes imparting a particular pigment to
the plant), or a gene encoding a detectable molecule, such as a
fluorescent protein, luciferase, or a unique polypeptide or nucleic
acid "tag" detectable by protein or nucleic acid detection methods,
respectively). Selectable markers are genes of interest of
particular utility in identifying successful processing of
constructs of the invention.
[0075] Genes of interest include those genes that may be described
as "target genes." The target gene can include a single gene or
part of a single gene that is targeted for suppression, or can
include, for example, multiple consecutive segments of a target
gene, multiple non-consecutive segments of a target gene, multiple
alleles of a target gene, or multiple target genes from one or more
species. The target gene can be translatable (coding) sequence, or
can be non-coding sequence (such as non-coding regulatory
sequence), or both. The transgene transcription unit can further
include 5' or 3' sequence or both as required for transcription of
the transgene. In other embodiments (e. g., where it is desirable
to suppress a target gene across multiple strains or species, for
instance of viruses), it may be desirable to design the recombinant
DNA construct to be processed to a mature miRNA for suppressing a
target gene sequence common to the multiple strains or species in
which the target gene is to be silenced. Thus, the miRNA processed
from the recombinant DNA construct can be designed to be specific
for one taxon (for example, specific to a genus, family, but not
for other taxa.
[0076] The nucleic acid molecules or fragments of the nucleic acid
molecules or other nucleic acid molecules in the sequence listing
are capable of specifically hybridizing to other nucleic acid
molecules under certain circumstances. As used herein, two nucleic
acid molecules are said to be capable of specifically hybridizing
to one another if the two molecules are capable of forming an
anti-parallel, double-stranded nucleic acid structure. A nucleic
acid molecule is said to be the complement of another nucleic acid
molecule if they exhibit complete complementarity. Two molecules
are said to be "minimally complementary" if they can hybridize to
one another with sufficient stability to permit them to remain
annealed to one another under at least conventional
"low-stringency" conditions. Similarly, the molecules are said to
be complementary if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions.
Conventional stringency conditions are described by Sambrook, et
al. (1989), and by Haymes et al. (1985).
[0077] Departures from complete complementarity are therefore
permissible for, as long as such departures do not completely
preclude the capacity of the molecules to form a double-stranded
structure. Thus, in order for a nucleic acid molecule or a fragment
of the nucleic acid molecule to serve as a primer or probe it needs
only be sufficiently complementary in sequence to be able to form a
stable double-stranded structure under the particular solvent and
salt concentrations employed.
[0078] Appropriate stringency conditions that promote DNA
hybridization are, for example, for applications requiring high
selectivity, a relatively low salt and/or high temperature
condition, such as provided by about 0.02 M to about 0.15 M NaCl at
temperatures of about 50.degree. C. to about 70.degree. C. A high
stringency condition, for example, is to wash the hybridization
filter at least twice with high-stringency wash buffer
(0.2.times.SSC or 1.times.SSC, 0.1% SDS, 65.degree. C.). Other
conditions, such as 6.0.times.sodium chloride/sodium citrate (SSC)
at about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C., are also known to those skilled in the art or can be
found in Current Protocols in Molecular Biology (1989). For
example, the salt concentration in the wash step can be selected
from a low stringency of about 2.0.times.SSC at 50.degree. C. to a
high stringency of about 0.2.times.SSC at 50.degree. C. In
addition, the temperature in the wash step can be increased from
low stringency conditions at room temperature, about 22.degree. C.,
to high stringency conditions at about 65.degree. C. Both
temperature and salt may be varied, or either the temperature or
the salt concentration may be held constant while the other
variable is changed. A nucleic acid for use in the present
invention may specifically hybridize to one or more of nucleic acid
molecules from a plant virus selected from the group consisting of
a tospovirus, a begomovirus, and a potexvirus, or complements
thereof under such conditions. In specific embodiments, a nucleic
acid for use in the present invention will exhibit at least from
about 80%, or at least from about 90%, or at least from about 95%,
or at least from about 98% or even about 100% sequence identity
with one or more nucleic acid molecules as set forth in the
sequence listing, or a complement thereof.
[0079] Nucleic acids of the present invention may also be
synthesized, either completely or in part, especially where it is
desirable to provide plant-preferred sequences, by methods known in
the art. Thus, all or a portion of the nucleic acids of the present
invention may be synthesized using codons preferred by a selected
host. Species-preferred codons may be determined, for example, from
the codons used most frequently in the proteins expressed in a
particular host species. Other modifications of the nucleotide
sequences may result in mutants having slightly altered
activity.
[0080] DsRNA or siRNA nucleotide sequences comprise double strands
of polymerized ribonucleotide and may include modifications to
either the phosphate-sugar backbone or the nucleoside.
Modifications in RNA structure may be tailored to allow specific
genetic inhibition. In one embodiment, the dsRNA molecules may be
modified through an enzymatic process so that siRNA molecules may
be generated. Alternatively, a construct may be engineered to
express a nucleotide segment for use in an miRNA- or siRNA-mediated
resistance approach. The siRNA can efficiently mediate the
down-regulation effect for some target genes in some pathogens.
This enzymatic process may be accomplished by utilizing an RNAse
III enzyme or a DICER enzyme, present in the cells of an insect, a
vertebrate animal, a fungus or a plant in the eukaryotic RNAi
pathway (Elbashir et al., 2001; Hamilton and Baulcombe, 1999). This
process may also utilize a recombinant DICER or RNAse III
introduced into the cells of a target insect through recombinant
DNA techniques that are readily known to the skilled in the art.
Both the DICER enzyme and RNAse III, being naturally occurring in a
pathogen or being made through recombinant DNA techniques, cleave
larger dsRNA strands into smaller oligonucleotides. The DICER
enzymes specifically cut the dsRNA molecules into siRNA pieces each
of which is about 19-25 nucleotides in length while the RNAse III
enzymes normally cleave the dsRNA molecules into 12-15 base-pair
siRNA. The siRNA molecules produced by the either of the enzymes
have 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3'
hydroxyl termini. The siRNA molecules generated by RNAse III enzyme
are the same as those produced by Dicer enzymes in the eukaryotic
RNAi pathway and are hence then targeted and degraded by an
inherent cellular RNA-degrading mechanism after they are
subsequently unwound, separated into single-stranded RNA and
hybridize with the RNA sequences transcribed by the target gene.
This process results in the effective degradation or removal of the
RNA sequence encoded by the nucleotide sequence of the target gene
in the pathogen. The outcome is the silencing of a particularly
targeted nucleotide sequence within the pathogen. Detailed
descriptions of enzymatic processes can be found in Hannon
(2002).
[0081] A nucleotide sequence of the present invention can be
recorded on computer readable media. As used herein, "computer
readable media" refers to any tangible medium of expression that
can be read and accessed directly by a computer. Such media
include, but are not limited to: magnetic storage media, such as
floppy discs, hard disc, storage medium, and magnetic tape:
[0082] optical storage media such as CD-ROM; electrical storage
media such as RAM and ROM; optical character recognition formatted
computer files, and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate that any of the presently known computer readable
mediums can be used to create a manufacture comprising computer
readable medium having recorded thereon a nucleotide sequence of
the present invention.
[0083] As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate media
comprising the nucleotide sequence information of the present
invention. A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII text file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0084] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium. Software that implements the BLAST
(Altschul et al., 1990) and BLAZE (Brutlag, et al., 1993) search
algorithms on a Sybase system can be used to identify open reading
frames (ORFs) within sequences such as the Unigenes and EST's that
are provided herein and that contain homology to ORFs or proteins
from other organisms. Such ORFs are protein-encoding fragments
within the sequences of the present invention and are useful in
producing commercially important proteins such as enzymes used in
amino acid biosynthesis, metabolism, transcription, translation,
RNA processing, nucleic acid and a protein degradation, protein
modification, and DNA replication, restriction, modification,
recombination, and repair.
[0085] As used herein, a "target," a "target structural motif," or
a "target motif," refers to any rationally selected sequence or
combination of sequences in which the sequences or sequence(s) are
chosen based on a three-dimensional configuration that is formed
upon the folding of the target motif or the nucleotide sequence
thereof, as appropriate. There are a variety of target motifs known
in the art.
C. Nucleic Acid Expression and Target Gene Suppression
[0086] The present invention provides, as an example, a transformed
host plant of a pathogenic target organism, transformed plant cells
and transformed plants and their progeny. The transformed plant
cells and transformed plants may be engineered to express one or
more of the dsRNA, miRNA, or mRNA sequences, under the control of a
heterologous promoter, described herein to provide a
pathogen-protective effect. These sequences may be used for gene
suppression in a pathogen, thereby reducing the level or incidence
of disease caused by the pathogen on a protected transformed host
organism. As used herein the words "gene suppression" are intended
to refer to any of the well-known methods for reducing the levels
of protein produced as a result of gene transcription to mRNA and
subsequent translation of the mRNA.
[0087] Gene suppression is also intended to mean the reduction of
protein expression from a gene or a coding sequence including
posttranscriptional gene suppression and transcriptional
suppression. Posttranscriptional gene suppression is mediated by
the homology between of all or a part of a mRNA transcribed from a
gene or coding sequence targeted for suppression and the
corresponding double stranded RNA used for suppression, and refers
to the substantial and measurable reduction of the amount of
available mRNA available in the cell for binding by ribosomes or
the prevention of translation by the ribosomes. The transcribed RNA
can be in the sense orientation to effect what is called
co-suppression, in the anti-sense orientation to effect what is
called anti-sense suppression, or in both orientations producing a
dsRNA to effect what is called RNA interference (RNAi).
[0088] Gene suppression can also be effective against target genes
in a plant virus that may contact plant material containing gene
suppression agents, specifically designed to inhibit or suppress
the expression of one or more homologous or complementary sequences
of the virus. Post-transcriptional gene suppression by anti-sense
or sense oriented RNA to regulate gene expression in plant cells is
disclosed in U.S. Pat. Nos. 5,107,065, 5,759,829, 5,283,184, and
5,231,020. The use of dsRNA to suppress genes in plants is
disclosed in WO 99/53050, WO 99/49029, U.S. Publication No.
2003/017596, U.S. Patent Application Publication 2004/0029283.
[0089] A beneficial method of post transcriptional gene suppression
versus a plant virus employs both sense-oriented and
anti-sense-oriented, transcribed RNA which is stabilized, e.g., as
a hairpin or stem and loop structure (e.g. U.S. Publication
2007/0259785). A DNA construct for effecting post transcriptional
gene suppression may be one in which a first segment encodes an RNA
exhibiting an anti-sense orientation exhibiting substantial
identity to a segment of a gene targeted for suppression, which is
linked to a second segment encoding an RNA exhibiting substantial
complementarity to the first segment. Such a construct forms a stem
and loop structure by hybridization of the first segment with the
second segment and a loop structure from the nucleotide sequences
linking the two segments (see WO94/01550, WO98/05770, US
2002/0048814, and US 2003/0018993). Co-expression with an
additional target gene segment may also be employed, as noted above
(e.g. WO05/019408).
