U.S. patent application number 14/960287 was filed with the patent office on 2016-07-07 for hypersensitive aba receptors.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Sean Cutler, Michael Nuccio, Quideng Que.
Application Number | 20160194653 14/960287 |
Document ID | / |
Family ID | 54073002 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160194653 |
Kind Code |
A1 |
Cutler; Sean ; et
al. |
July 7, 2016 |
HYPERSENSITIVE ABA RECEPTORS
Abstract
Hypersensitive PYR/PYL polypeptides, compositions, and methods
are provided.
Inventors: |
Cutler; Sean; (Riverside,
CA) ; Nuccio; Michael; (Research Triangle Park,
NC) ; Que; Quideng; (Research Triangle Park,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Family ID: |
54073002 |
Appl. No.: |
14/960287 |
Filed: |
December 4, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2015/047020 |
Aug 26, 2015 |
|
|
|
14960287 |
|
|
|
|
62042095 |
Aug 26, 2014 |
|
|
|
62098025 |
Dec 30, 2014 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/252.33; 435/254.2; 435/320.1; 435/325; 435/348; 435/349;
435/419; 536/23.6; 536/24.1; 800/298 |
Current CPC
Class: |
A01H 5/00 20130101; C07K
14/415 20130101; C12N 15/8213 20130101; C12N 15/8243 20130101; C12N
15/8271 20130101; C12N 15/00 20130101; C12N 15/8273 20130101; C12N
15/8293 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. 1258175 awarded by the National Science Foundation. The
Government has certain rights in this invention.
Claims
1. An isolated nucleic acid comprising a polynucleotide encoding a
mutated PYR/PYL receptor polypeptide comprising an amino acid
substitution corresponding to the amino acid F61, V81, I110, E141,
and A160 in PYR1 as set forth in SEQ ID NO:1, wherein the mutated
PYR/PYL receptor has increased sensitivity to abscisic acid
compared to a control PYR/PYL receptor lacking the
substitution.
2. The isolated nucleic acid of claim 1, wherein the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid F61.
3. The isolated nucleic acid of claim 2, wherein the amino acid
substitution is selected from L and M.
4. The isolated nucleic acid of claim 1, wherein the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid V81.
5. The isolated nucleic acid of claim 4, wherein the amino acid
substitution is selected from I and Y.
6. The isolated nucleic acid of claim 1, wherein the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid I110.
7. The isolated nucleic acid of claim 6, wherein the amino acid
substitution is selected from C and S.
8. The isolated nucleic acid of claim 1, wherein the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid E141.
9. The isolated nucleic acid of claim 8, wherein the amino acid
substitution is selected from C, I, L, M, N, T, V, W, and Y.
10. The isolated nucleic acid of claim 1, wherein the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid A160.
11. The isolated nucleic acid of claim 8, wherein the amino acid
substitution is selected from C, I, and V.
12. The isolated nucleic acid of claim 1, wherein the mutated
PYR/PYL receptor polypeptide is substantially identical to any of
SEQ ID NOs:1-119 or SEQ ID NOs:155-361 or comprises any of SEQ ID
NOs: 120-123.
13. The isolated nucleic acid of claim 1, wherein the
polynucleotide encodes a fusion protein, the fusion protein
comprising the mutated PYR/PYL receptor polypeptide and a fusion
partner protein.
14. The isolated nucleic acid of claim 13, wherein the fusion
partner protein is a transcriptional activation or modulation
domain.
15. The isolated nucleic acid of claim 14, wherein the
transcriptional activator is VP16 or VP64.
16. The isolated nucleic acid of claim 13, wherein the fusion
protein further comprises a nuclear localization signal
sequence.
17. A cell comprising a heterologous polynucleotide of claim 1.
18. The cell of claim 17, wherein the cell is a non-plant
eukaryotic cell.
19. A plant comprising the polynucleotide of claim 1.
20. A plant comprising an in situ mutated PYR/PYL receptor
polypeptide comprising an amino acid substitution corresponding to
the amino acid F61, V81, I110, E141, and A160 in PYR1 as set forth
in SEQ ID NO:1, wherein the mutated PYR/PYL receptor polypeptide
has increased sensitivity to abscisic acid compared to a control
PYR/PYL receptor lacking the substitution.
21. An expression cassette comprising a promoter operably linked to
the polynucleotide of claim 1, wherein introduction of the
expression cassette into a plant results in the plant having
increased sensitivity to abscisic acid compared to a control plant
lacking the expression cassette.
22. The expression cassette of claim 21, wherein the promoter is
heterologous to the polynucleotide.
23. The expression cassette of claim 21, wherein the promoter is
inducible.
24. The expression cassette of claim 21, wherein the promoter is a
stress-inducible promoter.
25. An expression vector comprising the expression cassette of
claim 21.
26. A plant comprising the expression cassette of claim 21, wherein
the plant has increased sensitivity to abscisic acid compared to a
control plant lacking the expression cassette.
27. A plant cell from the plant of claim 19.
28. A seed, flower, leaf, fruit, processed food, or food ingredient
from the plant of claim 19.
29. A method of producing a plant having increased sensitivity to
abscisic acid, the method comprising: introducing the expression
cassette of claim 21 into a plurality of plants; and selecting a
plant that expresses the polynucleotide from the plurality of
plants.
30. A method of producing a plant having increased sensitivity to
abscisic acid, the method comprising: introducing a mutation into a
polynucleotide encoding a PYR/PYL polypeptide, wherein the mutation
results in the polynucleotide of claim 1.
31. The method of claim 30, wherein the introducing occurs in situ
in the genome of a plant cell.
32. The method of claim 31, wherein the introducing comprises
clustered regularly interspaced short palindromic repeats
(CRISPR)/Cas genome editing.
33. A guide ribonucleic acid (gRNA) comprising: a) a CRISPR
ribonucleic acid (crRNA) that is substantially identical to SEQ ID
NOS: 363, 364, 365, 366, 367 or 369; and b) a transacting
ribonucleic acid (tracRNA), wherein the PYR/PYL mutation target
site comprises a nucleic acid that encodes for V89 of PYL-E or E149
of PYL-E.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present patent application is a continuation-in-part of
PCT/US2015/047020, filed Aug. 26, 2015, which claims benefit of
priority to U.S. Provisional Patent Application No. 62/042,095,
filed Aug. 26, 2014 and U.S. Provisional Patent Application No.
62/098,025, filed Dec. 30, 2014, which are incorporated by
reference for all purposes.
BACKGROUND OF THE INVENTION
[0003] Abscisic acid (ABA) is a plant hormone that regulates signal
transduction associated with abiotic stress responses Cutler, S.
R., et al. Annu. Rev. Plant Biol. 61, 651-679 (2010)). The ABA
signaling pathway has been exploited to improve plant stress
response and associated yield traits via numerous approaches (Wang,
Y., et al. Plant J. 43, 413-424 (2005)). The direct application of
ABA to plants improves their water use efficiency (Rademacher, W.,
Maisch, R., Liessegang, J., & Jung, J. (1987). Water
consumption and yield formation in crop plants under the influence
of synthetic analogues of abscisic acid. Plant growth regulators
for agricultural and amenity use. BCPC Monograph, (36), 53-66); for
this reason, the discovery of ABA agonists (Okamoto, M., et al.,
Proc. Natl. Acad. Sci. U.S.A 110, 12132-12137 (2013); Park, S.-Y.,
et al. Science 324, 1068-1071 (2009)) has received increasing
attention, as such molecules may be beneficial for improving crop
yield. A complementary approach to activating the ABA pathway
involves increasing a plant's sensitivity to ABA via genetic
methods. For example, conditional antisense of farnesyl transferase
beta subunit gene, which increases a plant's ABA sensitivity,
improves yield under moderate drought in both canola and
Arabidopsis (Wang et al., 2005).
[0004] It has recently been discovered that ABA elicits many of its
cellular responses by binding to a soluble family of receptors
called PYR/PYL proteins. PYR/PYL proteins belong to a large family
of ligand-binding proteins named the START superfamily (Iyer, L.
M., et al., Proteins Struct. Funct. Bioinforma. 43, 134-144, 2001;
Ponting, C. P., and Aravind, L. (1999). Trends Biochem. Sci. 24,
130-132 1999). These proteins contain a conserved three-dimensional
architecture consisting of seven anti-parallel beta sheets, which
surround a central alpha helix to form a "helix-grip" motif;
together, these structural elements form a ligand-binding pocket
for binding ABA or other agonists.
[0005] Structural and functional studies have uncovered a series of
conformational changes and critical contacts between PYR/PYL
receptors and type II C protein phosphatases (PP2Cs) that are
necessary for ABA-mediated PP2C inhibition by receptors. For
example, when ABA or another agonist binds within the
ligand-binding pockets of PYR/PYL proteins, it stabilizes a
conformational change that allows the receptors to bind and inhibit
a family of PP2Cs that normally repress ABA signaling (Weiner et
al., 2010). In particular, ABA binding leads to a large
rearrangement in a flexible "gate" loop that flanks the
ligand-binding pocket. Upon ABA binding, the gate loop adopts a
closed conformation that is stabilized by several direct contacts
between the loop and ABA. This agonist-bound, closed form of the
gate allows PYR/PYL proteins to dock into, and inhibit, the active
site of PP2Cs. The resulting inhibition in turn allows activation
of downstream kinases in the SnRK2 class, which are responsible for
the regulation of the activity of transcription factors, ion
channels and other proteins involved in ABA responses (Weiner, J.
J., et al. (2010) Curr. Opin. Plant Biol. 13, 495-5022010). Thus,
the stabilization of a closed gate conformation of the receptors
plays a role in their activation and PYR/PYL receptors are
molecular switches at the apex of a signaling cascade that
regulates diverse ABA responses.
[0006] In addition to the role that gate closure plays in receptor
activation, other structural rearrangements also occur. For
example, PYR1, PYL1, and PYL2 are homodimers in solution, but bind
to PP2Cs as monomers. The homodimer interface overlaps with the
PP2C binding interface and therefore an intact receptor homodimer
cannot bind to and inhibit the PP2C. Thus, dimer formation is
antagonistic to ABA signaling and receptor dimer-breaking is a
necessary step in receptor activation. Additionally, a recognition
module containing a central conserved tryptophan "lock" residue
located on the PP2C inserts into a small pore formed in the
ABA-bound receptors. Mutation of the tryptophan lock residue
abolishes receptor-mediated inactivation of PP2C activity,
demonstrating a role of the lock residue's insertion into the
receptor's pore.
[0007] Over-expression of wild type or mutant ABA receptors in
transgenic Arabidopsis thaliana, Solanum lycopersicum and Oryza
sativa improves drought tolerance (Gonzalez-Guzman, M., et al.
(2014). J. Exp. Bot. eru219, 2014; Kim et al., J. Exp. Bot. 63,
1013-1024 2012; Santiago et al., Plant J. 60, 575-588 (2009)). ABA
receptors with increased sensitivity relative to their wild type
counterparts can elicit greater ABA responses when expressed in
planta. Consistent with this, Pizzio et al., Plant Physiol. 163,
441-455 (2013) described the PYL4 mutation A194T mutant, which
requires lower concentrations of ABA to elicit measured ABA
responses in comparison to wild type PYL4. When this mutant is
over-expressed in transgenic Arabidopsis, the plants have increased
sensitivity to ABA relative to both wild type controls and PYL4
over-expression controls (Pizzio et al., Plant Physiol. 163,
441-455 (2013)). Moreover, the 35S::PYL4.sup.A194T lines display
better drought tolerance and water use than wild type or 35S::PYL4
overexpression lines. The A194T mutation is located in PYL4's
carboxyl terminus, which is a part of the receptors that is highly
variable in length and composition between receptors. This lack of
conservation makes it difficult to predict the mechanism by which
the mutation alters ABA sensitivity.
BRIEF SUMMARY OF THE INVENTION
[0008] Mutations in PYR/PYL receptor proteins have been identified
that result in the receptor proteins being hypersensitive to ABA.