[0090] According to one embodiment of the present invention, there
is provided an exogenous nucleotide sequence (i.e. not naturally
found in the genome of the host plant cell), for which expression
results in transcription of a RNA sequence that is substantially
similar in sequence to a RNA molecule of a targeted gene of a plant
virus, selected from the group consisting of a tospovirus, a
begomovirus, and a potexvirus, that comprises an RNA sequence
encoded by a nucleotide sequence within the genome of the virus. By
substantially similar is meant that the exogenous RNA sequence is
capable of effecting RNA-mediated gene suppression of a target
sequence in a viral genome. Thus, a down-regulation of the
expression of the nucleotide sequence corresponding to the target
gene is effected.
[0091] In certain embodiments of the invention, expression of a
fragment of at least 21 contiguous nucleotides of a nucleic acid
sequence of any of SEQ ID NOs:169-455, or complements thereof, may
be utilized, including expression of a fragment of up to 21, 36,
60, 100, 550, or 1000 contiguous nucleotides, or sequences
displaying 90-100% identity with such sequences, or their
complements. In specific embodiments, a nucleotide provided by the
invention may comprise a sequence selected from among SEQ ID NOs:
1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99,
113, 127, and 141. In other specific embodiments, a nucleotide
provided by the invention may comprise a sequence selected from
among SEQ ID NOs: 379-455. In yet other embodiments, a nucleotide
provided by the invention may be described as comprising one or
more of nucleotides 1-21, 22-50, 51-100, 101-150, 151-200, 201-250,
251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600,
601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950,
951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,
1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,
1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,
1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 23-75,
76-125, 126-175, 176-225, 226-275, 276-325, 326-375, 376-425,
426-475, 476-525, 526-575, 576-625, 626-675, 676-725, 726-775,
776-825, 826-875, 876-925, 926-975, 976-1025, 1026-1075, 1076-1125,
1126-1175, 1176-1225, 1226-1275, 1276-1325, 1326-1375, 1376-1425,
1426-1475, 1476-1525, 1526-1575, 1576-1625, 1626-1675, 1676-1725,
1726-1775, 1776-1825, 1826-1875, 1876-1925, 1926-1975, 1976-2025,
2026-2075, 2076-2125, 1-550, 200-750, 300-850, 400-950, 500-1050,
600-1150, 700-1250, 800-1350, 900-1450, 1000-1550, 1100-1650,
1200-1750, 1300-1850, 1400-1950, 1500-2050, up to the full length
of the sequence, of one or more of any of SEQ ID NOs:169-455. A
sequence complementary to all or a portion of any one or more of
SEQ ID NOs:169-455, or SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43,
47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141, wherein
expression of said sequence suppresses the expression of any one or
more of gene(s) encoded by a nucleotide sequence of SEQ ID
NOs:169-455, is contemplated. The sequences arrayed for expression
to produce dsRNA can be combined with: (1) sequences designed for
production of miRNA, including in stacked miRNA cassettes; and/or
(2) sequences for inhibition of viral assembly, in order to
synergistically inhibit target viruses. Methods for selecting
specific sub-sequences as targets for miRNA or siRNA-mediated gene
suppression are known in the art (e.g. Reynolds et al., 2004).
[0092] Inhibition of a target gene using dsRNA technology of the
present invention is sequence-specific in that nucleotide sequences
corresponding to the duplex region of the RNA are targeted for
suppression. RNA containing a nucleotide sequences identical to a
portion of the target gene transcript is usually preferred for
inhibition. RNA sequences with insertions, deletions, and single
point mutations relative to the target sequence have also been
found to be effective for inhibition. In performance of the present
invention, the inhibitory dsRNA and the portion of the target gene
may share at least from about 80% sequence identity, or from about
90% sequence identity, or from about 95% sequence identity, or from
about 99% sequence identity, or even about 100% sequence identity.
Alternatively, the duplex region of the RNA may be defined
functionally as a nucleotide sequence that is capable of
hybridizing with a portion of the target gene transcript. A less
than full length sequence exhibiting a greater homology compensates
for a longer less homologous sequence. The length of the identical
nucleotide sequences may be at least about 18, 21, 25, 50, 100,
200, 300, 400, 500 or at least about 1000 bases. Normally, a
sequence of greater than 20-100 nucleotides should be used, though
a sequence of greater than about 200-300 nucleotides may be
preferred, and a sequence of greater than about 500-1000
nucleotides may be especially preferred depending on the size of
the target gene. The invention has the advantage of being able to
tolerate sequence variations that might be expected due to genetic
mutation, strain polymorphism, or evolutionary divergence. The
introduced nucleic acid molecule may not need to be absolutely
homologous to the target sequence, and it may not need to be full
length relative to either the primary transcription product or
fully processed mRNA of the target gene. Therefore, those skilled
in the art need to realize that, as disclosed herein, 100% sequence
identity between the RNA and the target gene is not required to
practice the present invention.
[0093] Inhibition of target gene expression may be quantified by
measuring either the endogenous target RNA or the protein produced
by translation of the target RNA and the consequences of inhibition
can be confirmed by examination of the outward properties of the
cell or organism. Techniques for quantifying RNA and proteins are
well known to one of ordinary skill in the art.
[0094] In certain embodiments gene expression is inhibited by at
least 10%, by at least 33%, by at least 50%, or by at least 80%. In
particular embodiments of the invention gene expression is
inhibited by at least 80%, by at least 90%, by at least 95%, or by
at least 99% within host cells infected by the virus, such a
significant inhibition takes place. Significant inhibition is
intended to refer to sufficient inhibition that results in a
detectable phenotype (e.g., reduction of symptom expression, etc.)
or a detectable decrease in RNA and/or protein corresponding to the
target gene being inhibited.
[0095] DsRNA molecules may be synthesized either in vivo or in
vitro. The dsRNA may be formed by a single self-complementary RNA
strand or from two complementary RNA strands. Endogenous RNA
polymerase of the cell may mediate transcription in vivo, or cloned
RNA polymerase can be used for transcription in vivo or in vitro.
Inhibition may be targeted by specific transcription in an organ,
tissue, or cell type; stimulation of an environmental condition
(e.g., infection, stress, temperature, chemical inducers); and/or
engineering transcription at a developmental stage or age. The RNA
strands may or may not be polyadenylated; the RNA strands may or
may not be capable of being translated into a polypeptide by a
cell's translational apparatus.
[0096] As used herein, the term "disease control agent," or "gene
suppression agent" refers, in certain embodiments, to a particular
RNA molecule consisting of a first RNA segment and a second RNA
segment linked by a third RNA segment. The first and the second RNA
segments lie within the length of the RNA molecule and are
substantially inverted repeats of each other and are linked
together by the third RNA segment. The complementarity between the
first and the second RNA segments results in the ability of the two
segments to hybridize in vivo and in vitro to form a double
stranded molecule, i.e., a stem, linked together at one end of each
of the first and second segments by the third segment which forms a
loop, so that the entire structure forms into a stem and loop
structure, or even more tightly hybridizing structures may form
into a stem-loop knotted structure. The first and the second
segments correspond invariably, but not necessarily respectively,
to a sense and an antisense sequence homologous with respect to the
target RNA transcribed from the target gene in the target virus
that is intended to be suppressed by the dsRNA molecule.
[0097] As used herein, the term "genome" as it applies to a plant
virus or a host encompasses not only viral DNA or RNA, or
chromosomal DNA found within the nucleus, but organelle DNA found
within subcellular components of the cell. The DNA's of the present
invention introduced into plant cells can therefore be either
chromosomally integrated or organelle-localized. The term "genome"
as it applies to bacteria encompasses both the chromosome and
plasmids within a bacterial host cell. The DNA's of the present
invention introduced into bacterial host cells can therefore be
either chromosomally integrated or plasmid-localized.
[0098] It is envisioned that the compositions of the present
invention can be incorporated within the seeds of a plant species
either as a product of expression from a recombinant gene
incorporated into a genome of the plant cells. The plant cell
containing a recombinant gene is considered herein to be a
transgenic event.
D. Recombinant Vectors and Host Cell Transformation
[0099] A recombinant DNA vector may, for example, be a linear or a
closed circular plasmid. The vector system may be a single vector
or plasmid or two or more vectors or plasmids that together contain
the total DNA to be introduced into the genome of the bacterial
host. In addition, a bacterial vector may be an expression vector.
Nucleic acid molecules as set forth in the sequence listing, or
complements or fragments thereof, can, for example, be suitably
inserted into a vector under the control of a suitable promoter
that functions in one or more microbial hosts to drive expression
of a linked coding sequence or other DNA sequence. Many vectors are
available for this purpose, and selection of the appropriate vector
will depend mainly on the size of the nucleic acid to be inserted
into the vector and the particular host cell to be transformed with
the vector. Each vector contains various components depending on
its function (amplification of DNA or expression of DNA) and the
particular host cell with which it is compatible. The vector
components for bacterial transformation generally include, but are
not limited to, one or more of the following: a signal sequence, an
origin of replication, one or more selectable marker genes, and an
inducible promoter allowing the expression of exogenous DNA.
[0100] Expression and cloning vectors generally contain a selection
gene, also referred to as a selectable marker. This gene encodes a
protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Typical selection genes
encode proteins that (a) confer resistance to antibiotics or other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline,
(b) complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. Those cells that are successfully
transformed with a heterologous protein or fragment thereof produce
a protein conferring drug resistance and thus survive the selection
regimen.
[0101] An expression vector for producing a mRNA can also contain
an inducible promoter that is recognized by a host organism and is
operably linked to the nucleic acid. The term "operably linked," as
used in reference to a regulatory sequence and a structural
nucleotide sequence, means that the regulatory sequence causes
regulated expression of the linked structural nucleotide sequence.
"Regulatory sequences" or "control elements" refer to nucleotide
sequences located upstream (5' noncoding sequences), within, or
downstream (3' non-translated sequences) of a structural nucleotide
sequence, and which influence the timing and level or amount of
transcription, RNA processing or stability, or translation of the
associated structural nucleotide sequence. Regulatory sequences may
include promoters, translation leader sequences, introns,
enhancers, stem-loop structures, repressor binding sequences, and
polyadenylation recognition sequences and the like.
[0102] Construction of suitable vectors containing one or more of
the above-listed components employs standard recombinant DNA
techniques. Isolated plasmids or DNA fragments are cleaved,
tailored, and re-ligated in the form desired to generate the
plasmids required. Examples of available bacterial expression
vectors include, but are not limited to, the multifunctional E.
coli cloning and expression vectors such as Bluescript.TM.
(Stratagene, La Jolla, Calif.), in which, for example, a nucleic
acid, or fragment thereof may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke and Schuster, 1989); and the
like.
[0103] The present invention also contemplates transformation of a
nucleotide sequence of the present invention into a plant to
achieve virus-inhibitory levels of expression of one or more RNA
molecules. A transformation vector can be readily prepared using
methods available in the art. The transformation vector comprises
one or more nucleotide sequences that is/are capable of being
transcribed to an RNA molecule and that is/are substantially
homologous and/or complementary to one or more nucleotide sequences
encoded by the genome of the target virus or viruses, such that
upon contact of the RNA transcribed from the one or more nucleotide
sequences by the target plant-parasitic virus, there is
down-regulation of expression of at least one of the respective
nucleotide sequences of the genome of the virus.
[0104] The transformation vector may be termed a dsDNA construct
and may also be defined as a recombinant molecule, a disease
control agent, a genetic molecule or a chimeric genetic construct.
A chimeric genetic construct of the present invention may comprise,
for example, nucleotide sequences encoding one or more antisense
transcripts, one or more sense transcripts, one or more of each of
the aforementioned, wherein all or part of a transcript there from
is homologous to all or part of an RNA molecule comprising an RNA
sequence encoded by a nucleotide sequence within the genome of a
plant virus.
[0105] In one embodiment a plant transformation vector comprises an
isolated and purified DNA molecule comprising a heterologous
promoter operatively linked to one or more nucleotide sequences of
the present invention. The nucleotide sequence includes a segment
coding all or part of an RNA present within a targeted RNA
transcript and may comprise inverted repeats of all or a part of a
targeted RNA. The DNA molecule comprising the expression vector may
also contain a functional intron sequence positioned either
upstream of the coding sequence or even within the coding sequence,
and may also contain a five prime (5') untranslated leader sequence
(i.e., a UTR or 5'-UTR) positioned between the promoter and the
point of translation initiation.
[0106] A plant transformation vector may contain sequences from
more than one gene, thus allowing production of more than one
dsRNA, miRNA, or siRNA for inhibiting expression of genes of the
more than one target virus. For instance a vector or construct may
comprise up to about 8 or 10, or more, nucleic acid segments for
transcription of antiviral sequences, as shown in FIG. 10. One
skilled in the art will readily appreciate that segments of DNA
whose sequence corresponds to that present in different genes can
be combined into a single composite DNA segment for expression in a
transgenic plant. Alternatively, a plasmid of the present invention
already containing at least one DNA segment can be modified by the
sequential insertion of additional DNA segments between the
enhancer and promoter and terminator sequences. In the disease
control agent of the present invention designed for the inhibition
of multiple genes, the genes to be inhibited can be obtained from
the same plant viral strain or species in order to enhance the
effectiveness of the control agent. In certain embodiments, the
genes can be derived from different plant viruses in order to
broaden the range of viruses against which the agent(s) is/are
effective. When multiple genes are targeted for suppression or a
combination of expression and suppression, a polycistronic DNA
element can be fabricated as illustrated and disclosed in U.S.
Publication No. 2004/0029283.
[0107] A recombinant DNA vector or construct of the present
invention may comprise a selectable marker that confers a
selectable phenotype on plant cells. Selectable markers may also be
used to select for plants or plant cells that contain the exogenous
nucleic acids encoding polypeptides or proteins of the present
invention. The marker may encode biocide resistance, antibiotic
resistance (e.g., kanamycin, G418 bleomycin, hygromycin, etc.), or
herbicide resistance (e.g., glyphosate, etc.). Examples of
selectable markers include, but are not limited to, a neo gene
which codes for kanamycin resistance and can be selected for using
kanamycin, G418, etc., a bar gene which codes for bialaphos
resistance; a mutant EPSP synthase gene which encodes glyphosate
resistance; a nitrilase gene which confers resistance to
bromoxynil; a mutant acetolactate synthase gene (ALS) which confers
imidazolinone or sulfonylurea resistance; and a methotrexate
resistant DHFR gene. Multiple selectable markers are available that
confer resistance to ampicillin, bleomycin, chloramphenicol,
gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,
phosphinothricin, puromycin, spectinomycin, rifampicin, and
tetracycline, and the like. Examples of such selectable markers are
illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and
6,118,047.
[0108] A recombinant vector or construct of the present invention
may also include a screenable marker. Screenable markers may be
used to monitor expression. Exemplary screenable markers include a
.beta.-glucuronidase or uidA gene (GUS) which encodes an enzyme for
which various chromogenic substrates are known (Jefferson et al.,
1987); one or more of the various fluorescent proteins (FP) genes
such as green fluorescent protein (GFP), red fluorescent protein
(RFP) or any one of a large family of proteins which typically
fluoresce at a characteristic wavelength; an R-locus gene, which
encodes a product that regulates the production of anthocyanin
pigments (red color) in plant tissues (Dellaporta et al., 1988); a
.beta.-lactamase gene (Sutcliffe et al., 1978), a gene which
encodes an enzyme for which various chromogenic substrates are
known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene
(Ow et al., 1986) a xylE gene (Zukowski et al., 1983) which encodes
a catechol dioxygenase that can convert chromogenic catechols; an
.alpha.-amylase gene (Ikatu et al., 1990); a tyrosinase gene (Katz
et al., 1983) which encodes an enzyme capable of oxidizing tyrosine
to DOPA and dopaquinone which in turn condenses to melanin; an
.alpha.-galactosidase, which catalyzes a chromogenic
.alpha.-galactose substrate.
[0109] Plant transformation vectors for use with the present
invention may for instance include those derived from a Ti plasmid
of Agrobacterium tumefaciens (e.g. U.S. Pat. Nos. 4,536,475,
4,693,977, 4,886,937, 5,501,967 and EP 0 122 791). Agrobacterium
rhizogenes plasmids (or "Ri") are also useful and known in the art.
Other preferred plant transformation vectors include those
disclosed, e.g., by Herrera-Estrella (1983); Bevan (1983), Klee
(1985) and EP 0 120 516.
[0110] In certain embodiments, a functional recombinant DNA may be
introduced at a non-specific location in a plant genome. In special
cases it may be useful to insert a recombinant DNA construct by
site-specific integration. Several site-specific recombination
systems exist which are known to function in plants include cre-lox
as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in
U.S. Pat. No. 5,527,695.
[0111] Suitable methods for transformation of host cells for use
with the current invention are believed to include virtually any
method by which DNA can be introduced into a cell (see, for
example, Miki et al., 1993), such as by transformation of
protoplasts (U.S. Pat. No. 5,508,184; Omirulleh et al., 1993), by
desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985),
by electroporation (U.S. Pat. No. 5,384,253), by agitation with
silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos.
5,302,523; and 5,464,765), by Agrobacterium-mediated transformation
(U.S. Pat. Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877;
5,981,840; 6,384,301) and by acceleration of DNA coated particles
(U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208;
6,399,861; 6,403,865; Padgette et al. 1995), etc. Through the
application of techniques such as these, the cells of virtually any
species may be stably transformed. In the case of multicellular
species, the transgenic cells may be regenerated into transgenic
organisms.
[0112] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium (for example, Horsch et al.,
1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil
bacteria which genetically transform plant cells. The Ti and Ri
plasmids of A. tumefaciens and A. rhizogenes, respectively, carry
genes responsible for genetic transformation of the plant.
Descriptions of Agrobacterium vector systems and methods for
Agrobacterium-mediated gene transfer are provided by numerous
references, including Gruber et al. 1993; Miki et al., 1993,
Moloney et al., 1989, and U.S. Pat. Nos: 4,940,838 and 5,464,763.
Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium
that interact with plants naturally can be modified to mediate gene
transfer to a number of diverse plants. These plant-associated
symbiotic bacteria can be made competent for gene transfer by
acquisition of both a disarmed Ti plasmid and a suitable binary
vector (Broothaerts et al., 2005). Methods for introducing virus
sequences to plants may also be used (e.g. Grimsley, 1990, Boulton,
1996).
[0113] Methods for the creation of transgenic plants and expression
of heterologous nucleic acids in plants in particular are known and
may be used with the nucleic acids provided herein to prepare
transgenic plants that exhibit reduced susceptibility to
plant-pathogenic viruses. Plant transformation vectors can be
prepared, for example, by inserting the dsRNA producing nucleic
acids disclosed herein into plant transformation vectors and
introducing these into plants. One known vector system has been
derived by modifying the natural gene transfer system of
Agrobacterium tumefaciens. The natural system comprises large Ti
(tumor-inducing)-plasmids containing a large segment, known as
T-DNA, which is transferred to transformed plants. Another segment
of the Ti plasmid, the vir region, is responsible for T-DNA
transfer. The T-DNA region is bordered by terminal repeats. In the
modified binary vectors the tumor-inducing genes have been deleted
and the functions of the vir region are utilized to transfer
foreign DNA bordered by the T-DNA border sequences. The T-region
may also contain a selectable marker for efficient recovery of
transgenic plants and cells, and a multiple cloning site for
inserting sequences for transfer such as a dsRNA encoding nucleic
acid.
[0114] Protocols for transformation of tomato cells are known in
the art (e.g. McCormick, 1991). Alternate plant transformation
protocols are discussed in Boulton (1996), and Grimsley (1990).
Transformation and regeneration protocols for other plants, such as
pepper, are known in the art (e.g. Christopher and Rajam, 1996;
U.S. Pat. No. 5,262,316; Liu et al. 1990). For instance, such a
protocol for transformation of tomato could include well known
steps of seed sterilization, seed germination and growth,
explanting of seedlings, Agrobacterium culture growth and
preparation, co-cultivation, selection, and regeneration.
E. Transgenic Plants and Cells
[0115] A transgenic plant formed using Agrobacterium transformation
methods typically may contain a single simple recombinant DNA
sequence inserted into one chromosome, referred to as a transgenic
event. Such transgenic plants can be referred to as being
heterozygous for the inserted exogenous sequence. A transgenic
plant homozygous with respect to a transgene can be obtained by
sexually mating (selfing) an independent segregant transgenic plant
that contains a single exogenous gene sequence to itself, for
example an F0 plant, to produce F1 seed. One fourth of the F1 seed
produced will be homozygous with respect to the transgene.
Germinating F1 seed results in plants that can be tested for
heterozygosity, typically using a SNP assay or a thermal
amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay). Crossing a
heterozygous plant with itself or another heterozygous plant
results in heterozygous progeny, as well as homozygous transgenic
and homozygous null progeny.
[0116] In addition to direct transformation of a plant with a
recombinant DNA construct, transgenic plants can be prepared by
crossing a first plant having a recombinant DNA construct with a
second plant lacking the construct. For example, recombinant DNA
for gene suppression can be introduced into first plant line that
is amenable to transformation to produce a transgenic plant that
can be crossed with a second plant line to introgress the
recombinant DNA for gene suppression into the second plant
line.
F. Virus Resistance Screens
[0117] Inoculation and disease testing with viruses such as TSWV,
TYLCV, and PepMV was performed using either mechanical transmission
or agroinfection (e.g. Boulton, 1996; Grimsley, 1990). TSWV
infection was accomplished mechanically, essentially as described
by Kumar et al., (1993) with some modifications. Inoculation with
begomovirus (e.g. TYLCV) and disease testing of plants was
accomplished by agroinfection as follows: Seeds of tomato
(Lycopersicon esculentum) cvs. (Resistant (R): HP919; Intermediate
Resistant (IR): Hilario; Susceptible (S): Arletta) were sown and
approximately 20 plants for each inoculation, as well as a
non-inoculated or mock-inoculated control, were grown for 7-10
days, to the cotyledon stage with no primary leaf visible. The
lower sides of the cotyledons were then infiltrated using a
needleless syringe, and additional inoculations were subsequently
performed by injection into stems at the 2-3 true leaf stage,
approximately 1-2 weeks later. Infiltration or injection utilized a
transformed A. tumefaciens strain, induced with acetosyringone,
containing an infections clone of the virus that had been grown in
YEB broth with antibiotics for selection of the pBIN vector for
about 15 hours at 28.degree. C. on a shaker (170 RPM) from
inoculation of a fresh culture. Following growth at 28.degree. C.,
the culture was spun down and resuspended in 10 ml MMA (Per 1
liter: 20 g sucrose, 5 g MS salts, 1.95 g MES, pH 5.6 with NaOH,
and 1 ml of 200 mM acetosyringone stock; dH.sub.2O to 1 liter).