In some embodiments, nucleic acids (e.g., isolated) encoding such
proteins are provided. In some embodiments, the nucleic acids
comprises a polynucleotide encoding a mutated PYR/PYL receptor
polypeptide comprising an amino acid substitution corresponding to
the amino acid F61, V81, I110, E141, and A160 in PYR1 as set forth
in SEQ ID NO:1, wherein the mutated PYR/PYL receptor has increased
sensitivity to abscisic acid compared to a control PYR/PYL receptor
lacking the substitution.
[0009] In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to the amino
acid F61. In some embodiments, the amino acid substitution is
selected from L and M.
[0010] In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to the amino
acid V81. In some embodiments, the amino acid substitution is
selected from I and Y.
[0011] In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to the amino
acid I110. In some embodiments, the amino acid substitution is
selected from C and S.
[0012] In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to the amino
acid E141. In some embodiments, the amino acid substitution is
selected from C, I, L, M, N, T, V, W, and Y.
[0013] In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to the amino
acid A160. In some embodiments, the amino acid substitution is
selected from C, I, and V.
[0014] In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to:
F61L and A160C;
F61M and A160V;
F61M, I110S, and A160V; or
F61L, V81I, I110C and A160V.
[0015] In some embodiments, the mutated PYR/PYL receptor
polypeptide is substantially identical to (e.g., at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%,
97%, 98%, or 99% identical to) any of SEQ ID NOs:1-119 or SEQ ID
NOs:124-154 (e.g., 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 151, 152, 153, or 154), 155-361 (e.g.,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,
181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,
246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,
259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,
272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,
285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,
298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,
311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,
324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,
337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,
350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, or 361) or
comprises any of SEQ ID NOs: 120-123.
[0016] Also provided is a plant (e.g. a transgenic or
non-transgenic plant) comprising a polynucleotide encoding a
PYR/PYL receptor polypeptide as described above or elsewhere
herein, e.g., comprising an amino acid substitution corresponding
to the amino acid F61, V81, I110, E141, and A160 in PYR1 as set
forth in SEQ ID NO:1. In some embodiments, the plant will have
increased sensitivity to ABA compared to a control plant lacking
the polypeptide. In some embodiments, the polynucleotide is
operably linked to a heterologous promoter. In some embodiments,
the polynucleotide is operably linked to a native
(non-heterologous) promoter. In some embodiments, the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to: F61L and A160C; F61M and A160V; F61M, I110S, and
A160V; or F61L, V81I, I110C and A160V. In some embodiments, the
encoded PYR/PYL receptor polypeptide only has one (or in some
embodiments, only 2, 3, or 4) amino acid substitution compared to
the plant's native PYR/PYL receptor polypeptide. In some
embodiments, the plant's native PYR/PYL receptor polypeptide coding
sequence has been modified (e.g., by CRISPR) to contain the 1, 2,
3, or 4 substitutions. In some embodiments, the PYR/PYL receptor
polypeptide comprises an amino acid substitution corresponding to
the amino acid F61. In some embodiments, the amino acid
substitution is selected from L and M. In some embodiments, the
PYR/PYL receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid V81. In some embodiments, the amino
acid substitution is selected from I and Y. In some embodiments,
the PYR/PYL receptor polypeptide comprises an amino acid
substitution corresponding to the amino acid I110. In some
embodiments, the amino acid substitution is selected from C and S.
In some embodiments, the PYR/PYL receptor polypeptide comprises an
amino acid substitution corresponding to the amino acid E141. In
some embodiments, the amino acid substitution is selected from C,
I, L, M, N, T, V, W, and Y. In some embodiments, the PYR/PYL
receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid A160. In some embodiments, the
amino acid substitution is selected from C, I, and V. In some
embodiments, the PYR/PYL receptor polypeptide comprises an amino
acid substitution corresponding to: F61L and A160C; F61M and A160V;
F61M, I110S, and A160V; or F61L, V81I, I110C and A160V.
[0017] Also provided is a plant (e.g., including but not limited to
a maize plant) comprising an in situ mutated PYR/PYL receptor
polypeptide comprising an amino acid substitution corresponding to
the amino acid F61, V81, I110, E141, and A160 in PYR1 as set forth
in SEQ ID NO:1, wherein the mutated PYR/PYL receptor polypeptide
has increased sensitivity to abscisic acid compared to a control
PYR/PYL receptor lacking the substitution. In some embodiments, the
PYR/PYL receptor polypeptide comprises an amino acid substitution
corresponding to the amino acid F61. In some embodiments, the amino
acid substitution is selected from L and M. In some embodiments,
the PYR/PYL receptor polypeptide comprises an amino acid
substitution corresponding to the amino acid V81. In some
embodiments, the amino acid substitution is selected from I and Y.
In some embodiments, the PYR/PYL receptor polypeptide comprises an
amino acid substitution corresponding to the amino acid I110. In
some embodiments, the amino acid substitution is selected from C
and S. In some embodiments, the PYR/PYL receptor polypeptide
comprises an amino acid substitution corresponding to the amino
acid E141. In some embodiments, the amino acid substitution is
selected from C, I, L, M, N, T, V, W, and Y. In some embodiments,
the PYR/PYL receptor polypeptide comprises an amino acid
substitution corresponding to the amino acid A160. In some
embodiments, the amino acid substitution is selected from C, I, and
V. In some embodiments, the PYR/PYL receptor polypeptide comprises
an amino acid substitution corresponding to: F61L and A160C; F61M
and A160V; F61M, I110S, and A160V; or F61L, V81I, I110C and A160V.
In some embodiments, the mutated PYR/PYL receptor polypeptide is
substantially identical to (e.g., at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%
identical to) any of SEQ ID NOs:1-119 or SEQ ID NOs:124-154 (e.g.,
124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,
137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,
150, 151, 152, 153, or 154), 155-361 (e.g., 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,
172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,
185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,
224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,
237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,
250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,
263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,
276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,
302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,
315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327,
328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,
341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,
354, 355, 356, 357, 358, 359, 360, or 361) or comprises any of SEQ
ID NOs: 120-123.
[0018] Also provided are expression cassettes comprising a promoter
operably linked to the polynucleotide encoding a PYR/PYL receptor
polypeptide as described above or elsewhere herein, e.g.,
comprising an amino acid substitution corresponding to the amino
acid F61, V81, I110, E141, and A160 in PYR1 as set forth in SEQ ID
NO:1, wherein introduction of the expression cassette into a plant
results in the plant having increased sensitivity to abscisic acid
compared to a control plant lacking the expression cassette.
[0019] In some embodiments, the promoter is heterologous to the
polynucleotide. In some embodiments, the promoter is inducible. In
some embodiments, the promoter is a stress-inducible promoter.
[0020] Also provided is an expression vector comprising the
expression cassette as described above or elsewhere herein.
[0021] Also provided are plants comprising an expression cassette
as described above or elsewhere herein, wherein the plant has
increased sensitivity to abscisic acid compared to a control plant
lacking the expression cassette. Also provided is a plant cell from
the plant.
[0022] Also provided is a seed, flower, leaf, fruit, processed
food, or food ingredient from a plant comprising a hypersensitive a
PYR/PYL receptor polypeptide as described herein.
[0023] Also provided is a method of producing a plant having
increased sensitivity to abscisic acid. In some embodiments, the
method comprises: introducing the expression cassette encoding a
hypersensitive a PYR/PYL receptor polypeptide as described herein
into a plurality of plants; and selecting a plant that expresses
the polynucleotide from the plurality of plants.
[0024] In some embodiments, the method comprises: introducing a
mutation into a polynucleotide encoding a hypersensitive PYR/PYL
polypeptide as described herein, e.g., wherein the mutation results
in a polynucleotide encoding an amino acid substitution
corresponding to the amino acid F61, V81, I110, E141, and A160 in
PYR1 as set forth in SEQ ID NO:1. In some embodiments, the
introducing occurs in situ in the genome of a plant cell. In some
embodiments, the introducing comprises clustered regularly
interspaced short palindromic repeats (CRISPR)/Cas genome
editing.
[0025] Provided herein are methods and reagents for producing a
plant (e.g., a maize plant) having increased sensitivity to
abscisic acid, the method includes introducing a mutation into a
polynucleotide encoding a PYR/PYL polypeptide, where the mutation
is introduced in situ in the genome of the plant using RNA directed
genome modification methods.
[0026] In one aspect, provided herein is a guide ribonucleic acid
(gRNA). In certain embodiments the gRNA includes a CRISPR
ribonucleic acid (crRNA) that is substantially identical to SEQ ID
NOS: 363, 364, 365, 366, 367 or 369; and a transacting ribonucleic
acid (tracRNA), where the PYR/PYL mutation target site comprises a
nucleic acid that encodes for V89 of PYL-E or E149 of PYL-E.
[0027] In some embodiments of the gRNA, the PYR/PYL mutation target
site includes a nucleic acid that encodes for V89 of PYL-E. In some
embodiments, the PYR/PYL mutation target site has the sequence of
SEQ ID NO:362.
[0028] In some embodiments, the PYR/PYL mutation target site
includes a nucleic acid that encodes for E149 of PYL-E. In certain
embodiments, the PYR/PYL mutation target site has the sequence of
SEQ ID NO:368.
[0029] In certain embodiments, the tracRNA is linked to the 3' end
of the gRNA. In specific embodiments, the tracRNA is encoded by a
nucleotide having a sequence that is substantially identical to SEQ
ID NO: 370.
[0030] In another aspect, provided herein is an isolated nucleic
acid that includes a polynucleotide encoding any one of the gRNAs
described herein.
[0031] In another aspect, provided herein is an expression cassette
that includes an RNA polymerase promoter operably linked to a
polynucleotide encoding any one of the gRNAs described herein. In
certain embodiments, the RNA polymerase promoter is an RNA
polymerase III (polIII) promoter. In specific embodiments, the
polIII promoter is a U3 promoter or a U6 promoter. In some
embodiments, the expression cassette has the sequence of any one of
SEQ ID NOS:371-373.
[0032] In another aspect, provided herein is an expression vector
that includes an expression cassette, where the expression cassette
includes an RNA polymerase promoter operably linked to a
polynucleotide encoding any one of the gRNAs described herein.
[0033] In another aspect, provided herein is an expression vector
that includes a first expression cassette and a second expression
cassette. In certain embodiments, the first expression cassette is
an expression cassette that includes an RNA polymerase promoter
operably linked to a polynucleotide encoding any one of the gRNAs
described herein and the second expression cassette is an
expression cassette comprising a promoter operably linked to a
polynucleotide encoding a CRISPR-associated endonuclease 9 (Cas9).
In some embodiments, the expression vector includes a third
expression cassette, wherein the third expression cassette is an
expression cassette that includes an RNA polymerase promoter
operably linked to a polynucleotide encoding any one of the gRNAs
described herein, and the third expression cassette is different
than the first expression cassette.
[0034] In some embodiments, the expression vector includes a first,
second and third expression cassette, where the first expression
cassette is an expression cassette that includes a promoter
operably linked to a polynucleotide encoding a CRISPR-associated
endonuclease 9 (Cas9), the second expression cassette has a
sequence that is substantially identical to SEQ ID NO: 371 or SEQ
ID NO:372, and the third expression cassette has a sequence that is
substantially identical to SEQ ID NO:373. In certain embodiments,
the promoter operably linked to the polynucleotide encoding Cas9 is
an ubiquitin-1 promoter (prUbi-10).
[0035] In another aspect, provided herein is a cell that includes
any of the expression vectors described above or elsewhere
herein.
[0036] Also provided is a plant that includes an expression vector
as described above or elsewhere herein. In some embodiments, the
plant is a maize plant.
[0037] Also provided is a plant cell from the plant described above
or elsewhere herein.
[0038] In another aspect, provided herein is a seed, flower, leaf,
fruit, processed food, or food ingredient from the plant described
above or elsewhere herein. In certain embodiments, the introduction
of the expression vector into the plant described above or
elsewhere herein results in the plant having increased sensitivity
to abscisic acid compared to a control plant lacking the expression
cassette.
[0039] In another aspect, provided herein is a method of producing
a plant having a mutation at a genomic PYR/PYL mutation target
site. In some embodiments the method includes introducing into
plant cells an expression vector that includes a polynucleotide
encoding a gRNA and a Cas9 as described above or elsewhere herein
and at least one repair nucleic acid comprising the mutation. In
certain embodiments, the mutation is introduced in the genomic
PYR/PYLR mutation target site by a homologous recombination upon a
Cas9 cleavage event in the genomic PYR/PYLR mutation target site.
In some embodiments, the method further includes selecting plant
cells having the mutation; thereby producing the plant. In some
embodiments, the introducing occurs in situ in the genome of a
plant cell. In some embodiments, the mutation is introduced by
introducing into a plant embryo cell the expression vector and at
least one repair nucleic acid, where the genome of the plant embryo
comprises the PYR/PYL mutation target site and where the repair
nucleic acid comprises the mutation and introduces the mutation at
the PYR/PYL mutation target site by homologous recombination upon a
Cas9 cleavage event in the PYL-E mutation target site.
[0040] In some embodiments, the repair nucleic acid has a sequence
that is substantially identical to any one of the sequence of SEQ
ID NOS:375 to 387 In certain embodiments, the repair nucleic acid
has a sequence that is substantially identical to SEQ ID NO:377. In
some embodiments, the repair nucleic acid has a sequence that is
substantially identical to the sequence of SEQ ID NO:387. In other
embodiments, two repair nucleic acids are introduced, and wherein
the repair nucleic acids have sequences that are substantially
identical to SEQ ID NO:377 and SEQ ID NO:379. In specific
embodiments, the plant is a maize plant.
[0041] In another aspect, provided herein is a kit that includes an
expression vector of that includes a polynucleotide encoding a gRNA
and a polynucleotide encoding a Cas9 as described above or
elsewhere herein and at least one repair nucleic acid, wherein the
repair nucleic acid comprises a PYL-E mutation and is capable of
introducing the PYL-E mutation in situ in a plant cell genome by
homologous recombination upon a Cas9 cleavage event. In some
embodiments, the at least one repair nucleic acid has a sequence
that is substantially identical to SEQ ID NOS::374 to 386.
[0042] In another aspect, provided herein is an isolated nucleic
acid comprising a polynucleotide encoding a mutated PYR/PYL
receptor polypeptide comprising an amino acid substitution
corresponding to the amino acid V89 in PYL-E, wherein the amino
acid substitution is A (SEQ ID NO:389). In some embodiments, the
mutated PYR/PYL receptor polypeptide further comprises an amino
acid substitution corresponding to the amino acid E149. In certain
embodiments the amino acid substitution corresponding to the amino
acid E149 is L (SEQ ID NO:390).
[0043] In yet another aspect, provided herein is an isolated
nucleic acid comprising a polynucleotide encoding a fusion protein
comprising a mutated PYR/PYL receptor polypeptide and a fusion
partner polypeptide, wherein the mutated PYR/PYL receptor
polypeptide comprises an amino acid substitution corresponding to
the amino acid V89 in PYL-E, wherein the amino acid substitution is
A. In certain embodiments, the mutated PYR/PYL receptor polypeptide
further comprises an amino acid substitution corresponding to the
amino acid E149. In specific embodiments, the amino acid
substitution corresponding to the amino acid E149 is L.
[0044] In some embodiments, the fusion partner polypeptide includes
a transcription activation domain or a transcription modulation
domain. In certain embodiments, the transcription activation domain
is VP16 or VP64. In certain embodiments, the fusion protein further
comprises a nuclear localization signal sequence. In some
embodiments, the mutated PYR/PYL receptor polypeptide has increased
sensitivity to abscisic acid compared to a control PYR/PYL receptor
polypeptide lacking the substitution.
[0045] Provided herein is a cell comprising a polynucleotide as
described above or elsewhere herein. In certain embodiments, the
polynucleotide is a heterologous polypeptide. In some embodiments,
the cell is a non-plant eukaryotic cell.
[0046] In yet another embodiment, provided herein is a plant that
includes a polynucleotide as described above or elsewhere herein.
In certain embodiments, the plant is a maize plant.
[0047] In another embodiment, provided herein is an expression
cassette comprising a promoter operably linked to a polynucleotide
as described above or elsewhere herein. In some embodiments, the
promoter is heterologous to the polynucleotide. In certain
embodiments, the promoter is inducible. In some embodiments, the
promoter is a stress-inducible promoter.
[0048] In another embodiment, provided herein is an expression
vector comprising the expression cassette as described above or
elsewhere herein.
[0049] In another aspect, provided herein is a plant that includes
the expression cassette as described above or elsewhere herein. In
another aspect, provided herein is a plant cell from the plant as
described above or elsewhere herein. In yet another aspect,
provided herein is a seed, flower, leaf, fruit, processed food, or
food ingredient from the plant as described above or elsewhere
herein.
[0050] Other aspects of the invention are described elsewhere
herein.
DEFINITIONS
[0051] The term "PYR/PYL receptor polypeptide" refers to a protein
characterized in part by the presence of one or more or all of a
polyketide cyclase domain 2 (PF10604), a polyketide cyclase domain
1 (PF03364), and a Bet V I domain (PF03364), which in wild-type
form mediates abscisic acid (ABA) and ABA analog signaling. A wide
variety of PYR/PYL receptor polypeptide sequences are known in the
art. In some embodiments, a PYR/PYL receptor polypeptide comprises
a polypeptide that is substantially identical to PYR1 (SEQ ID
NO:1), PYL1 (SEQ ID NO:2), PYL2 (SEQ ID NO:3), PYL3 (SEQ ID NO:4),
PYL4 (SEQ ID NO:5), PYL5 (SEQ ID NO:6), PYL6 (SEQ ID NO:7), PYL7
(SEQ ID NO:8), PYL8 (SEQ ID NO:9), PYL9 (SEQ ID NO:10), PYL10 (SEQ
ID NO:11), PYL11 (SEQ ID NO:12), PYL12 (SEQ ID NO:13), or PYL13
(SEQ ID NO:14), or to any of SEQ ID NOs:15-119.
[0052] A "wild-type PYR/PYL receptor polypeptide" refers to a
naturally occurring PYR/PYL receptor polypeptide that mediates
abscisic acid (ABA) and ABA analog signaling.
[0053] A "mutated PYR/PYL receptor polypeptide" refers to a PYR/PYL
receptor polypeptide that is a variant from a naturally-occurring
(i.e., wild-type) PYR/PYL receptor polypeptide. As used herein, a
mutated PYR/PYL receptor polypeptide comprises one, two, three,
four, or more amino acid substitutions relative to a corresponding
wild-type PYR/PYL receptor polypeptide while retaining
ABA-responsiveness of the receptor. In this context, a "mutated"
polypeptide can be generated by any method for generating non-wild
type nucleotide sequences. In some embodiments, a mutated PYR/PYL
receptor polypeptide is hypersensitive, meaning the mutant receptor
polypeptide is activated by ABA more strongly than a corresponding
homologous wildtype receptor (or at least compared to an otherwise
identical PYR/PYL polypeptide having the wildtype amino acid at the
mutated position described herein) would be activated by the same
concentration of ABA, or that the mutant receptor polypeptide is
activated by a lower (e.g., half or less of the) concentration of
ABA than activates the corresponding homologous wild-type receptor,
or both. In some embodiments, the mutant receptor polypeptide can
be determined visually in a HAB1 yeast two-hybrid assay to respond
to 0.25 .mu.M or less ABA.
[0054] An "amino acid substitution" refers to replacing the
naturally occurring amino acid residue in a given position (e.g.,
the naturally occurring amino acid residue that occurs in a
wild-type PYR/PYL receptor polypeptide) with an amino acid residue
other than the naturally-occurring residue. For example, the
naturally occurring amino acid residue at position 60 of the
wild-type PYR1 receptor polypeptide sequence (SEQ ID NO:1) is
histidine (H60); accordingly, an amino acid substitution at H60
refers to replacing the naturally occurring histidine with any
amino acid residue other than histidine.
[0055] An amino acid residue "corresponding to an amino acid
residue [X] in [specified sequence," or an amino acid substitution
"corresponding to an amino acid substitution [X] in [specified
sequence]" refers to an amino acid in a polypeptide of interest
that aligns with the equivalent amino acid of a specified sequence.
Generally, as described herein, the amino acid corresponding to a
position of a specified PYR/PYL receptor polypeptide sequence can
be determined using an alignment algorithm such as BLAST. In some
embodiments of the present invention, "correspondence" of amino
acid positions is determined by aligning to a region of the PYR/PYL
receptor polypeptide comprising SEQ ID NO:1, as discussed further
herein. When a PYR/PYL receptor polypeptide sequence differs from
SEQ ID NO:1 (e.g., by changes in amino acids or addition or
deletion of amino acids), it may be that a particular mutation
associated with hypersensitive activity of the PYR/PYL receptor
will not be in the same position number as it is in SEQ ID NO:1.
For example, amino acid position V85 of PYL2 (SEQ ID NO:3) aligns
with amino acid position V81 of PYR1 (SEQ ID NO:1), as can be
readily illustrated in an alignment of the two sequences. In this
example, amino acid position 85 in SEQ ID NO:3 corresponds to
position 81 in SEQ ID NO:1. Examples of corresponding positions are
shown in FIG. 2
[0056] Two nucleic acid sequences or polypeptides are said to be
"identical" if the sequence of nucleotides or amino acid residues,
respectively, in the two sequences is the same when aligned for
maximum correspondence as described below. The terms "identical" or
percent "identity," in the context of two or more nucleic acids or
polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same, when compared
and aligned for maximum correspondence over a comparison window, as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. When percentage of
sequence identity is used in reference to proteins or peptides, it
is recognized that residue positions that are not identical often
differ by conservative amino acid substitutions, where amino acids
residues are substituted for other amino acid residues with similar
chemical properties (e.g., charge or hydrophobicity) and therefore
do not change the functional properties of the molecule. Where
sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to correct for the
conservative nature of the substitution. Means for making this
adjustment are well known to those of skill in the art. Typically
this involves scoring a conservative substitution as a partial
rather than a full mismatch, thereby increasing the percentage
sequence identity. Thus, for example, where an identical amino acid
is given a score of 1 and a non-conservative substitution is given
a score of zero, a conservative substitution is given a score
between zero and 1. The scoring of conservative substitutions is
calculated according to, e.g., the algorithm of Meyers &
Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif., USA).
[0057] The phrase "substantial identity" or "substantially
identical," used in the context of two nucleic acids or
polypeptides, refers to a sequence that has at least 50% sequence
identity with a reference sequence. Alternatively, percent identity
can be any integer from 50% to 100%. In some embodiments, a
sequence is substantially identical to a reference sequence if the
sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to
the reference sequence as determined using the methods described
herein; preferably BLAST using standard parameters, as described
below. Embodiments of the present invention provide for nucleic
acids encoding polypeptides that are substantially identical to any
of SEQ ID NO:1-119 or SEQ ID NOs:155-361.
[0058] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0059] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection.
[0060] Algorithms that are suitable for determining percent
sequence identity and sequence similarity are the BLAST and BLAST
2.0 algorithms, which are described in Altschul et al. (1990)J.
Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids
Res. 25: 3389-3402, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (NCBI) web site. The algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al, supra).
These initial neighborhood word hits acts as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Cumulative scores
are calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a word size (W) of 28, an
expectation (E) of 10, M=1, N=-2, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
word size (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)).
[0061] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.01, more preferably
less than about 10.sup.-5, and most preferably less than about
10.sup.-20.