Plants were grown at 20-25.degree. C. (day/night), 16 hours light
in the greenhouse or growth chamber. After 6 weeks, symptoms were
scored as follows:
[0118] 1 no symptoms visible (resistant)
[0119] 3. very mild symptoms (resistant)
[0120] 5. medium symptoms
[0121] 7. strong symptoms
[0122] 9. very strong symptoms
[0123] Inoculation with the potexvirus Pepino mosaic virus (PepMV)
was accomplished as follows: Virus inoculum was prepared by
mechanically infecting tomato seedlings (susceptible cv. Apollo,
9-12 days old) following dusting of leaves with carborundum powder.
Infected tissues were harvested and 1 g of infected tissue was
homogenized with 5-10 ml of phosphate buffer (pH 9). This prepared
inoculum was then used to mechanically inoculate experimental
plants at the cotyledon stage (7-10 days after sowing). Inoculated
plants were grown in the greenhouse at 19-23.degree. C.
(day/night), with 16 hours of light per 24 hours, in greenhouse
with 70% relative humidity. Plants were evaluated at 11-21 days
after inoculation, and scored as follows:
[0124] 1. no symptoms visible (resistant)
[0125] 3. some foliar chlorosis
[0126] 5. chlorosis in foliar veins and/or foliar mosaic
[0127] 7. foliar vein chlorosis and spiky leaves
[0128] 9. foliar vein chlorosis and spiky leaves and yellow
mosaic
G. RNA Extraction and siRNA Northern Blots
[0129] RNA was extracted for small RNA northern blotting using
Trizol.RTM. essentially according to the manufacturer's directions
(Invitrogen). Briefly, approximately 100-200 mg of fresh leaf
tissue was ground in liquid nitrogen in 1.5 ml centrifuge tube
placed on dry ice. Sample was removed from dry ice and 1 ml of
Trizol was added under a fume hood. This was mixed well and
incubated at room temperature (RT) for 10 minutes. Next, 0.2 ml of
chloroform was added, and the sample was shaken by hand for 30
seconds and centrifuged at 13000 rpm in a refrigerated table top
centrifuge for 15 minutes at 4.degree. C.
[0130] The aqueous phase was transferred to a new tube, 0.5 ml of
isopropanol was added, and tubes were inverted a few times, and
then incubated for 10 min at RT followed by centrifugation at 13000
rpm in a refrigerated table top centrifuge for 15 min at 4.degree.
C. The supernatant was discarded and the pellet was washed by
adding 0.75 ml of 75% ethanol, then centrifuged at 13000 rpm for 10
min at 4.degree. C. The supernatant was discarded and RNA was air
dried for 10 minutes at room temperature.
[0131] The pellet was resuspended by adding 100 .mu.l of RNAse free
ddH2O and was vortexed for a few seconds. The samples were frozen
on dry ice and the RNA concentrated by the use of a speed vacuum
for about 20 minutes: this step removes all traces of ethanol. RNA
was quantified by spectrophotometer (usually about 80-100 .mu.g are
recovered from tomato leaf). About 7 .mu.g of total RNA were loaded
for siRNA analysis.
[0132] siRNA Northern blot with DIG labeled probe: Pre-run the 15%
TBE-Urea gel (Invitrogen EC68855BOX) for about 30' at 110 volts.
Samples were prepared for loading by adding 7 .mu.l of Novex.RTM.
TBE-Urea Sample Buffer 2.times. (Invitrogen LC6876) to 7 .mu.l of
total RNA (5-7 .mu.g), they are denatured for about 10' at
94.degree. C. and immediately cooled on ice. If sample
visualization under UV light was needed, ethidium bromide was added
to the sample buffer (1 .mu.l of EtBr @0.624 mg/ml for every 100
.mu.l of buffer). Samples were briefly spun down before loading
into the gel wells and electrophoresis was carried on for about 1.5
hrs at 180 Volts in 0.5.times.TBE until the blue dye reached the
bottom of the gel. After the run was terminated, the gel was
observed under UV lights.
[0133] Transfer of the gel to Nytran Supercharge Nylon membrane
(VWR 28151-318) was done in a Transblot semi dry transfer cell
(BioRad 170-3940): the membrane was pre-wet in water and
equilibrated in 0.5.times.TBE together with 2 pieces of extra-thick
paper (BioRad 170-3968). The transfer was set up according to
manufacturer's instruction (anode-blotting
paper-membrane-gel-blotting paper-cathode) and carried on for 50'
at 380 mAmp. After transfer the membrane was left to air dry for
about 10' and then the RNA was cross-linked to it in a Stratalinker
1800 (Stratagene).
[0134] Hybridization: The membrane was pre-hybridized at 42.degree.
C. for 1 hr with 10 ml of PerfectHyb solution (Sigma H7033) in a
hybridization oven. 200 ng of DIG labeled probe, prepared with the
PCR DIG labeling mix (Roche 11585550910) following manufacturer's
instruction, was denatured for 10' at 94.degree. C., cooled on ice,
and added to 10 ml of fresh hybridization solution, which was added
to the pre-hybridized membrane and incubated overnight at
42.degree. C. in the hybridization oven.
[0135] Washes and detection: After discarding the hybridization
solution, the membranes was briefly rinsed in 2.times.SSC 0.1% SDS;
and then washed 2 times in pre-warmed 2.times.SSC 0.1% SDS for 20'
at 50.degree. C., 2 times in pre-warmed 1.times.SSC 0.1% SDS for
20' at 50.degree. C., 2 times in pre-warmed 0.5.times.SSC 0.1% SDS
for 20' at 50.degree. C. Detection was performed following the
instructions provided with the DIG Wash and Block Buffer Set (Roche
11585762001). Briefly, the membrane was rinsed for 2' in
1.times.DIG Washing Buffer before incubating for 1 hr at RT in 100
mL DIG Blocking Solution (10 mL 10.times. Blocking buffer+10 mL
10.times. Maleic Acid+80 mL water). It was then incubated for 1 hr
at room temperature in 100 mL fresh DIG Blocking Solution to which
10 .mu.l of Anti-Digoxigenin-AP antibody (Roche 11093274910) had
been added. The membrane was washed 15' at RT twice 100 mL
1.times.DIG Washing Buffer and equilibrated with 100 mL of
1.times.DIG Detection Buffer. About 1 mL of CDP-star solution
(Roche 12041677001) was distributed across the top of the membrane,
which was placed between 2 plastic sheets, incubated at RT for 5'
and exposed to a film for variable amounts of time before
developing.
H. Validation of Constructs in Transgenic Tomato Plants
[0136] The engineered sequences for generating dsRNA or miRNA were
cloned into binary vectors for tomato transformation. R.sub.0
transgenic plants were assayed for the production of the specific
.about.21mers and/or virus resistance efficacy by small RNA
northern blot analysis and resistance assays as outlined above and
as described in Example 7. Other selected dsRNA-generating
sequences or miRNA-generating sequences, for instance from among
any of SEQ ID NOs:1-154, or from within SEQ ID NOs:169-455 or
portions thereof, may be utilized to create additional efficacious
constructs. Thus, for instance, MIR backbone sequence(s), for
instance MON1 from soybean, MON5 from soybean, MON13 from rice,
MON18 from maize, miR159a from maize, or mi167g from maize (SEQ ID
NOs:155, 157, 159, 161, 163, or 165) may be utilized to create
dsRNA- or miRNA-generating sequence(s). In certain embodiments, the
dsRNA-generating sequence or miRNA-generating sequence may comprise
any of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 55, 59, 63, 67,
71, 85, 99, 113, 127, 141, or 379-455. In particular embodiments,
the sequence may comprise any of SEQ ID NOs:156, 158, 160, 162,
164, 166, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,
374, or 375.
[0137] The present invention includes combinations with other
disease control traits in a plant, including non-transgenic
approaches, to achieve desired traits for enhanced control of plant
disease. Combining disease control traits that employ distinct
modes-of-action can provide protected transgenic plants with
superior durability over plants harboring a single control trait
because of the reduced probability that resistance will develop in
the field. Thus, in certain embodiments, at least two or at least
three modes of action are employed to confer virus resistance,
wherein such modes of action are selected from the group consisting
of expression of dsRNA, expression of miRNA, inhibition of virion
assembly, phenotypic expression of a non-transgenic virus
resistance trait, and transgenic protein expression. In particular
embodiments, transgenic protein expression comprises expression of
a coat protein-encoding nucleic acid sequence, expression of a
movement protein-encoding sequence or expression of a
replicase-encoding nucleic acid sequence.
[0138] The invention also relates to commodity products containing
one or more of the sequences of the present invention, and produced
from a recombinant plant or seed containing one or more of the
nucleotide sequences of the present invention are specifically
contemplated as embodiments of the present invention. A commodity
product containing one or more of the sequences of the present
invention is intended to include, but not be limited to, meals,
oils, crushed or whole grains or seeds of a plant, or any food
product comprising any meal, oil, or crushed or whole grain of a
recombinant plant or seed containing one or more of the sequences
of the present invention. The detection of one or more of the
sequences of the present invention in one or more commodity or
commodity products contemplated herein is defacto evidence that the
commodity or commodity product is composed of a transgenic plant
designed to express one or more of the nucleotides sequences of the
present invention for the purpose of controlling plant disease
using dsRNA mediated gene suppression methods.
I. Nucleic Acid Compositions
[0139] The present invention provides methods for obtaining a
nucleic acid comprising a nucleotide sequence for producing a
dsRNA, miRNA, or siRNA. In one embodiment, such a method comprises:
(a) analyzing one or more target gene(s) for their expression,
function, and phenotype upon dsRNA-mediated, or mi-RNA- or
siRNA-mediated suppression of a gene of a plant pathogenic virus;
(b) probing a nucleic acid library with a hybridization probe
comprising all or a portion of a nucleotide sequence, or a homolog
thereof, from a targeted virus that displays an altered phenotype
in a dsRNA-mediated suppression analysis; (c) identifying a DNA
clone that hybridizes with the hybridization probe; (d) isolating
the DNA clone identified in step (b); and (e) sequencing the
nucleic acid fragment that comprises the clone isolated in step (d)
wherein the sequenced nucleic acid molecule transcribes all or a
substantial portion of the RNA nucleotide acid sequence or a
homolog thereof. The RNA-mediated resistance approach utilizing
dsRNA, miRNA, or siRNA may be supplemented by placing antiviral
sequences into the loop of a dsRNA-encoding sequence, or by
inserting a sequence that encodes an efficacious dsRNA or miRNA
into an intron of a polypeptide expression cassette (intronic
dsRNA/miRNA; Frizzi et al., 2008) (e.g. FIG. 9). For example, viral
protein(s) such as coat protein and/or replicase may be expressed
in a transgenic plant that also expresses an efficacious dsRNA,
miRNA, or siRNA, without use of an additional transgene cassette,
by inserting protein coding sequence into the loop of a dsRNA.