[0062] The term "promoter," as used herein, refers to a
polynucleotide sequence capable of driving transcription of a
coding sequence in a cell. Thus, promoters used in the
polynucleotide constructs of the invention include cis-acting
transcriptional control elements and regulatory sequences that are
involved in regulating or modulating the timing and/or rate of
transcription of a gene. For example, a promoter can be a
cis-acting transcriptional control element, including an enhancer,
a promoter, a transcription terminator, an origin of replication, a
chromosomal integration sequence, 5' and 3' untranslated regions,
or an intronic sequence, which are involved in transcriptional
regulation. These cis-acting sequences typically interact with
proteins or other biomolecules to carry out (turn on/off, regulate,
modulate, etc.) gene transcription. A "plant promoter" is a
promoter capable of initiating transcription in plant cells. A
"constitutive promoter" is one that is capable of initiating
transcription in nearly all tissue types, whereas a
"tissue-specific promoter" initiates transcription only in one or a
few particular tissue types.
[0063] A polynucleotide sequence is "heterologous" to an organism
or a second polynucleotide sequence if it originates from a foreign
species, or, if from the same species, is modified from its
original form. For example, when a promoter is said to be operably
linked to a heterologous coding sequence, it means that the coding
sequence is derived from one species whereas the promoter sequence
is derived another, different species; or, if both are derived from
the same species, the coding sequence is not naturally associated
with the promoter (e.g., is a genetically engineered coding
sequence, e.g., from a different gene in the same species, or an
allele from a different ecotype or variety).
[0064] An "expression cassette" refers to a nucleic acid construct
that, when introduced into a host cell, results in transcription
and/or translation of an RNA or polypeptide, respectively.
Antisense or sense constructs that are not or cannot be translated
are expressly included by this definition. In the case of both
expression of transgenes and suppression of endogenous genes (e.g.,
by antisense, or sense suppression) one of skill will recognize
that the inserted polynucleotide sequence need not be identical,
but may be only substantially identical to a sequence of the gene
from which it was derived. As explained herein, these substantially
identical variants are specifically covered by reference to a
specific nucleic acid sequence.
[0065] The term "host cell" refers to any cell capable of
replicating and/or transcribing and/or translating a heterologous
polynucleotide. Thus, a "host cell" refers to any prokaryotic cell
(including but not limited to E. coli) or eukaryotic cell
(including but not limited to yeast cells, mammalian cells, avian
cells, amphibian cells, plant cells, fish cells, and insect cells),
whether located in vitro or in vivo. For example, host cells may be
located in a transgenic animal or transgenic plant prokaryotic cell
(including but not limited to E. coli) or eukaryotic cells
(including but not limited to yeast cells, mammalian cells, avian
cells, amphibian cells, plant cells, fish cells, and insect cells).
Host cells can be for example, transformed with the heterologous
polynucleotide.
[0066] The term "plant" includes whole plants, shoot vegetative
organs and/or structures (e.g., leaves, stems and tubers), roots,
flowers and floral organs (e.g., bracts, sepals, petals, stamens,
carpels, anthers), ovules (including egg and central cells), seed
(including zygote, embryo, endosperm, and seed coat), fruit (e.g.,
the mature ovary), seedlings, plant tissue (e.g., vascular tissue,
ground tissue, and the like), cells (e.g., guard cells, egg cells,
trichomes and the like), and progeny of same. The class of plants
that can be used in the method of the invention is generally as
broad as the class of higher and lower plants amenable to
transformation techniques, including angiosperms (monocotyledonous
and dicotyledonous plants), gymnosperms, ferns, and multicellular
algae. It includes plants of a variety of ploidy levels, including
aneuploid, polyploid, diploid, haploid, and hemizygous.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] FIG. 1 provides signal in a yeast two-hybrid assay with ABA
concentration shown at the top and the identity of the mutants
shown on the left side.
[0068] FIG. 2 provides the corresponding naturally-occurring amino
acid at the five positions described herein for a number of
different PYR/PYL proteins.
[0069] FIG. 3 provides an alignment of the middle portion a number
of PYR/PYL proteins. (SEQ ID NOs:142-154)
[0070] FIG. 4 provides signal in a yeast two-hybrid assay and
includes multiple mutations within PYR1.
[0071] FIG. 5 depicts a biolistic transformation vector
pZmPYLE-V89A carrying expression cassettes for maize-optimized Cas9
and ZmPYLE-V89A gRNA to mediate cleavage at the ZmPYL-E target
sequence (5'-CGCGA CGTCA ACGTC AAGAC-3' (SEQ ID NO:362))
[0072] FIG. 6 provides a schematic map of binary vector
pZmPYLE-E149L used for delivery with Agrobacterium-mediated
transformation.
[0073] FIG. 7 provides a schematic map of plasmid vector
pZmPYLE-V89A-E149L carrying expression cassettes for 2 different
gRNAs and Cas9.
[0074] FIG. 8 provides a schematic map of binary plant
transformation vector 23190 carrying expression cassettes for Cas9,
gRNA and selectable marker gene PMI for mediating ZmPYL-E E149L
mutagenesis.
[0075] FIG. 9A-B provide a schematic map of binary plant
transformation vectors 23136 and 23189 carrying expression
cassettes for Cas9, gRNA and selectable marker gene PMI for
mediating ZmPYL-D E169L mutagenesis.
[0076] FIG. 10A-10B provide a schematic map of binary plant plant
transformation vectors 22981 and 23191 carrying expression
cassettes for Cas9, gRNA and selectable marker gene PMI for
mediating ZmPYL-F E164L mutagenesis.
[0077] FIG. 11 provides a schematic map of binary plant plant
transformation vector 23192 carrying expression cassettes for Cas9,
gRNA and selectable marker gene PMI for mediating ZmPYL-E
E148L.
[0078] FIG. 12 provides a schematic drawing of end point assay
example to detect specific DNA sequence change (GA to CT) in
ZmPYL-F that results in E164L amino acid residue mutation.
[0079] FIG. 13 shows sequence alignment (SEQ ID NOS:414-430) of
targeted mutations in ZmPYL-F mediated by gRNA-Cas9 expressed from
vector 22981.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0080] To identify mutations causing increased receptor ABA
sensitivity, we screened for mutants that lower the concentration
of ABA required to induce a detectable interaction between PYR1 and
HAB1 using a collection of PYR1 mutants that contain all possible
single amino acid substitutions residues located in close proximity
to ABA. Based on these results, we describe mutations in
highly-conserved residues that substantially increase receptor ABA
sensitivity.
[0081] Mutations in PYR/PYL receptor polypeptides have been
discovered that result in hypersensitive forms of the PYR/PYL
receptor, i.e., the mutated receptors are more responsive to the
ABA compared to a corresponding wildtype PYR/PYL polypeptide.
[0082] Expression in a plant of one or more hypersensitive mutant
PYR/PY1 receptor polypeptides as described here will result in a
plant with increased ABA-sensitivity, and in some embodiments,
higher stress tolerance or other phenotypes associated with
ABA-responsiveness.
[0083] Also provided herein are methods and reagents for producing
a plant (e.g., a maize plant) having increased sensitivity to
abscisic acid, the method includes introducing a mutation into a
polynucleotide encoding a PYR/PYL polypeptide, where the mutation
is introduced in situ in the genome of the plant using RNA directed
genome modification methods.
II. Hypersensitive PYR/PYL Receptor Polypeptides
[0084] A wide variety of wild-type (naturally occurring) PYR/PYL
polypeptide sequences are known in the art. Although PYR1 was
originally identified as an abscisic acid (ABA) receptor in
Arabidopsis, in fact PYR1 is a member of a group of at least 14
proteins (PYR/PYL proteins) in the same protein family in
Arabidopsis that also mediate ABA signaling. This protein family is
also present in other plants (see, e.g., SEQUENCE LISTING) and is
characterized in part by the presence of one or more or all of a
polyketide cyclase domain 2 (PF10604), a polyketide cyclase domain
1 (PF03364), and a Bet V I domain (PF03364). START/Bet v 1
superfamily domain are described in, for example, Radauer, BMC
Evol. Biol. 8:286 (2008). In some embodiments, a wild-type PYR/PYL
receptor polypeptide comprises any of SEQ ID NOs:1-119. In some
embodiments, a wild-type PYR/PYL receptor polypeptide is
substantially identical to (e.g., at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%
identical to) any of SEQ ID NOs:1-119.
[0085] PYR/PYL receptor proteins have a conserved START-domain
ligand-binding pocket flanked by two loops called the "gate" and
the "latch" (Melcher, K. et al., Nature 462 (2009)). ABA binds to a
PYR/PYL receptor protein at the ligand-binding pocket and ABA
binding induces closure of the loops to seal ABA inside the
ligand-binding pocket. The ligand-binding pocket of a PYR/PYL
receptor polypeptide comprises amino acid residues that are in
close proximity (e.g., within about 5 .ANG.) to a PYR/PYL ligand
(e.g., ABA) or a ligand-contacting water molecule when the ligand
is bound to the PYR/PYL receptor. There are 25 residues that make
up the PYR1 ligand-binding pocket. The residues of the
ligand-binding pocket are also highly conserved among other PYR/PYL
family members.
[0086] PYR/PYL receptor proteins directly bind to type 2 protein
phosphatases (PP2Cs) and thus also contain a PP2C binding
interface. The PP2C binding interface of a PYR/PYL receptor
polypeptide comprises amino acid residues that are in close
proximity (e.g., within about 5 .ANG.) to PP2C when PP2C, the
PYR/PYL receptor, and ABA are all bound together in a ternary
complex. There are 25 residues that make up the PYR1 PP2C binding
interface. The residues of the PP2C binding interface are also
highly conserved among other PYR/PYL family members.
[0087] Hypersensitive PYR/PYL receptor polypeptides are
non-naturally-occurring variants from naturally occurring (i.e.,
wild-type) PYR/PYL receptor polypeptides, wherein the variant
(mutant) PYR/PYL receptor polypeptide is able to bind to and/or
inhibit the activity of a PP2C in the presence of abscisic acid to
a greater extent than a control PYR/PYL receptor polypeptide in the
presence of the same concentration of ABA. Hypersensitive active
PYR/PYL receptor polypeptides as described herein comprise one or
more amino acid substitutions compared to a wild type PYR/PYL
receptor polypeptide. In some embodiments, a hypersensitive PYR/PYL
receptor polypeptide is substantially identical to (e.g., at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%
95%, 96%, 97%, 98%, or 99% identical to) any of SEQ ID NO:1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, or 119 and comprises 1, 2, 3, 4, or more mutations (e.g.,
amino acid substitutions) as described herein. In some embodiments,
a hypersensitive PYR/PYL receptor polypeptide comprises SEQ ID
NO:120, 121, 122, or 123 and comprises 1, 2, 3, 4, or more
mutations (e.g., amino acid substitutions) as described herein:
TABLE-US-00001 (SEQ ID NO: 120)
CxSxxxxxxxAPxxxxWxxxxxFxxPxxxxxFxxxC (SEQ ID NO: 121)
GxxRxVxxxSxxPAxxSxExLxxxD (SEQ ID NO: 122) GGxHRLxNYxS (SEQ ID NO:
123) ESxxVDxPxGxxxxxTxxFxxxxxxxNLxxL.
[0088] As shown in the Examples, it has been discovered that
mutations can be made at any of several positions in PYR/PYL
receptor polypeptides result in hypersensitivity to ABA. These
positions are (corresponding to their position in Arabidopsis PYR1
(SEQ ID NO:1)): F61, V81, I110, E141, and A160. In some
embodiments, a mutated PYR/PYL receptor polypeptide comprises one
or more (e.g., one, two, three, or four) amino acid substitutions
corresponding to these positions. For example, in some embodiments,
the mutated PYR/PYL receptor polypeptide comprises at least the
following corresponding mutations:
F61L and A160C;
F61M and A160V;
F61M, I110S, and A160V; or
F61L, V81I, I110C and A160V.
TABLE-US-00002 [0089] SEQ ID NO: 1; Arabidopsis wildtype PYR1 Met
Pro Ser Glu Leu Thr Pro Glu Glu Arg Ser Glu 1 5 10 Leu Lys Asn Ser
Ile Ala Glu Phe His Thr Tyr Gln 15 20 Leu Asp Pro Gly Ser Cys Ser
Ser Leu His Ala Gln 25 30 35 Arg Ile His Ala Pro Pro Glu Leu Val
Trp Ser Ile 40 45 Val Arg Arg Phe Asp Lys Pro Gln Thr Tyr Lys His
50 55 60 Phe Ile Lys Ser Cys Ser Val Glu Gln Asn Phe Glu 65 70 Met
Arg Val Gly Cys Thr Arg Asp Val Ile Val Ile 75 80 Ser Gly Leu Pro
Ala Asn Thr Ser Thr Glu Arg Leu 85 90 95 Asp Ile Leu Asp Asp Glu
Arg Arg Val Thr Gly Phe 100 105 Ser Ile Ile Gly Gly Glu His Arg Leu
Thr Asn Tyr 110 115 120 Lys Ser Val Thr Thr Val His Arg Phe Glu Lys
Glu 125 130 Asn Arg Ile Trp Thr Val Val Leu Glu Ser Tyr Val 135 140
Val Asp Met Pro Glu Gly Asn Ser Glu Asp Asp Thr 145 150 155 Arg Met
Phe Ala Asp Thr Val Val Lys Leu Asn Leu 160 165 Gln Lys Leu Ala Thr
Val Ala Glu Ala Met Ala Arg 170 175 180 Asn Ser Gly Asp Gly Ser Gly
Ser Gln Val Thr 185 190
[0090] For position F61 (corresponding to the position in SEQ ID
NO:1), hypersensitive mutations will include F61L or F61M. For
position V81 (corresponding to the position in SEQ ID NO:1),
hypersensitive mutations will include V81I or V81Y. For position
I110 (corresponding to the position in SEQ ID NO:1), hypersensitive
mutations will include I110C or MOS. As some native PYR/PYL
polypeptides have a valine at the position corresponding to I110 of
SEQ ID NO:1, in some embodiments where position I110 is mutated,
the native amino acid will be valine, subsequently mutated to C or
S. For position E141 (corresponding to the position in SEQ ID
NO:1), hypersensitive mutations will include E141C, E141I, E141L,
E141M, E141N, E141T, E141V, E141W, or E141Y. For position A160
(corresponding to the position in SEQ ID NO:1), hypersensitive
mutations will include A160C, A160I or A160V. As some native
PYR/PYL polypeptides have a valine at the position corresponding to
A160 of SEQ ID NO:1, in some embodiments where position A160 is
mutated, the native amino acid will be valine, subsequently mutated
to C or I.
[0091] Any of the mutations described herein can be made in any
wildtype PYR/PYL polypeptide, for example, in the polypeptides of
any of SEQ ID NOs:1-119 or in polypeptides substantially identical
to any of SEQ ID NOs:1-119 or comprising any of SEQ ID NOs:
120-123. Analogous amino acid substitutions can be made, for
example, in PYR/PYL receptors other than PYR1 by aligning the
PYR/PYL receptor polypeptide sequence to be mutated with the PYR1
receptor polypeptide sequence as set forth in SEQ ID NO:1. As a
non-limiting example, an amino acid substitution in PYL2 that is
analogous to the amino acid substitution V81I in PYR1 as set forth
in SEQ ID NO:1 can be determined by aligning the amino acid
sequences of PYL2 (SEQ ID NO:3) and PYR1 (SEQ ID NO:1) and
identifying position V85 in PYL2 as aligning with amino acid
position V81 of PYR1 (SEQ ID NO:1). Analogous amino acid positions
in PYR/PYL receptors are shown in FIGS. 2 and 3. As an example, SEQ
ID NOS:155-361 represent maize PYR/PYL polypeptides containing the
hypersensitive mutations described herein. It will be appreciated
that the polypeptides can be further mutated (e.g., with
conservative mutations, e.g., outside active sites) without
substantially affecting activity. Accordingly, in some embodiments,
the hypersensitive polypeptides as described herein comprise a
sequence substantially (e.g., at least 70%, 75%, 80%, 85%, 90%,
95%, 98%) identical to the entire sequence of one of SEQ ID NOs:
155-361.
[0092] The extent to which one or more amino acid substitutions in
the PYR/PYL receptor activity renders the receptor hypersensitive
to ABA can be quantitatively measured, for example by assaying
phosphatase activity in the presence of ABA and the PYR/PYL
receptor comprising one or more amino acid substitutions and
comparing the phosphatase activity to that of a control PYR/PYL
receptor. A control PYR/PYL receptor will typically be the wildtype
PYR/PYL polypeptide most similar to the mutated a PYR/PYL
polypeptide. In some embodiments, e.g., when the starting protein
is not a wildtype PYR/PYL polypeptide, the control PYR/PYL
polypeptide can be substantially identical (e.g., at least 90, 95,
or 98% identical) to the test PYR/PYL polypeptide (i.e., suspected
of being hypersensitive) and having the wildtype amino acid at the
corresponding position. For example, if the mutant PYR/PYL receptor
has a mutation of F61X, where X is any non-F amino acid, the
control would have F61 at the same position but would otherwise be
identical to the mutant PYR/PYL receptor. If the mutant PYR/PYL
receptor has a mutation of V81X, where X is any non-V amino acid,
the control would have V81 at the same position but would otherwise
be identical to the mutant PYR/PYL receptor. If the mutant PYR/PYL
receptor has a mutation of I110X, where X is any non-I, non-V amino
acid, the control would have I110 or V at the same position but
would otherwise be identical to the mutant PYR/PYL receptor. If the
mutant PYR/PYL receptor has a mutation of E141X, where X is any
non-E amino acid, the control would have E141 at the same position
but would otherwise be identical to the mutant PYR/PYL receptor. If
the mutant PYR/PYL receptor has a mutation of A160X, where X is any
non-A, non-V amino acid, the control would have A160 or valine at
the same position but would otherwise be identical to the mutant
PYR/PYL receptor.
[0093] In some embodiments, a mutated PYR/PYL receptor polypeptide
comprises two or more amino acid substitutions as described herein.
In some embodiments, the two or more amino acid substitutions
corresponding to, F61X, V81X, I110X, E141X, and A160X, in PYR1 as
set forth in SEQ ID NO:1, where X is the amino acid indicated
herein as resulting in hypersensitivity.
[0094] Embodiments of the present invention provide for use of the
above proteins and/or nucleic acid sequences, encoding such
polypeptides, in the methods and compositions (e.g., expression
cassettes, transgenic plants, plants with in situ PYR/PYL
modifications, etc.) of the present invention. The isolation of a
polynucleotide sequence encoding a plant wild-type PYR/PYL receptor
(e.g., from plants where PYR/PYL sequences have not yet been
identified) may be accomplished by a number of techniques. For
instance, oligonucleotide probes based on the PYR/PYL coding
sequences disclosed (e.g., as listed in the SEQUENCE LISTING) here
can be used to identify the desired wild-type PYR/PYL gene in a
cDNA or genomic DNA library. To construct genomic libraries, large
segments of genomic DNA are generated by random fragmentation,
e.g., using restriction endonucleases, and are ligated with vector
DNA to form concatemers that can be packaged into the appropriate
vector. To prepare a cDNA library, mRNA is isolated from the
desired tissue, such as a leaf from a particular plant species, and
a cDNA library containing the gene transcript of interest is
prepared from the mRNA. Alternatively, cDNA may be prepared from
mRNA extracted from other tissues in which PYR/PYL gene is
expressed.
[0095] The cDNA or genomic library can then be screened using a
probe based upon the sequence of a PYR/PYL gene disclosed here.
Probes may be used to hybridize with genomic DNA or cDNA sequences
to isolate homologous genes in the same or different plant species.
Alternatively, antibodies raised against a polypeptide can be used
to screen an mRNA expression library.
[0096] Alternatively, the nucleic acids encoding PYR/PYL can be
amplified from nucleic acid samples using amplification techniques.
For instance, polymerase chain reaction (PCR) technology can be
used to amplify the coding sequences of PYR/PYL directly from
genomic DNA, from cDNA, from genomic libraries or cDNA libraries.
PCR and other in vitro amplification methods may also be useful,
for example, to clone polynucleotide sequences encoding PYR/PYL to
be expressed, to make nucleic acids to use as probes for detecting
the presence of the desired mRNA in samples, for nucleic acid
sequencing, or for other purposes. For a general overview of PCR
see PCR Protocols: A Guide to Methods and Applications (Innis, M.,
Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San
Diego (1990). Appropriate primers and probes for identifying
sequences from plant tissues are generated from comparisons of the
sequences provided here with other related genes.
[0097] In some embodiments, the partial or entire genome of a
number of plants has been sequenced and open reading frames
identified. By a BLAST search, one can identify the coding sequence
for wild-type PYR/PYL in various plants.
III. Methods of Making Hypersensitive PYR/PYL Receptor
Polypeptides
[0098] In another aspect, the present invention provides for
methods of making ABA hypersensitive PYR/PYL receptor polypeptides
comprising one or more amino acid substitutions. In some
embodiments, the method comprises mutagenizing a wild-type PYR/PYL
receptor and determining whether the mutagenized PYR/PYL receptor
is hypersensitive to ABA.
[0099] Mutated PYR/PYL receptor polypeptides can be constructed by
mutating the DNA sequences that encode the corresponding wild-type
PYR/PYL receptor polypeptide (e.g., a wild-type PYR/PYL polypeptide
of any of SEQ ID NOs:1-119, having any of SEQ ID NO:s 120-123, or a
corresponding variant from which the mutant PYR/PYL receptor
polypeptide of the invention is derived), such as by using
site-directed or random mutagenesis. Nucleic acid molecules
encoding the wild-type PYR/PYL receptor polypeptide can be mutated
by a variety of polymerase chain reaction (PCR) techniques
well-known to one of ordinary skill in the art. (See, e.g., PCR
Strategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds.,
1995, Academic Press, San Diego, Calif.) at Chapter 14; PCR
Protocols: A Guide to Methods and Applications (M. A. Innis, D. H.
Gelfand, J. J. Sninsky, and T. J. White eds., Academic Press, N Y,
1990).
[0100] As a non-limiting example, mutagenesis may be accomplished
using site-directed mutagenesis, in which point mutations,
insertions, or deletions are made to a DNA template. Kits for
site-directed mutagenesis are commercially available, such as the
QuikChange Site-Directed Mutagenesis Kit (Stratagene). Briefly, a
DNA template to be mutagenized is amplified by PCR according to the
manufacturer's instructions using a high-fidelity DNA polymerase
(e.g., Pfu Turbo.TM.) and oligonucleotide primers containing the
desired mutation. Incorporation of the oligonucleotides generates a
mutated plasmid, which can then be transformed into suitable cells
(e.g., bacterial or yeast cells) for subsequent screening to
confirm mutagenesis of the DNA.
[0101] As another non-limiting example, mutagenesis may be
accomplished by means of error-prone PCR amplification (ePCR),
which modifies PCR reaction conditions (e.g., using error-prone
polymerases, varying magnesium or manganese concentration, or
providing unbalanced dNTP ratios) in order to promote increased
rates of error in DNA replication. Kits for ePCR mutagenesis are
commercially available, such as the GeneMorph.RTM. PCR Mutagenesis
kit (Stratagene) and Diversify.RTM. PCR Random Mutagenesis Kit
(Clontech). Briefly, DNA polymerase (e.g., Taq polymerase), salt
(e.g., MgCl2, MgSO4, or MnSO4), dNTPs in unbalanced ratios,
reaction buffer, and DNA template are combined and subjected to
standard PCR amplification according to manufacturer's
instructions. Following ePCR amplification, the reaction products
are cloned into a suitable vector to construct a mutagenized
library, which can then be transformed into suitable cells (e.g.,
yeast cells) for subsequent screening (e.g., via a two-hybrid
screen) as described below.
[0102] Alternatively, mutagenesis can be accomplished by
recombination (i.e. DNA shuffling). Briefly, a shuffled mutant
library is generated through DNA shuffling using in vitro
homologous recombination by random fragmentation of a parent DNA
followed by reassembly using PCR, resulting in randomly introduced
point mutations. Methods of performing DNA shuffling are known in
the art (see, e.g., Stebel, S. C. et al., Methods Mol Biol
352:167-190 (2007)).