[0140] In another embodiment, a method of the present invention for
obtaining a nucleic acid fragment comprising a nucleotide sequence
for producing a substantial portion of a dsRNA or siRNA comprises:
(a) synthesizing a first and a second oligonucleotide primers
corresponding to a portion of one of the nucleotide sequences from
a targeted pathogen; and (b) amplifying a nucleic acid insert
present in a cloning vector using the first and second
oligonucleotide primers of step (a) wherein the amplified nucleic
acid molecule transcribes a substantial portion of the a
substantial portion of a dsRNA or siRNA of the present
invention.
[0141] In practicing the present invention, target genes may be
derived from a begomovirus, tospovirus, or potexvirus. It is
contemplated that several criteria may be employed in the selection
of preferred target genes. Such sequences may be identified by
aligning, for instance, begomovirus or tospovirus sequences from
multiple strains and/or species. A bioinformatics approach has
identified numerous 21-mers that are mostly conserved in more than
100 begomovirus strains and species. In some instances, mismatches
within a particular 21-mer are allowed for broader targeting.
[0142] As used herein, the term "derived from" refers to a
specified nucleotide sequence that may be obtained from a
particular specified source or species, albeit not necessarily
directly from that specified source or species.
[0143] In one embodiment, a gene is selected that results in
suppression of viral replication and/or symptomatology. Other
target genes for use in the present invention may include, for
example, those that play important roles in viral transmission,
movement within a plant, or virion assembly (e.g. tospovirus
terminal sequences, comprising the terminal repeat sequences).
According to one aspect of the present invention for virus control,
the target sequences may essentially be derived from the targeted
plant viruses. Some of the exemplary target sequences cloned from a
begomovirus, tospovirus, or potexvirus may be found in the Sequence
Listing, for instance in SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43,
47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141, as well as
within SEQ ID NOs:169-455.
[0144] For the purpose of the present invention, the dsRNA
molecules may be obtained by polymerase chain (PCR) amplification
of a target gene sequences derived from a gDNA or cDNA library or
portions thereof. The DNA library may be prepared using methods
known to the ordinary skilled in the art and DNA/RNA may be
extracted. Genomic DNA or cDNA libraries generated from a target
organism may be used for PCR amplification for production of the
dsRNA or siRNA.
[0145] The target gene sequences may be then be PCR amplified and
sequenced using the methods readily available in the art. One
skilled in the art may be able to modify the PCR conditions to
ensure optimal PCR product formation. The confirmed PCR product may
be used as a template for in vitro transcription to generate sense
and antisense RNA with the included minimal promoters. In one
embodiment, the present invention comprises isolated and purified
nucleotide sequences that may be used as plant-virus control
agents. The isolated and purified nucleotide sequences may comprise
those as set forth in the sequence listing.
[0146] As used herein, the phrase "coding sequence," "structural
nucleotide sequence" or "structural nucleic acid molecule" refers
to a nucleotide sequence that is translated into a polypeptide,
usually via mRNA, when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a translation start codon at the 5'-terminus and a
translation stop codon at the 3'-terminus. A coding sequence can
include, but is not limited to, genomic DNA, cDNA, EST and
recombinant nucleotide sequences.
[0147] The term " recombinant DNA" or "recombinant nucleotide
sequence" refers to DNA that contains a genetically engineered
modification through manipulation via mutagenesis, restriction
enzymes, and the like.
EXAMPLES
[0148] The inventors herein have identified a means for controlling
virus infections in plants by incorporating into plants engineered
miRNAs, ta-siRNAs and/or phased siRNAs. Any one or any combination
of these attributes can result in an effective inhibition of plant
infection, inhibition of plant disease, and/or reduction in
severity of disease symptoms.
Example 1
Targets for Multivirus Resistance
[0149] Sequences of targeted viruses were assembled from Genbank.
FIG. 1 schematically shows the genome organization of
representative targeted viruses. For targeted begomoviruses, 58
isolates of Tomato yellow leaf curl virus (TYLCV), 30 isolates of
Tomato leaf curl New Delhi virus (ToLCNDV), 5 isolates of Tomato
severe leaf curl virus (ToSLCV), 5 isolates of Pepper golden mosaic
virus (PepGMV), and 2 isolates of Pepper huasteco yellow vein virus
(PHYVV) were analyzed. For targeted tospoviruses, Tomato spotted
wilt virus (TSWV), Groundnut bud necrosis virus (GBNV) and Capsicum
chlorosis virus (CaCV), 6 L segments (TSWV: 4; GBNV: 1; CaCV: 1),
23 M segments (TSWV: 20; GBNV: 2; CaCV: 1), and 45 S segments
(TSWV: 41; GBNV: 2; CaCV: 2) were analyzed. For isolates of the
targeted potexvirus, 13 isolates of Pepino mosaic virus (PepMV)
were analyzed (e.g. Lopez et al., 2005; Cotillon et al., 2002). A
bioinformatics approach was utilized to identify approximately
20-24 nucleotide sequences to be used as artificial miRNAs to
suppress as many of the targeted viruses as possible.
[0150] The following Table (Table 1) lists sequences analyzed for
possible use in designing transgene constructs for generating
engineered miRNAs, as well as for identifying sequences for use in
dsRNA-mediated viral resistance approaches:
TABLE-US-00001 TABLE 1 Exemplary sequences used for bioinformatics
analysis (SEQ ID NOs: 169-362) Virus type Virus name GenBank
Accession Begomo- Tomato yellow leaf AF024715, X15656, X76319,
virus curl virus (TYLCV) AB014346, AB014347, AB110217, AB110218,
AB116629, AB16630, AB116631, AB116632, AB116633, AB116634,
AB116635, AB116636, AF071228, AF105975, AF271234, AJ132711,
AJ223505, AJ489258, AJ519441, AJ812277, AJ865337, AM282874,
AM409201, AM698117, AM698118, AM698119, AY044138, AY134494,
AY227892, AY502934, AY530931, AY594174, AY594175. DQ144621,
DQ358913, DQ631892, DQ644565, EF051116, EF054893, EF054894,
EF060196, EF101929, EF107520, EF110890, EF158044, EF185318,
EF210554, EF210555, EF433426, EF523478, EF539831, EU031444,
EU143745 Begomo- Tomato leaf curl NC_004611, U15015, U15016, virus
New Delhi virus Y16421, AB330079, AB368447, (ToLCNDV) AB368448,
AF102276, AF448058, AF448059, AJ620187, AJ875157, AM286433,
AM286434, AM292302, AM850115, AY286316, AY428769, AY939926,
DQ116880, DQ116883, DQ116885, DQ169056, EF035482, EF043230,
EF043231, EF063145, EF068246, EF450316, EF620534, EU309045 Begomo-
Tomato severe AF130415, AJ508784, AJ508785, virus leaf curl
DQ347946, DQ347947 virus (ToSLCV) Begomoi- Pepper Huasteco
NC_001359, X70418, AY044162 virus yellow vein virus (PHYVV) Begomo-
Pepper golden NC_004101, U57457, AF149227, virus mosaic virus
AY928512, AY928514, AY928516 (PepGMV) Tospo- Tomato spotted
NC_002052, D10066, AB190813, virus wilt wirus AB198742, AY070218
(TSWV) (L segment) Tospo- Tomato spotted NC_002050, S48091,
AB010996, virus wilt wirus AB190818, AF208497, AF208498, (TSWV)
AY744481, AY744482, AY744483, (M segment) AY744484, AY744485,
AY744486, AY744487, AY744488, AY744489, AY744490, AY744491,
AY744492, AY744493, AY870389, AY870390 Tospo- Tomato spotted
NC_002051, D00645, AB088385, virus wilt wirus AB 190819, AF020659,
AF020660, (TSWV) AJ418777, AJ418778, AJ418779, (S segment)
AJ418780, AJ418781, AY744468, AY744469, AY744470, AY744471,
AY744472, AY744473, AY744474, AY744475, AY744476, AY744477,
AY744478, AY744479, AY744480, AY870391, AY870392, DQ376177,
DQ376178, DQ376179, DQ376180, DQ376181, DQ376182, DQ376183,
DQ376184, DQ376185, DQ398945, DQ431237, DQ431238, DQ915946,
DQ915947, DQ915948 Tospo- Groundnut bud NC_003614, AF025538 virus
necrosis virus (GBNV) (L segment) Tospo- Groundnut bud NC_003620,
U42555, AY871097 virus necrosis virus (GBNV) (M segment) Tospo-
Groundnut bud NC_003619, U27809, AY871098 virus necrosis virus
(GBNV) (S segment) Tospo- Capsicum NC_008302, DQ256124 virus
chlorosis virus (CaCV) (L segment) Tospo- Capsicum DQ256125 virus
chlorosis virus (CaCV) (M segment) Tospo- Capsicum DQ355974,
DQ256123 virus chlorosis virus (CaCV) (S segment) Potex- Pepino
mosaic EF599605, AY509926, AJ438767, virus virus (PepMV) DQ000984,
DQ000985, AY509927, EF408821, AM491606, AJ606360, AJ606359,
AJ606361, AF484251, AM109896
Example 2
Virus Segments for RNAi
[0151] Selected viral genomes were divided to .about.350-500 bp
fragments, partially overlapping by about 50 bp (e.g. FIG. 2A-2D).
Efficacy data was collected for the ability of single sequences to
control particular virus species when expressed individually in
transformed plants. In initial studies, segments corresponding to
approximately 2.3 kB out of the 2.7 kB geminivirus DNA-A genome
were screened (FIG. 2B; segments 1-8). Likewise, as shown in FIG.
2C, segments 1-8 representing approximately 4 kB out of the 6.5 kB
PepMV (potexvirus) genome were tested. FIG. 2D shows the portions
of the tripartite tospovirus genome that were tested for efficacy:
approximately 1.5 kB out of the 8.9 kB L vRNA (i.e. virus RNA)
(segments 1-3); approximately 1.5 kB of the 5.4 kB M vRNA (segments
4-6); and approximately 1 kB of the 2.9 kB S vRNA (segments
7-8).
[0152] In one representative study, a nucleic acid segment
corresponding to a part of the TSWV coat protein (nucleocapsid) was
tested for efficacy when expressed as an inverted repeat (FIG. 3).
Virus resistance was found to correlate with siRNA production in
tomato plants. Also in the TYLCV resistance screen, R.sub.1 plants
were first examined for the presence of the transgene and then
infected with the TYLCV infectious clone. As a control, a
non-transformed (wild-type) tomato line was included; these
wild-type plants typically have less than 5% "resistance", i.e.
less than 5% of plants escape infection by inoculation in this
assay. In contrast, among CP4 positive plants (containing the
selectable marker gene specifying resistance to glyphosate, which
is linked to the expression cassette with viral sequence), 56% of
R.sub.1 plants comprising an introduced nucleic acid segment
targeting the replication protein (3 constructs), 27% comprising an
introduced nucleic acid segment targeting the coat protein (3
constructs), and 38% comprising an introduced nucleic acid segment
targeting the rest of the coding regions (2 construct) showed
resistance to TYLCV.