[0103] Optionally, multiple rounds of mutagenesis may be performed
in order to improve the efficiency of mutant proteins isolated.
Thus, in some embodiments, PYR/PYL mutants isolated from ePCR and
subsequent screening may be pooled and used as templates for later
rounds of mutagenesis.
[0104] In some embodiments, the variants are generated by exposing
a plant of plant seeds or cells to a mutagen selecting the plant or
cell carrying a hypersensitive PYR/PYL polypeptide as described
herein by phenotype or genotype. Examples of mutagens include,
e.g., chemical mutagens (e.g., EMS) or radiological mutagens.
Variants having a desired mutation can be selected based on
phenotype of genotype (e.g., by using TILLING techniques).
[0105] In some embodiments, the method comprises mutagenizing a
wild-type PYR/PYL receptor in situ and determining whether the
mutagenized PYR/PYL receptor is hypersensitive to ABA. Mutated
PYR/PYL receptor polypeptides can be constructed by mutating the
DNA sequences that encode the corresponding wild-type PYR/PYL
receptor polypeptide (e.g., a wild-type PYR/PYL polypeptide of any
of SEQ ID NOs:1-119, having any of SEQ ID NO:s 120-123, or a
corresponding variant from which the mutant PYR/PYL receptor
polypeptide of the invention is derived), such as by using
site-directed or random mutagenesis.
IV. Screening for Hypersensitive PYR/PYL Receptor Polypeptides
[0106] The hypersensitivity of the mutant PYR/PYL receptors
described herein can be measured in several alternative ways. When
expressed in yeast, most wild-type PYR/PYL receptors will only bind
to the type 2 protein phosphatase (PP2C) HAB1 (or other PP2Cs) when
the appropriate yeast cells are grown in the presence of ABA. Thus,
in some embodiments, hypersensitivity can be measured by
determining the ability of a PYR/PYL mutant receptor, expressed in
yeast, to bind to and inactivate PP2C in yeast to a greater extent
than a control PYR/PYL receptor expressed in yeast. In some
embodiments, the hypersensitive mutant PYR/PYL receptor comprises
mutations that result in the mutated receptor inhibiting the
activity of the PP2C in a phosphatase assay in the presence of ABA
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80% or more as compared to a wild-type or
other control PYR/PYL receptor in the presence of the same
concentration of ABA. Several test concentrations ranging from low
nM to low .mu.M could be conducted to infer IC.sub.50 values and
the IC.sub.50 values of hypersensitive mutants are substantially
lower than appropriate wild type controls.
[0107] Alternatively, cell-based or plant-based methods of
screening can be used. For example, cells that naturally express a
wild-type PYR/PYL receptor polypeptide or that recombinantly
express a wild-type or mutated PYR/PYL receptor polypeptide can be
used. In some embodiments, the cells used are plant cells, animal
cells, bacterial cells, fungal cells, including but not limited to
yeast cells, insect cells, or mammalian cells. In general terms,
the screening methods involve comparing the activity of a mutated
PYR/PYL receptor polypeptide to the activity of a wild-type PYR/PYL
receptor polypeptide in the presence of ABA, e.g., by comparing
ABA-regulated gene expression in the wild-type and mutant PYR/PYL
receptor-expressing cells or plants.
[0108] One exemplary assay involves testing whether a mutated
PYR/PYL receptor can bind to a type 2 protein phosphatase (PP2C)
(e.g., Homology to ABI1 (HAB1)) in the presence of ABA. Binding
assays can involve contacting a mutated PYR/PY1 receptor
polypeptide with a PP2C and allowing sufficient time for the
PYR/PYL receptor and PP2C to form a binding complex. Any binding
complexes formed can be detected using any of a number of
established analytical techniques. Protein binding assays include,
but are not limited to, methods that measure co-precipitation or
co-migration on non-denaturing SDS-polyacrylamide gels, and
co-migration on Western blots (see, e.g., Bennet, J. P. and
Yamamura, H. I. (1985) "Neurotransmitter, Hormone or Drug Receptor
Binding Methods," in Neurotransmitter Receptor Binding (Yamamura,
H. I., et al., eds.), pp. 61-89. Other binding assays involve the
use of mass spectrometry or NMR techniques to identify molecules
bound to the PYR/PYL polypeptide. The PYR/PYL polypeptide protein
utilized in such assays can be naturally expressed, cloned or
synthesized.
[0109] In some embodiments, mammalian or yeast two-hybrid
approaches (see, e.g., Bartel, P. L. et. al. Methods Enzymol,
254:241 (1995)) can be used to identify polypeptides or other
molecules that interact or bind when expressed together in a cell.
In some embodiments, a hypersensitive PYR/PYL polypeptide is
identified in a two-hybrid assay between a PYR/PYL polypeptide and
a PP2C polypeptide, wherein the PYR/PYL polypeptide and the PP2C
bind in the presence of ABA.
[0110] In another exemplary assay, the level of basal activity of a
mutated PYR/PYL receptor polypeptide (i.e., level of activity in
the absence of ABA) can be determined using an enzymatic
phosphatase assay, in which the PYR/PYL receptor and PP2C are
incubated in the presence of ABA. In this type of assay, a decrease
in phosphatase activity in the presence of ABA to a greater extent
than occurred for a control PYR/PYL receptor is indicative of
hypersensitive PYR/PYL receptor. A decrease in phosphatase activity
can be determined and quantified using any detection reagent known
in the art, e.g., a colorimetric detection reagent such as
para-nitrophenylphosphate.
[0111] Hypersensitive PYR/PYL receptor polypeptides that are
initially identified by any of the foregoing screening methods can
be further tested to validate the apparent activity and/or
determine other biological effects of the hypersensitive PYR/PYL
receptor polypeptide. In some cases, the PYR/PYL receptor
polypeptide is tested for the ability to affect plant stress (e.g.,
drought tolerance and/or high salt tolerance), seed germination, or
another phenotype affected by ABA. A number of such assays and
phenotypes are known in the art and can be employed according to
the methods of the invention.
V. Recombinant Expression Vectors
[0112] Once a polynucleotide encoding a mutated PYR/PYL receptor
polypeptide is obtained, it can also be used to prepare an
expression cassette for expressing the mutated PYR/PYL receptor
polypeptide in a transgenic plant, directed by a heterologous
promoter. Increased expression of mutated PYR/PYL polynucleotide is
useful, for example, to produce plants that selectively activate
PYR/PYL receptors, thus enhancing stress tolerance.
[0113] Any of a number of means well known in the art can be used
to drive mutated PYR/PYL activity or expression in plants. Any
organ can be targeted, such as shoot vegetative organs/structures
(e.g. leaves, stems and tubers), roots, flowers and floral
organs/structures (e.g. bracts, sepals, petals, stamens, carpels,
anthers and ovules), seed (including embryo, endosperm, and seed
coat) and fruit. Alternatively, the mutated PYR/PYL polynucleotide
can be expressed specifically in certain cell and/or tissue types
within one or more organs (e.g., guard cells in leaves using a
guard cell-specific promoter). Alternatively, the mutated PYR/PYL
polynucleotide can be expressed constitutively (e.g., using the
CaMV 35S promoter).
[0114] To use a polynucleotide sequence for a mutated PYR/PYL
receptor polypeptide in the above techniques, recombinant DNA
vectors suitable for transformation of plant cells are prepared.
Techniques for transforming a wide variety of higher plant species
are well known and described in the technical and scientific
literature. See, e.g., Weising et al. Ann. Rev. Genet. 22:421-477
(1988). A DNA sequence coding for the mutated PYR/PYL receptor
polypeptide preferably will be combined with transcriptional and
translational initiation regulatory sequences which will direct the
transcription of the sequence from the gene in the intended tissues
of the transformed plant.
[0115] For example, a plant promoter fragment may be employed to
direct expression of the mutated PYR/PYL polynucleotide in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumafaciens, and other transcription initiation regions from
various plant genes known to those of skill.
[0116] Alternatively, the plant promoter may direct expression of
the mutated PYR/PYL receptor protein in a specific tissue
(tissue-specific promoters) or may be otherwise under more precise
environmental control (inducible promoters). Examples of
tissue-specific promoters under developmental control include
promoters that initiate transcription only in certain tissues, such
as leaves or guard cells (including but not limited to those
described in WO 2005/085449; U.S. Pat. No. 6,653,535; Li et al.,
Sci China C Life Sci. 2005 April; 48(2):181-6; Husebye, et al.,
Plant Physiol, April 2002, Vol. 128, pp. 1180-1188; and Plesch, et
al., Gene, Volume 249, Number 1, 16 May 2000, pp. 83-89(7)).
Examples of environmental conditions that may affect transcription
by inducible promoters include anaerobic conditions, elevated
temperature, or the presence of light.
[0117] If proper protein expression is desired, a polyadenylation
region at the 3'-end of the coding region should be included. The
polyadenylation region can be derived from a naturally occurring
PYR/PYL gene, from a variety of other plant genes, or from
T-DNA.
[0118] The vector comprising the sequences (e.g., promoters or
PYR/PYL coding regions) will typically comprise a marker gene that
confers a selectable phenotype on plant cells. For example, the
marker may encode biocide resistance, particularly antibiotic
resistance, such as resistance to kanamycin, G418, bleomycin,
hygromycin, or herbicide resistance, such as resistance to
chlorosluforon or Basta.
[0119] In some embodiments, the mutated PYR/PYL nucleic acid
sequence is expressed recombinantly in plant cells. A variety of
different expression constructs, such as expression cassettes and
vectors suitable for transformation of plant cells, can be
prepared. Techniques for transforming a wide variety of higher
plant species are well known and described in the technical and
scientific literature. See, e.g., Weising et al. Ann. Rev. Genet.
22:421-477 (1988). A DNA sequence coding for a PYR/PYL protein can
be combined with cis-acting (promoter) and transacting (enhancer)
transcriptional regulatory sequences to direct the timing, tissue
type and levels of transcription in the intended tissues of the
transformed plant. Translational control elements can also be
used.
[0120] Embodiments of the present invention also provide for a
mutated PYR/PYL nucleic acid operably linked to a promoter which,
in some embodiments, is capable of driving the transcription of the
PYR/PYL coding sequence in plants. The promoter can be, e.g.,
derived from plant or viral sources. The promoter can be, e.g.,
constitutively active, inducible, or tissue specific. In
construction of recombinant expression cassettes, vectors,
transgenics, of the invention, a different promoters can be chosen
and employed to differentially direct gene expression, e.g., in
some or all tissues of a plant or animal.
Constitutive Promoters
[0121] A fragment can be employed to direct expression of a mutated
PYR/PYL nucleic acid in all transformed cells or tissues, e.g., as
those of a regenerated plant. The term "constitutive regulatory
element" means a regulatory element that confers a level of
expression upon an operatively linked nucleic molecule that is
relatively independent of the cell or tissue type in which the
constitutive regulatory element is expressed. A constitutive
regulatory element that is expressed in a plant generally is widely
expressed in a large number of cell and tissue types. Promoters
that drive expression continuously under physiological conditions
are referred to as "constitutive" promoters and are active under
most environmental conditions and states of development or cell
differentiation.
[0122] A variety of constitutive regulatory elements useful for
ectopic expression in a transgenic plant are well known in the art.
The cauliflower mosaic virus 35S (CaMV 35S) promoter, for example,
is a well-characterized constitutive regulatory element that
produces a high level of expression in all plant tissues (Odell et
al., Nature 313:810-812 (1985)). The CaMV 35S promoter can be
particularly useful due to its activity in numerous diverse plant
species (Benfey and Chua, Science 250:959-966 (1990); Futterer et
al., Physiol. Plant 79:154 (1990); Odell et al., supra, 1985). A
tandem 35S promoter, in which the intrinsic promoter element has
been duplicated, confers higher expression levels in comparison to
the unmodified 35S promoter (Kay et al., Science 236:1299 (1987)).
Other useful constitutive regulatory elements include, for example,
the cauliflower mosaic virus 19S promoter; the Figwort mosaic virus
promoter; and the nopaline synthase (nos) gene promoter (Singer et
al., Plant Mol. Biol. 14:433 (1990); An, Plant Physiol. 81:86
(1986)).
[0123] Additional constitutive regulatory elements including those
for efficient expression in monocots also are known in the art, for
example, the pEmu promoter and promoters based on the rice Actin-1
5' region (Last et al., Theor. Appl. Genet. 81:581 (1991); Mcelroy
et al., Mol. Gen. Genet. 231:150 (1991); Mcelroy et al., Plant Cell
2:163 (1990)). Chimeric regulatory elements, which combine elements
from different genes, also can be useful for ectopically expressing
a nucleic acid molecule encoding a mutated PYR/PYL receptor protein
(Comai et al., Plant Mol. Biol. 15:373 (1990)).
[0124] Other examples of constitutive promoters include the 1'- or
2'-promoter derived from T-DNA of Agrobacterium tumafaciens (see,
e.g., Mengiste (1997) supra; O'Grady (1995) Plant Mol. Biol.
29:99-108); actin promoters, such as the Arabidopsis actin gene
promoter (see, e.g., Huang (1997) Plant Mol. Biol. 1997
33:125-139); alcohol dehydrogenase (Adh) gene promoters (see, e.g.,
Millar (1996) Plant Mol. Biol. 31:897-904); ACT11 from Arabidopsis
(Huang et al. Plant Mol. Biol. 33:125-139 (1996)), Cat3 from
Arabidopsis (GenBank No. U43147, Zhong et al., Mol. Gen. Genet.
251:196-203 (1996)), the gene encoding stearoyl-acyl carrier
protein desaturase from Brassica napus (Genbank No. X74782,
Solocombe et al. Plant Physiol. 104:1167-1176 (1994)), GPc1 from
maize (GenBank No. X15596, Martinez et al. J. Mol. Biol 208:551-565
(1989)), Gpc2 from maize (GenBank No. U45855, Manjunath et al.,
Plant Mol. Biol. 33:97-112 (1997)), other transcription initiation
regions from various plant genes known to those of skill. See also
Holtorf Plant Mol. Biol. 29:637-646 (1995).
Inducible Promoters
[0125] Alternatively, a plant promoter may direct expression of the
mutated PYR/PYL polynucleotide under the influence of changing
environmental conditions or developmental conditions. Examples of
environmental conditions that may affect transcription by inducible
promoters include anaerobic conditions, elevated temperature,
drought, or the presence of light. Such promoters are referred to
herein as "inducible" promoters. In some embodiments, an inducible
promoter is one that is induced by one or more environmental
stressors, including but not limited to, drought, freezing cold,
and high salt. For example, the invention can incorporate a
drought-specific promoter such as a drought-inducible promoter of
maize (e.g., the maize rab17 drought-inducible promoter (Vilardell
et al. (1991) Plant Mol. Biol. 17:985-993; Vilardell et al. (1994)
Plant Mol. Biol. 24:561-569)); or alternatively a cold, drought,
and high salt inducible promoter from potato (Kirch (1997) Plant
Mol. Biol. 33:897-909) or from Arabidopsis (e.g., the rd29A
promoter (Kasuga et al. (1999) Nature Biotechnology 17:287-291).
Other environmental stress-inducible promoters include promoters
from the following genes: Rab21, Wsi18, Lea3, Uge1, Dip1, and R1G1B
in rice (Yi et al. (2010) Planta 232:743-754).
[0126] In some embodiments, a plant promoter is a stress-inducible
promoter (e.g., a drought-, cold-, or salt-inducible promoter) that
comprises a dehydration-responsive element (DRE) and/or an
ABA-responsive element (ABRE), including but not limited to the
rd29A promoter.
[0127] Alternatively, plant promoters which are inducible upon
exposure to plant hormones, such as auxins, are used to express the
mutated PYR/PYL polynucleotide. For example, the invention can use
the auxin-response elements E1 promoter fragment (AuxREs) in the
soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407);
the auxin-responsive Arabidopsis GST6 promoter (also responsive to
salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10:
955-966); the auxin-inducible parC promoter from tobacco (Sakai
(1996) 37:906-913); a plant biotin response element (Streit (1997)
Mol. Plant Microbe Interact. 10:933-937); and, the promoter
responsive to the stress hormone abscisic acid (Sheen (1996)
Science 274:1900-1902).
[0128] Plant promoters inducible upon exposure to chemicals
reagents that may be applied to the plant, such as herbicides or
antibiotics, are also useful for expressing the mutated PYR/PYL
polynucleotide. For example, the maize In2-2 promoter, activated by
benzenesulfonamide herbicide safeners, can be used (De Veylder
(1997) Plant Cell Physiol. 38:568-577); application of different
herbicide safeners induces distinct gene expression patterns,
including expression in the root, hydathodes, and the shoot apical
meristem. A PYR/PYL coding sequence can also be under the control
of, e.g., a tetracycline-inducible promoter, e.g., as described
with transgenic tobacco plants containing the Avena sativa L. (oat)
arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);
or, a salicylic acid-responsive element (Stange (1997) Plant J.
11:1315-1324; Uknes et al., Plant Cell 5:159-169 (1993); Bi et al.,
Plant J. 8:235-245 (1995)).
[0129] Examples of useful inducible regulatory elements include
copper-inducible regulatory elements (Mett et al., Proc. Natl.
Acad. Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717
(1988)); tetracycline and chlor-tetracycline-inducible regulatory
elements (Gatz et al., Plant J. 2:397-404 (1992); Roder et al.,
Mol. Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell Biol.
50:411-424 (1995)); ecdysone inducible regulatory elements
(Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318
(1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24
(1994)); heat shock inducible regulatory elements (Takahashi et
al., Plant Physiol. 99:383-390 (1992); Yabe et al., Plant Cell
Physiol. 35:1207-1219 (1994); Ueda et al., Mol. Gen. Genet.
250:533-539 (1996)); and lac operon elements, which are used in
combination with a constitutively expressed lac repressor to
confer, for example, IPTG-inducible expression (Wilde et al., EMBO
J. 11:1251-1259 (1992)). An inducible regulatory element useful in
the transgenic plants of the invention also can be, for example, a
nitrate-inducible promoter derived from the spinach nitrite
reductase gene (Back et al., Plant Mol. Biol. 17:9 (1991)) or a
light-inducible promoter, such as that associated with the small
subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et
al., Mol. Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471
(1990)).
Tissue-Specific Promoters
[0130] Alternatively, the plant promoter may direct expression of
the mutated PYR/PYL polynucleotide in a specific tissue
(tissue-specific promoters). Tissue specific promoters are
transcriptional control elements that are only active in particular
cells or tissues at specific times during plant development, such
as in vegetative tissues or reproductive tissues.
[0131] Examples of tissue-specific promoters under developmental
control include promoters that initiate transcription only (or
primarily only) in certain tissues, such as vegetative tissues,
e.g., roots or leaves, or reproductive tissues, such as fruit,
ovules, seeds, pollen, pistols, flowers, or any embryonic tissue,
or epidermis or mesophyll. Reproductive tissue-specific promoters
may be, e.g., ovule-specific, embryo-specific, endosperm-specific,
integument-specific, seed and seed coat-specific, pollen-specific,
petal-specific, sepal-specific, or some combination thereof. In
some embodiments, the promoter is cell-type specific, e.g., guard
cell-specific.
[0132] Epidermal-specific promoters include, for example, the
Arabidopsis LTP1 promoter (Thoma et al. (1994) Plant Physiol.
105(1):35-45), the CER1 promoter (Aarts et al. (1995) Plant Cell
7:2115-27), and the CER6 promoter (Hooker et al. (2002) Plant
Physiol 129:1568-80), and the orthologous tomato LeCER6 (Vogg et
al. (2004) J. Exp Bot. 55:1401-10).
[0133] Guard cell-specific promoters include, for example, the DGP1
promoter (Li et al. (2005) Science China C Life Sci.
48:181-186).
[0134] Other tissue-specific promoters include seed promoters.
Suitable seed-specific promoters are derived from the following
genes: MAC1 from maize (Sheridan (1996) Genetics 142:1009-1020);
Cat3 from maize (GenBank No. L05934, Abler (1993) Plant Mol. Biol.
22:10131-1038); vivparous-1 from Arabidopsis (Genbank No. U93215);
atmyc1 from Arabidopsis (Urao (1996) Plant Mol. Biol. 32:571-57;
Conceicao (1994) Plant 5:493-505); napA from Brassica napus
(GenBank No. J02798, Josefsson (1987) JBL 26:12196-1301); and the
napin gene family from Brassica napus (Sjodahl (1995) Planta
197:264-271).
[0135] A variety of promoters specifically active in vegetative
tissues, such as leaves, stems, roots and tubers, can also be used
to express polynucleotides encoding mutated PYR/PYL receptor
polypeptides. For example, promoters controlling patatin, the major
storage protein of the potato tuber, can be used, see, e.g., Kim
(1994) Plant Mol. Biol. 26:603-615; Martin (1997) Plant J.
11:53-62. The ORF13 promoter from Agrobacterium rhizogenes that
exhibits high activity in roots can also be used (Hansen (1997)
Mol. Gen. Genet. 254:337-343. Other useful vegetative
tissue-specific promoters include: the tarin promoter of the gene
encoding a globulin from a major taro (Colocasia esculenta L.
Schott) corm protein family, tarin (Bezerra (1995) Plant Mol. Biol.
28:137-144); the curculin promoter active during taro corm
development (de Castro (1992) Plant Cell 4:1549-1559) and the
promoter for the tobacco root-specific gene TobRB7, whose
expression is localized to root meristem and immature central
cylinder regions (Yamamoto (1991) Plant Cell 3:371-382).
[0136] Leaf-specific promoters, such as the ribulose biphosphate
carboxylase (RBCS) promoters, can also be used. For example, the
tomato RBCS1, RBCS2 and RBCS3A genes are expressed in leaves and
light-grown seedlings, only RBCS1 and RBCS2 are expressed in
developing tomato fruits (Meier (1997) FEBS Lett. 415:91-95). A
ribulose bisphosphate carboxylase promoters expressed almost
exclusively in mesophyll cells in leaf blades and leaf sheaths at
high levels, described by Matsuoka (1994) Plant J. 6:311-319, can
be used. Another leaf-specific promoter is the light harvesting
chlorophyll a/b binding protein gene promoter, see, e.g., Shiina
(1997) Plant Physiol. 115:477-483; Casal (1998) Plant Physiol.
116:1533-1538. The Arabidopsis thaliana myb-related gene promoter
(Atmyb5) described by Li (1996) FEBS Lett. 379:117-121, is
leaf-specific. The Atmyb5 promoter is expressed in developing leaf
trichomes, stipules, and epidermal cells on the margins of young
rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA
appears between fertilization and the 16 cell stage of embryo
development and persists beyond the heart stage. A leaf promoter
identified in maize by Busk (1997) Plant J. 11:1285-1295, can also
be used.
[0137] Another class of useful vegetative tissue-specific promoters
are meristematic (root tip and shoot apex) promoters. For example,
the "SHOOTMERISTEMLESS" and "SCARECROW" promoters, which are active
in the developing shoot or root apical meristems, described by Di
Laurenzio (1996) Cell 86:423-433; and, Long (1996) Nature
379:66-69; can be used. Another useful promoter is that which
controls the expression of 3-hydroxy-3-methylglutaryl coenzyme A
reductase HMG2 gene, whose expression is restricted to meristematic
and floral (secretory zone of the stigma, mature pollen grains,
gynoecium vascular tissue, and fertilized ovules) tissues (see,
e.g., Enjuto (1995) Plant Cell. 7:517-527). Also useful are
kn1-related genes from maize and other species which show
meristem-specific expression, see, e.g., Granger (1996) Plant Mol.