[0153] Additional studies scanning these and additional portions of
the tospovirus genome were then undertaken, in order to identify
and define portions of the viral genome which can mediate
dsRNA-related virus resistance to representative viruses. These
sequences were tested as inverted repeat segments in double
stranded (dsRNA)-generating constructs, wherein tested constructs
comprised a promoter operably linked to a given sequence in an
antisense orientation (e.g. SEQ ID NOs:379-442 as described below),
followed by a loop sequence, and then the given sequence in a sense
orientation, and a transcriptional terminator. After transcription
and base pairing of the inverted repeat sequences, the double
stranded RNA regions are cleaved by a Dicer or Dicer-like RNAse to
generate the specific antiviral siRNAs which target and interfere
with viral gene expression.
[0154] Capsicum chlorosis virus (CaCV), Groundnut bud necrosis
virus (GBNV), and Tomato spotted wilt virus (TSWV), among other
tospoviruses. Virus resistance tests of transgenic R.sub.1 plants
demonstrated that all regions of the tospovirus genome are equally
as effective as the target of dsRNA. Thus, significant virus
resistance was seen following expression of transcripts which
disrupt expression of tospovirus coat protein (CP), as well as the
glycoprotein-encoding virus genome segments GP1, GP2, GP3, and
RNA-dependent-RNA polymerase (RdRP) protein segments. FIG. 11A-B
describes resistance results seen from transgenic plants. With each
bar representing an individual transgenic event and target region,
for instance 50-80% of R.sub.1 plants expressing a dsRNA targeting
the CP region were resistant to CaCV, while .about.25-90% of
R.sub.1 plants expressing a dsRNA targeting a virus glycoprotein
showed virus resistance, and about 25-80% of R.sub.1 plants
expressing a dsRNA targeting a virus RdRP showed resistance (e.g.
FIG. 11B). dsRNA-mediated resistance to TSWV was particularly
effective, with 100% of R.sub.1 plants displaying virus resistance
from all tested events with constructs targeting the coat protein
(FIG. 11A). Representative TSWV, CaCV, and GBNV sequences selected
for use in generation of dsRNAs targeting tospovirus gene
expression are listed in SEQ ID NOs:419-436 (in antisense
orientation as listed).
[0155] Additional studies demonstrated that all regions of the
PepMV genome are equally effective in generating dsRNA-mediated
resistance against this potexvirus. FIG. 12 shows that in most
cases, 95-100% of all transgenic R.sub.1 plants comprising
virus-encoded segments targeting CP (coat protein), CP/MOV (Coat
Protein and Movement Protein sequences), RdRP, TGB (Triple Gene
Block motif protein implicated in viral cell-to-cell and long
distance movement in a host plant), or TGB/RdRP were resistant to
PepMV. Representative PepMV sequences selected for use in
generation of dsRNAs targeting potexvirus gene expression are
listed in SEQ ID NOs:437-442, in antisense orientation as
listed.
[0156] Likewise, regions of the begomovirus (geminivirus) genome
were tested for their ability to generate dsRNA-mediated resistance
against this virus group. TYLCV, ToSLCV, PepGMV, PHYVV, and ToLCNDV
were tested in bioassays as representative begomoviruses. FIGS.
13A-B shows that up to about 70% of transgenic R.sub.1 plants
displayed resistance to a given virus when CP was the dsRNA target,
while up to about 100% of transgenic R.sub.1 plants displayed
resistance when a nucleic acid segment encoding viral replication
protein was the dsRNA target. Representative tospovirus sequences
selected for use in generation of dsRNAs targeting begomovirus gene
expression are listed in SEQ ID NOs:379-418, in antisense
orientation as listed.
[0157] Together, numerous effective dsRNA targets for viruses in
these three genera (i.e. Tospovirus, Potexvirus, Begomovirus) were
identified.
[0158] Based on these RNAi results, sequences targeting efficacious
target regions were selected for each virus and fused into two
transgenic cassettes on a nucleic acid construct, to target
multiple viruses in multiple virus families. An example of this
approach is shown in FIG. 14. For begomoviruses, a Rep region was
used, while for tospovirus and potexvirus, CP regions were selected
since the CP region is relatively more conserved among different
strains of the same viruses. Some of the sequences utilized in the
cassettes were further modified, in view of expected G::U base
pairings, to allow for broadening of protection to related viral
strains. Representative sequences selected for use in generation of
dsRNAs targeting virus gene expression in the cassettes are listed
in SEQ ID NOs:443-446 for "cassette 1" of FIG. 14, and in SEQ ID
NOs:447-451 for "cassette 2" of FIG. 14, each in antisense
orientation as listed in the sequence listing. Thus, for instance,
the nucleic acid segment targeting GBNV-CP1 which is found in
cassette 1 (SEQ ID NO:443) is similar to the segment targeting
GBNV-CP1 as found in SEQ ID NO:429, but may have modification as
noted above, and likewise for the other nucleic acid segments used
in the cassettes, relative to the target segments as initially
tested for relative efficacy.
Example 3
Artificial dsRNA Fusion Constructs for Multivirus Resistance
[0159] Nucleotide segments for dsRNA expression, from two or three
of the different virus groups (geminivirus, tospovirus,
potexvirus), were also combined in a single expression cassette and
plants were generated from transformed cells and analyzed for
resistance to more than one virus. This was accomplished by fusing
viral genomic fragments that tested effective for generating siRNAs
conferring virus resistance, for instance as shown in Example 2.
Analogously to Example 2, these sequences were tested as inverted
repeat segments in double stranded (dsRNA)-generating constructs,
wherein tested constructs comprised a promoter operably linked to a
given sequence in an antisense orientation (e.g. SEQ ID NOs:452-454
as described below), followed by a loop sequence, and then the
given sequence in a sense orientation, and a transcriptional
terminator. After transcription and base pairing of the inverted
repeat sequences, the double stranded RNA regions are cleaved by a
Dicer or Dicer-like RNAse to generate the specific antiviral siRNAs
which target and interfere with viral gene expression. In this
approach, specific dsRNA segments for each of the targeted virus
were used for multiple virus resistance (FIG. 4A).
[0160] Alternatively, conserved regions of the various viral
genomes may provide a broader spectrum of resistance against
different variants within the species or closely related species.
Additionally, inverted repeats comprising sequences that are highly
similar to, but less than 100% identical to, targeted segments of
viral genomes may be designed and tested for efficacy. This
approach, i.e. partially similar or "imperfect" inverted repeats,
may broaden RNA-mediated protection to related virus strains and
species. For instance, since G-U basepairing at the RNA level has a
comparable degree of thermodynamic stability as some typical
Watson-Crick baseparing (e.g. A-U), some imperfect repeats may
nonetheless activate RISC and guide cleavage of both perfect and
imperfect viral targets.
[0161] In this instance, such partially similar inverted repeats
may also comprise one or more sub-sequences of at least 21
nucleotides in length that display near 100% identity to one or
more viral target sequences, in order to stimulate RNAi-mediated
virus resistance. Thus for instance, the overall identity of an
imperfect repeat segment to one or more viral target(s) may be as
low as 75%, but with 2-4 or more sub-sequences of 21-24 nucleotides
that display 100% identity to a viral target sequence.
Additionally, the presence of such "imperfect" dsRNA may help to
increase accumulation of dsRNA in plants and prevent the triggering
of transcriptional gene silencing. Thus, nucleotide segments from
more than one strain of a virus, or from more than one virus, may
be utilized in design and preparation of a fusion construct for
dsRNA expression.
[0162] Such sequences may be identified by aligning, for instance,
geminivirus or tospovirus sequences from multiple strains and/or
species. Table 2 shows percent similarities between geminivirus
targets at the whole genome level. Table 3 shows percent
similarities between tospovirus targets at the whole genome
level.
TABLE-US-00002 TABLE 2 Percent similarities between geminivirus
targets (whole genome). TYLCV ToLCNDV PHYVV ToSLCV PepGMV TYLCV --
71 64 63 60 ToLCNDV 71 -- 62 60 59 PHYVV 64 62 -- ToSLCV 63 60 67
-- 76 PepGMV 60 59 66 76 --
TABLE-US-00003 TABLE 3 Percent similarities between tospovirus
targets (whole genome). Segment Virus CapCV GBNV TSWV L vRNA
CapCV_L -- 44 45 8.8 kb GBNV_L 44 -- 58 TSWV_L 45 58 -- M vRNA
CapCV_M -- 78 53 4.8-5.4 kB GBNV_M 78 -- 54 TSWV_M 53 54 -- S vRNA
CapCV_S -- 65 42 2.9-3.3 kB GBNV_S 73 -- 48 TSWV_S 49 49 --
[0163] FIG. 4B schematically illustrates exemplary fusion
constructs for dsRNA expression. Coding sequences, including
sequences lacking significant identity with human and host plant
genomes, were selected for generation of inverted repeats.
Viral-derived segments may be oriented, for instance,
"antisense-loop-sense" for dsRNA expression. This approach can
reduce the size of a transgenic dsRNA cassette required for
multiple virus resistance. The schematically described artificial
fusion constructs of FIG. 4B are listed as SEQ ID NOs: 452-453. SEQ
ID NO:452 artificial coat protein construct comprises sequences as
follows: bp 1-157: TYLCV precoat protein for the loop; bp 158-507
(i.e. 350 bp segment) targeting TYLCV and ToLCNDV CP; bp 508-851
(i.e. 344 bp segment) targeting ToSLCV, PepGMV, and PHYVV CP
expression. SEQ ID NO:453 comprises segments as follows: bp 1-160
targeting TSWV coat protein (CP) for the loop; bp 161-456 (i.e. 296
bp segment) targeting TSWV CP; bp 457-687 (i.e. 231 bp segment)
targeting the PepMV CP; bp 688-919 (i.e. 232 bp segment) targeting
the CaCV and GBNV CP. Efficacy results for SEQ ID NO:453, the
artificial CP sequence against multiple tospoviruses and PepMV, are
shown in FIG. 15. An additional artificial dsRNA fusion construct
targeting Rep proteins of five geminiviruses (TYLCV, ToLCNDV,
ToSLCV, PepGMV, and PHYVV) is given as SEQ ID NO:454. In this
sequence, bp 1-150 target TYLCV Rep protein for the loop; bp
151-500 (i.e. 350 bp segment) target TYLCV and ToLCNDV Rep protein;
bp 501-860 (i.e. 360 bp segment) target ToSLCV, PepGMV, and PHYVV
Rep protein.
Example 4
Engineered miRNA Approach: Selection of Suitable Sequences
[0164] The bioinformatics approach described in Example 1 was used
to identify several suitable sequences, approximately 21 nt in
length, that target geminivirus sequences thought to be useful for
miRNA-mediated suppression of geminivirus replication and/or
symptom expression (Table 4; FIG. 5).
TABLE-US-00004 TABLE 4 Suitable 21 nt sequences against targeted
Geminiviruses. SEQ ID NO: Name miRNA Sequence (5'.fwdarw.3') Target
1 Gemini 1 TGTCATCAATGACGTTGTACT Rep 7 Gemini 2
TGGACTTTACATGGGCCTTCA Coat Protein 13 Gemini 3
TACATGCCATATACAATAGCA Coat Protein 19 Gemini 4
TCATAGAAGTAGATCCGGATT Coat Protein 25 Gemini 5
TTCCCCTGTGCGTGAATCCGT C2/C3 31 Gemini 6 TTCCGCCTTTAATTTGGATTG Rep
37 Gemini 7 TTACATGGGCCTTCACAGCCT Coat Protein
[0165] The bioinformatics approach described in Example 1 was used
to identify several suitable sequences, approximately 21 nt in
length, that target Tospovirus sequences thought to be useful for
miRNA-mediated suppression of Tospovirus replication and/or symptom
expression (Table 5; FIG. 6).
TABLE-US-00005 TABLE 5 Suitable 21 nt sequences for each genomic
segment against three targeted Tospoviruses. SEQ ID NO: Name miRNA
Sequence (5'.fwdarw.3') Target 43 TospoL1-1 TTTAGGCATCATATAGATAGCT
RdRP 47 Tospo L1-2 TGATTTAGGCATCATATAGAT RdRP 51 Tospo M1
TATCTATATTTTCCATCTACC GP 55 Tospo M2 TTAGTTTGCAGGCTTCAATTA NSm 59
Tospo M3 TTGCATGCTTCAATGAGAGCT NSm 63 Tospo S2
TTGACGTTAGACATGGTGTTT N 67 Tospo S3 TAGAAAGTTTTGAAGTTGAAT N
[0166] The bioinformatics approach described in Example 1 was used
to identify several suitable sequences, approximately 21 nt in
length, that target Potexvirus sequences thought to be useful for
miRNA-mediated suppression of Tospovirus replication and/or symptom
expression (Table 6; FIG. 7A-7B).
TABLE-US-00006 TABLE 6 Suitable ~21 nt sequences against targeted
potexvirus. SEQ ID NO: Name miRNA Sequence (5'.fwdarw.3') Target 71
PepMV1 TCTTCATTGTAGTTAATGGAG RdPR 85 PepMV2 TTGGAAGAGGAAAAGGTGGTT
RdPR 99 Pep MV3 TCAATCATGCACCTCCAGTCG RdPR 113 PepMV4
TAAGTAGCAAGGCCTAGGTGA TGBp1 127 PepMV5 TTTGGAAGTAAATGCAGGCTG TGBp2
141 PepMV6 TAACCCGTTCCAAGGGGAGAAG TGBp3
Example 5
Incorporation of miRNA-Generating Sequences in a MIR Gene
Backbone
[0167] The miRNA-generating sequences of Tables 4-6 were
incorporated into transgene constructs by gene synthesis. For
instance, the wildtype MIR gene backbone "MON1" (see U.S.
Publication 2007/0300329) from soybean with the wildtype 21nt
miRNA-generating sequence (SEQ ID NO:155 of the present
application) was altered to replace the wildtype miRNA-generating
sequence with the Gemini1 target (SEQ ID NO:1), resulting in the
Gemini1/MON1 construct (SEQ ID NO:156). Thus, a MIR backbone (e.g.
the MON1 backbone of SEQ ID NO:155) can be altered by replacing the
wildtype 21nt miRNA-generating sequence with any of the miRNA
sequences of Tables 4-6.
[0168] Similarly, MONS from soybean (SEQ ID NO:157), MON13 from
rice (SEQ ID NO:159), MON18 from maize (SEQ ID NO:161), miR159a
from maize (SEQ ID NO:163), or miR167g (SEQ ID NO:165), were used
as backbone sequences with miRNA-generating sequences of Tables
4-6. Thus, the following sequences were created (Table 7):
TABLE-US-00007 TABLE 7 Exemplary sequences incorporated into MIR
gene backbone. miRNA-generating MIR backbone Incorporated construct
sequence (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) Gemini 1 MON1
Gemini1/MON1 (SEQ ID NO: 1) (SEQ ID NO: 155) (SEQ ID NO: 156)
Gemini 4 MON5 Gemini 4/MON5 (SEQ ID NO: 19) (SEQ ID NO: 157) (SEQ
ID NO: 158) Gemini 7 MON13 Gemini 7/MON13 (SEQ ID NO: 37) (SEQ ID
NO: 159) (SEQ ID NO: 160) PepMV5 MON18 PepMV5/MON18 (SEQ ID NO:
127) (SEQ ID NO: 161) (SEQ ID NO: 162) Gemini 6 miR159a Gemini
6/miR159a (SEQ ID NO: 31) (SEQ ID NO: 163) (SEQ ID NO: 164) PepMV6
miR167g PepMV6/miR167g (SEQ ID NO: 141) (SEQ ID NO: 165) (SEQ ID
NO: 166) Gemini 2 MON13 Gemini 2/MON13 (SEQ ID NO: 7) (SEQ ID NO:
159) (SEQ ID NO: 367) Gemini 3 MON1 Gemini3/MON1 (SEQ ID NO: 13)
(SEQ ID NO: 155) (SEQ ID NO: 368) Gemini 5 miR159a Gemini 5/MiR159a
(SEQ ID NO: 25) (SEQ ID NO: 163) (SEQ ID NO: 369) PepMV1 miR159a
PepMV1/miR159a (SEQ ID NO: 71) (SEQ ID NO: 163) (SEQ ID NO: 363)
PepMV2 MON5 PepMV2/MON5 (SEQ ID NO: 85) (SEQ ID NO: 157) (SEQ ID
NO: 364) PepMV3 MON13 PepMV3/MON13 (SEQ ID NO: 99) (SEQ ID NO: 159)
(SEQ ID NO: 365) PepMV4 MON1 PepMV4/MON1 (SEQ ID NO: 113) (SEQ ID
NO: 155) (SEQ ID NO: 366) TospoL1-1 miR167g TospoL1-1/miR167g (SEQ
ID NO: 43) (SEQ ID NO: 165) SEQ ID NO: 370) TospoL1-2 MON1
TospoL1-2/MON1 (SEQ ID NO: 47) (SEQ ID NO: 155) (SEQ ID NO: 371)
TospoM2 MON1 TospoM2/MON1 (SEQ ID NO: 55) (SEQ ID NO: 155) (SEQ ID
NO: 372) TospoM3 MON13 TospoM3/MON13 (SEQ ID NO: 59) (SEQ ID NO:
159) (SEQ ID NO: 373) TospoS2 MON5 TospoS2/MON5 (SEQ ID NO: 63)
(SEQ ID NO: 157) (SEQ ID NO: 374) TospoS3 MON13 TospoS3/MON13 (SEQ
ID NO: 67) (SEQ ID NO: 159) (SEQ ID NO: 375)
Other selected miRNA-generating sequences, for instance from among
any of SEQ ID NOs:1-154, or from within SEQ ID NOs:169-362 or
portions thereof, may be utilized with MIR backbone sequences, for
instance MON1 from soybean, MONS from soybean, MON13 from rice,
MON18 from maize, miR159a from maize, or mi167g from maize (SEQ ID
NOs:155, 157, 159, 161, 163, or 165) to create additional
efficacious miRNA-generating constructs. Additionally, the MIR
backbone sequences may be modified for instance by replacing the
portion of the sequence of the plant-derived backbone sequence
which specifies an miRNA with a selected virus-derived or
artificial (e.g. consensus) sequence, and/or shortened, e.g. a
deletion made at the 3' and/or 5' end, for instance as reflected in
the miRNA-generating sequences and MIR backbone portions of SEQ ID
NOs:158, 160, 162, 164, 166, 363, 364, 365, 367, 369, 370, 373,
374, and 375, described below.
[0169] 21-mers identified from within SEQ ID NOs:169-362, for
instance among or within SEQ ID NOs:1-154, are tested individually,
or two or more at a time while present in one expression cassette,
for efficacy in transgenic tomato plants against appropriate
viruses such as TYLCV and TSWV. Multiple effective 21-mers are
expressed in one transgenic cassette using a polycistronic or a
phased siRNA backbone (e.g. FIG. 8). Results of virus resistance
assays from such testing of 21-mers are given in Example 7.
[0170] Once the efficacious antiviral miRNAs are identified,
multiple miRNAs are deployed by expressing as a single transgenic
trait, to achieve multiple virus resistance in transgenic plants.
This is accomplished by fusing engineered MIR gene(s) described
above together (e.g. to create SEQ ID NOs:156, 158, 160, 162, 164,
166, 363-375), or using a ta-siRNA or a phased siRNA gene structure
that can deliver multiple antiviral .about.21nt sequences (e.g.
FIG. 8). The later approaches are accomplished via a new round of
gene synthesis to replace wild-type siRNAs with the efficacious
antiviral 21nt sequences in the ta-siRNA or phased siRNA
backbone.
Example 6
Supplementing RNA-Based Resistance
[0171] The RNA-mediated resistance approach utilizing dsRNA or
miRNA can be supplemented by placing antiviral sequences, e.g. ones
that encode a protein, into the loop of a dsRNA-encoding sequence,
or by inserting a sequence that encodes an efficacious dsRNA or
miRNA into an intron of a polypeptide expression cassette (intronic
dsRNA/miRNA; Frizzi et al., 2008) (e.g. FIG. 9). For example, viral
protein(s) such as coat protein and/or replicase may be expressed
in a transgenic plant that also expresses an efficacious dsRNA,
miRNA, or siRNA, without use of an additional transgene cassette,
by inserting protein coding sequence into the loop of a dsRNA. A
sequence that encodes a peptide aptamer that interferes with
geminivirus replication may also be employed (e.g. Lopez-Ochoa et
al., 2006).
[0172] Thus, artificial sequences may be expressed along with the
dsRNA, miRNA, or siRNA in order to augment the virus resistant
phenotype. For instance, an artificial nucleotide loop engineered
from the tospovirus genome may be used. The tospovirus (ssRNA)
genome comprises a "panhandle" structure due to the presence of
conserved terminal repeats of 8-11 nt at both ends of each genome
component. These terminal repeats from each component of the genome
(SEQ ID NOs:167-168), for instance from GBNV (Groundnut bud
necrosis virus) or CaCV (Capsicum chlorosis virus), may be fused
and used as part of a loop-forming sequence in a plant
transformation construct. Such an artificial sequence may comprise,
for instance, SEQ ID NOs:376-378, or SEQ ID NO:455, and can serve
as an artificial substrate competing for reverse transcriptase, may
interfere with proper circularization of replicating viral genome
components, may themselves generate nucleotide segments efficacious
via RNAi, or one or more of the above, thus interfering with virion
assembly.
[0173] FIG. 16 demonstrates that inclusion of tospovirus terminal
sequences in a construct for generating dsRNA results in measurable
resistance to a virus such as CaCV. In the tested construct, SEQ ID
NOs:376-378, themselves comprising SEQ ID NOs:168, were fused to
create SEQ ID NO:455 (i.e. bp 1-247 of SEQ ID NO:455 comprise SEQ
ID NO:376; bp 248-303 comprise SEQ ID NO:377, and bp 304-369
comprise SEQ ID NO:378). The construct was tested in both sense and
antisense orientations, and the level of virus resistance was
compared to that demonstrated by a control tomato plant,
non-transgenic but otherwise isogenic.
[0174] Sequences encoding, for instance, coat protein or replicase,
as well as artificial sequences described above, may be embedded
within an intron sequence as well. Thus, multiple modes of action
may be deployed by being engineered into a single expression
cassette (e.g. FIG. 9), or more than one expression cassette.
Example 7
Results of Exemplary Virus Resistance Assays Using Engineered
miRNAs
[0175] Engineered miRNA-producing constructs as listed in Table 7
were tested for efficacy. Bioassay results for geminivirus and
potexvirus assays are shown in Tables 8-9. Virus resistance was
observed due to miRNA expression, and correlating to expression and
proper processing of a given transgene transcript. For instance,
for SEQ ID NO:166, proper processing of PepMV6/mir167g was not
observed, nor was virus resistance seen for this construct, thus
correlating miRNA production with virus resistance in transgenic
R.sub.1 plants. Regarding tospovirus experiments, expression and
proper processing of transcripts from SEQ ID NOs:370, 371, 373, and
375 was observed, targeting, respectively, the RdRP, RdRP, NsM, and
N genes. CP4-positive R.sub.1 plants displayed reduced symptoms
when infected with TSWV. For SEQ ID NOs:372 and 374, targeting,
respectively, the NsM and N genes, proper processing of miRNA was
not observed, and no reduction in symptoms was noted.
[0176] The synthetic miRNAs were most effective against PepMV, and
gave delayed or reduced virus infection symptoms against
geminiviruses and tospovirus. The constructs can be combined in
stacked miRNA cassettes in order to synergistically inhibit the
target viruses. Since multiple miRNAs can be expressed from a
single expression cassette, constructs expressing, for instance, 8
or 10 antiviral miRNAs can be created (as shown in FIG. 10A-10B).
As an additional approach, antiviral miRNA expression cassettes can
be inserted as the "loop" sequence (e.g. FIG. 9) of a dsRNA
expression cassette, to combine the dsRNA and miRNA mechanisms for
viral control (see FIG. 10C).
TABLE-US-00008 TABLE 8 Results of geminivirus resistance assays
utilizing engineered sequences incorporated into MIR gene
backbones. Incorporated construct % Virus Resistance.sup.b Target
(SEQ ID NO) miRNA Expression.sup.a TYLCV ToLCNDV PepGMV PHYVV
Region SEQ ID NO: 156 Gemini1/MON1 yes .sup. 16 .+-. 16% 31 .+-. 17
37 .+-. 13 29 .+-. 16 Rep SEQ ID NO: 367 Gemini 2/MON13 yes 34 .+-.
27 27 .+-. 14 29 .+-. 4 32 .+-. 16 CP SEQ ID NO: 368 Gemini3/MON1
yes 13 .+-. 8 23 .+-. 7 34 .+-. 11 14 .+-. 19 CP SEQ ID NO: 158
Gemini4/MON5 no -- -- -- -- CP SEQ ID NO: 369 Gemini5/miR159a yes
14 .+-. 19 30 .+-. 20 51 .+-. 16 28 .+-. 37 C2/C3 SEQ ID NO: 164
Gemini6/miR159a yes 20 .+-. 13 23 .+-. 12 36 .+-. 10 23 .+-. 19 Rep
SEQ ID NO: 160 Gemini 7-1/MON13 yes 7 28 40 11 CP .sup.aproper
processing of the miRNA detected in transgenic plants .sup.baverage
percentage .+-. s.d. of R1 CP4-positive segregants for resistance
to tomato yellow leaf curl virus (TYLCV), Tomato leaf curl New
Delhi virus (ToLCNDV), Pepper golden mosaic virus (PePGMV), or
Pepper Huasteco yellow vein virus (PHYVV.
TABLE-US-00009 TABLE 9 Results of Pepino mosaic virus (PepMV)
resistance assays utilizing engineered sequences incorporated into
MIR gene backbones. Incorporated construct (SEQ ID NO) miRNA
Expression.sup.a % Virus Resistance.sup.b Target Region 363
PepMV1/miR159a yes Not tested RdRP 364 PepMV2/MON5 yes (weak) 10
.+-. 14 RdRP 365 PepMV3/MON13 yes 100 .+-. 0 RdRP 366 PepMV4/MON1
yes (weak) 44 .+-. 52 TGBp1 162 PepMV5/MON18 yes 99 .+-. 3 TGBp2
166 PepMV6/miR167g no -- TGBp3 .sup.aproper processing of the miRNA
detected in transgenic plants .sup.baverage percentage .+-. s.d. of
R1 CP4-positive segregants for resistance to PepMV.
[0177] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of the foregoing
illustrative embodiments, it will be apparent to those of skill in
the art that variations, changes, modifications, and alterations
may be applied to the composition, methods, and in the steps or in
the sequence of steps of the methods described herein, without
departing from the true concept, spirit, and scope of the
invention. More specifically, it will be apparent that certain
agents that are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope, and concept of the invention as defined
by the appended claims.
REFERENCES
[0178] The references listed below are incorporated herein by
reference to the extent that they supplement, explain, provide a
background, or teach methodology, techniques, and/or compositions
employed herein. [0179] U.S. Pat. Nos. 4,536,475; 4,693,977;
4,886,937; 4,940,838; 4,959,317; 5,015,580; 5,107,065; 5,110,732;
5,231,020; 5,262,316; 5,283,184; 5,302,523; 5,384,253; 5,464,763;
5,464,765; 5,501,967; 5,508,184; 5,527,695; 5,459,252; 5,538,880;
5,550,318; 5,563,055; 5,591,616; 5,693,512; 5,633,435; 5,759,829;
5,981,840; 6,118,047; 6,160,208; 6,229,067; 6,384,301; 6,399,861;
6,403,865; 6,852,907. [0180] U.S. Publication 2002/0048814; U.S.
Publication 2003/017596; U.S. Publication 2003/018993; U.S.
Publication 2004/0029283; U.S. Publications 2005/0120415; U.S.
Publication 2005/144669; U.S. Publication 2006/0200878; U.S.
Publication 2006/0174380; U.S. Publication 2007/0259785; U.S.
Publication 2007/0300329; U.S. Publication 2008/0066206. [0181]
Allen et al., Cell 121:207-221, 2005. [0182] Altschul et al., J.
Mol. Biol. 215:403-410, 1990. [0183] Ambros et al., RNA 9:277-279,
2003. [0184] Bartel, Cell 116:281-297, 2004. [0185] Bevan, Nature
304:184-187, 1983. [0186] Boulton, "Agrobacterium-Mediated Transfer
of geminiviruses to Plant Tissues," in "Methods in Molec. Biol.,
vol. 49: Plant Gene Transfer and Expression Protocols, ed. H.
Jones, Humana Press, Totowa, N.J., 1996. [0187] Brodersen and
Voinnet, Trends Genetics, 22:268-280, 2006. [0188] Broothaerts et
al., Nature, 433:629-633, 2005. [0189] Brutlag et al., Comp. Chem.
17: 203-207, 1993. [0190] Christopher and Rajam, Pl. Cell Tiss.
Org. Cult. 46:245-250, 1996. [0191] Chuang et al., PNAS,
97:4985-4990, 2000. [0192] Cotillon et al., Arch. Virol.
147:2225-2230, 2002. [0193] Dellaporta et al., Stadler Symposium
11:263-282, 1988. [0194] Elbashir et al., Genes & Devel.,
15:188-200, 2002. [0195] Frizzi et al., Plant Biotechnol. J.,
6:13-21, 2008. [0196] Griffiths-Jones, Nucleic Acids Res.,
31:439-441, 2003. [0197] Grimsley, Physiol. Plantarum, 79:147-153,
1990. [0198] Gruber et al., In: Vectors for Plant Transformation,
Glick and Thompson (Eds.), CRC Press, Inc., Boca Raton, 89-119,
1993. [0199] Hamilton and Baulcombe, Science, 286:950-952, 1999.
[0200] Hannon, Nature 418:244-251, 2002. [0201] Haymes et al.,
Nucleic Acid Hybridization, A Practical Approach, IRL Press,
Washington, D.C. (1985). [0202] Herrera-Estrella Nature
303:209-213, 1983. [0203] Hirel et al., Plant Molecular Biology,
20:207-218, 1992. [0204] Horsch et al., Science, 227:1229, 1985.
[0205] Ikatu et al., Bio/Technol. 8:241-242, 1990. [0206] Jefferson
et al., EMBO J. 6:3901-3907, 1987. [0207] Jones-Rhoades et al.
Annu. Rev. Plant Biol., 57:19-53, 2006. [0208] Kaeppler et al.,
Plant Cell Reports, 9:415-418, 1990. [0209] Katz et al., J. Gen.
Microbiol. 129:2703-2714, 1983. [0210] Kim, Nature Rev. Mol. Cell
Biol., 6:376-385, 2005. [0211] Klee, Bio/Technol. 3:637-642, 1985.
[0212] Kumar et al., Plant Dis. 77:938-941, 1993. [0213] Liu et
al., Plant Cell Rep. 9:360-364, 1990. [0214] Lopez et al., Arch.
Virol. 150:619-627, 2005. [0215] Lopez-Ochoa et al., J. Virol.,
80:5841, 2006. [0216] McCormick, Plant Tissue Culture Manual
B6:1-9, 1991. [0217] Miki et al., In: Methods in Plant Molecular
Biology and Biotechnology, Glick and Thompson (Eds.), CRC Press,
67-88, 1993. [0218] Moloney et al., Plant Cell Reports, 8:238,
1989. [0219] Niu et al., Nature Biotechnology 24:1420-1428, 2006.
[0220] Odell et al., Nature 313:810-812, 1985. [0221] Omirulleh et
al., Plant Mol. Biol., 21:415-28, 1993. [0222] Ow et al., Science
234:856-859, 1986. [0223] Padidam et al., J. Gen. Virol.
73:1609-1616, 1999. [0224] Potrykus et al., Mol. Gen. Genet.,
199:183-188, 1985. [0225] Redenbaugh et al. in "Safety Assessment
of Genetically Engineered Fruits and Vegetables", CRC Press, 1992.
[0226] Reynolds et al., Nat Biotechnol. 22:326-30, 2004. [0227]
Sambrook et al., (ed.), Molecular Cloning, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. [0228] Sutcliffe
et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741, 1978. [0229]
Tang, Trends Biochem. Sci., 30:106-14, 2005. [0230] Tomari and
Zamore, Genes & Dev., 19:517-529, 2005. [0231] Tomari et al.
Curr. Biol., 15:R61-64, 2005. [0232] Van Heeke and Schuster, J.
Biol. Chem. 264:5503-5509, 1989. [0233] Wesley et al., Plant J.,
27:581-590, 2001. [0234] WO 98/05770; WO 99/053050; WO 99/049029;
WO94/01550; WO 05/019408
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190249189A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190249189A1).
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