Biol. 31:373-378; Kerstetter (1994) Plant Cell 6:1877-1887; Hake
(1995) Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51. For
example, the Arabidopsis thaliana KNAT1 promoter (see, e.g.,
Lincoln (1994) Plant Cell 6:1859-1876).
[0138] One of skill will recognize that a tissue-specific promoter
may drive expression of operably linked sequences in tissues other
than the target tissue. Thus, as used herein a tissue-specific
promoter is one that drives expression preferentially in the target
tissue, but may also lead to some expression in other tissues as
well.
[0139] In another embodiment, the mutated PYR/PYL polynucleotide is
expressed through a transposable element. This allows for
constitutive, yet periodic and infrequent expression of the
constitutively active polypeptide. The invention also provides for
use of tissue-specific promoters derived from viruses including,
e.g., the tobamovirus subgenomic promoter (Kumagai (1995) Proc.
Natl. Acad. Sci. USA 92:1679-1683; the rice tungro bacilliform
virus (RTBV), which replicates only in phloem cells in infected
rice plants, with its promoter which drives strong phloem-specific
reporter gene expression; the cassava vein mosaic virus (CVMV)
promoter, with highest activity in vascular elements, in leaf
mesophyll cells, and in root tips (Verdaguer (1996) Plant Mol.
Biol. 31:1129-1139).
VI. Production of Plants Comprising Hypersensitive Mutations
[0140] In another aspect, the present invention provides for
transgenic plants comprising recombinant expression cassettes for
expressing a hypersensitive PYR/PYL receptor protein as described
herein in a plant. In some embodiments, a transgenic plant is
generated that contains a complete or partial sequence of a
polynucleotide that is derived from a species other than the
species of the transgenic plant. It should be recognized that
transgenic plants encompass the plant or plant cell in which the
expression cassette is introduced as well as progeny of such plants
or plant cells that contain the expression cassette, including the
progeny that have the expression cassette stably integrated in a
chromosome.
[0141] A recombinant expression vector comprising a PYR/PYL coding
sequence driven by a heterologous promoter may be introduced into
the genome of the desired plant host by a variety of conventional
techniques. For example, the DNA construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the DNA construct can be introduced directly to
plant tissue using ballistic methods, such as DNA particle
bombardment. Alternatively, the DNA construct may be combined with
suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. While transient expression of the
constitutively active PYR/PYL receptor is encompassed by the
invention, generally expression of construction of the invention
will be from insertion of expression cassettes into the plant
genome, e.g., such that at least some plant offspring also contain
the integrated expression cassette.
[0142] Microinjection techniques are also useful for this purpose.
These techniques are well known in the art and thoroughly described
in the literature. The introduction of DNA constructs using
polyethylene glycol precipitation is described in Paszkowski et al.
EMBO J. 3:2717-2722 (1984). Electroporation techniques are
described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824
(1985). Ballistic transformation techniques are described in Klein
et al. Nature 327:70-73 (1987).
[0143] Agrobacterium tumefaciens-mediated transformation
techniques, including disarming and use of binary vectors, are well
described in the scientific literature. See, for example, Horsch et
al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad.
Sci. USA 80:4803 (1983).
[0144] Transformed plant cells derived by any of the above
transformation techniques can be cultured to regenerate a whole
plant that possesses the transformed genotype and thus the desired
phenotype such as enhanced abiotic stress resistance. Such
regeneration techniques rely on manipulation of certain
phytohormones in a tissue culture growth medium, typically relying
on a biocide and/or herbicide marker which has been introduced
together with the desired nucleotide sequences. Plant regeneration
from cultured protoplasts is described in Evans et al., Protoplasts
Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176,
MacMillilan Publishing Company, New York, 1983; and Binding,
Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press,
Boca Raton, 1985. Regeneration can also be obtained from plant
callus, explants, organs, or parts thereof. Such regeneration
techniques are described generally in Klee et al. Ann. Rev. of
Plant Phys. 38:467-486 (1987).
[0145] One of skill will recognize that after the expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0146] The expression cassettes of the invention can be used to
confer abiotic stress resistance on essentially any plant. Thus,
the invention has use over a broad range of plants, including
species from the genera Asparagus, Atropa, Avena, Brassica, Citrus,
Citrullus, Capsicum, Cucumis, Cucurbita, Daucus, Fragaria, Glycine,
Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca,
Linum, Lolium, Lycopersicon, Malus, Manihot, Majorana, Medicago,
Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus,
Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum,
Trigonella, Triticum, Vitis, Vigna, and, Zea. In some embodiments,
the plant is selected from the group consisting of rice, maize,
wheat, soybeans, cotton, canola, turfgrass, and alfalfa. In some
embodiments, the plant is an ornamental plant. In some embodiment,
the plant is a vegetable- or fruit-producing plant.
[0147] Those of skill will recognize that a number of plant species
can be used as models to predict the phenotypic effects of
transgene expression in other plants. For example, it is well
recognized that both tobacco (Nicotiana) and Arabidopsis plants are
useful models of transgene expression, particularly in other
dicots.
[0148] In some embodiments, the plants of the invention have
enhanced ABA-mediated phenotypes, for example enhanced seed
dormancy, as compared to plants that are otherwise identical except
for expression of the hypersensitive PYR/PYL receptor polypeptide.
Those of skill in the art will recognize that ABA is a well-studied
plant hormone and that ABA mediates many changes in
characteristics, any of which can be monitored to determine changes
in phenotype. In some embodiments, an enhanced ABA-mediated
phenotype is manifested by altered timing of seed germination or
altered stress (e.g., drought, freezing cold, and/or salt)
tolerance.
[0149] Abiotic stress resistance can be assayed according to any of
a number of well-known techniques. For example, for drought
tolerance, plants can be grown under conditions in which less than
optimum water is provided to the plant. Drought resistance can be
determined by any of a number of standard measures including turgor
pressure, growth, yield, and the like. In some embodiments, a
transgenic plant expressing a mutated PYR/PYL receptor as described
herein has enhanced drought tolerance if the loss of turgor in the
transgenic plant is reduced by at least about 10%, 15%, 20%, 25%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a
non-transgenic control plant over a defined period of time (e.g.,
over the course of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more,
e.g., 3, 4, 5 weeks or more).
[0150] In some embodiments, the enhanced ABA-mediated phenotype is
enhanced tolerance to moderate or high salinity. Salinity tolerance
can be determined by any of a number of standard measures,
including germination, growth, yield, or plant survival, leaf
injury, premature loss of chlorophyll, and the like. In some
embodiments, transgenic plants expressing a mutated PYR/PYL
receptor as described herein have enhanced salt tolerance if the
survival of the transgenic plants under moderate-salt or high-salt
conditions (e.g., about 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300
mM NaCl or higher) is increased by at least about 10%, 15%, 20%,
25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to a
non-transgenic control plant over a defined period of time (e.g.,
over the course of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more,
e.g., 3, 4, 5 weeks or more).
[0151] Plant gene manipulations can now be precisely tailored in
non-transgenic organisms using the CRISPR/Cas9 genome editing
method. In this bacterial antiviral and transcriptional regulatory
system, a complex of two small RNAs--the CRISPR-RNA (crRNA) and the
transactivating crRNA (tracrRNA)--directs the nuclease (Cas9) to a
specific DNA sequence complementary to the crRNA (Jinek, M., et al.
Science 337, 816-821 (2012)). Binding of these RNAs to Cas9
involves specific sequences and secondary structures in the RNA.
The two RNA components can be simplified into a single element, the
single guide-RNA (sgRNA), which is transcribed from a cassette
containing a target sequence defined by the user (Jinek, M., et al.
Science 337, 816-821 (2012)). This system has been used for genome
editing in humans, zebrafish, Drosophila, mice, nematodes,
bacteria, yeast, and plants (Hsu, P. D., et al., Cell 157,
1262-1278 (2014)). In this system the nuclease creates double
stranded breaks at the target region programmed by the sgRNA. These
can be repaired by non-homologous recombination, which often yields
inactivating mutations. The breaks can also be repaired by
homologous recombination, which enables the system to be used for
gene targeted gene replacement (Li, J.-F., et al. Nat. Biotechnol.
31, 688-691, 2013; Shan, Q., et al. Nat. Biotechnol. 31, 686-688,
2013). The hypersensitive mutations described in this application
can be introduced into plants using the CAS9/CRISPR system.
[0152] Accordingly, in some embodiments, instead of generating a
transgenic plant, a native PYR/PYR coding sequence in a plant or
plant cell can be altered in situ to generate a plant or plant cell
carrying a polynucleotide encoding a hypersensitive PYR/PYL
polypeptide as described herein. For example, in some embodiments,
CRISPR technology is used to introduce one or more nucleotide
changes into a PYR/PYL coding sequence in situ to change the
appropriate codon to make a change corresponding to F61X, V81X,
I110X, or V163X of SEQ ID NO:1. The CRISPR/Cas system has been
modified for use in prokaryotic and eukaryotic systems for genome
editing and transcriptional regulation. The "CRISPR/Cas" system
refers to a widespread class of bacterial systems for defense
against foreign nucleic acid. CRISPR/Cas systems are found in a
wide range of eubacterial and archaeal organisms. CRISPR/Cas
systems include type I, II, and III sub-types. Wild-type type II
CRISPR/Cas systems utilize the RNA-mediated nuclease, Cas9 in
complex with guide and activating RNA to recognize and cleave
foreign nucleic acid. Cas9 homologs are found in a wide variety of
eubacteria, including, but not limited to bacteria of the following
taxonomic groups: Actinobacteria, Aquificae,
Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi,
Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and
Thermotogae. An exemplary Cas9 protein is the Streptococcus
pyogenes Cas9 protein. Additional Cas9 proteins and homologs
thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013
May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6):
467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep. 24;
110(39):15644-9; Sampson et al., Nature. 2013 May 9;
497(7448):254-7; and Jinek, et al., Science. 2012 Aug. 17;
337(6096):816-21.
[0153] Accordingly, in one aspect, a method is provided of using
CRISPR/CAS9 to introduce at least one of the mutation described
herein into a plant cell is performed. In some embodiments, a
method of altering a (e.g., native) nucleic acid encoding PYR/PYL
polypeptide in a plant is provided. In some embodiments, the method
comprises introducing into the plant cell containing and expressing
a DNA molecule having a target nucleic acid encoding PYR/PYL
polypeptide an engineered, non-naturally occurring Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR
associated (Cas) (CRISPR-Cas) system. In some embodiments, the
CRISPR-Cas system comprises one or more vectors comprising: a) a
first regulatory element operable in a plant cell operably linked
to at least one nucleotide sequence encoding a CRISPR-Cas system
guide RNA that hybridizes with the target sequence, and b) a second
regulatory element operable in a plant cell operably linked to a
nucleotide sequence encoding a Type-II Cas9 protein, wherein
components (a) and (b) are located on same or different vectors of
the system, whereby the guide RNA targets the target sequence and
the Cas9 protein cleaves the DNA molecule, whereby at least one of
the hypersensitive mutations described herein is introduced into
the target nucleic acid encoding the PYR/PYL polypeptide. In some
embodiments, the PRY/PYL polypeptide is selected from any of SEQ ID
NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, or 119 or a substantially identical polypeptide. In
some embodiments, the plant is from a genus selected from
Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum,
Cucumis, Cucurbita, Daucus, Fragaria, Glycine, Gossypium,
Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum,
Lolium, Lycopersicon, Malta, Manihot, Majorana, Medicago,
Nicotiana, Oryza, Panieum, Pannesetum, Persea, Pisum, Pyrus,
Prunus, Raphanus, Secale, Senecio, Sinapis, Solanum, Sorghum,
Trigonella, Triticum, Vitis, Vigna, and, Zea. In some embodiments,
the plant is selected from the group consisting of rice, maize,
wheat, and soybean. In some embodiments, the hypersensitive
mutation introduced to the target nucleic acid is (corresponding to
their position in Arabidopsis PYR1 (SEQ ID NO:1)): F61, V81, I110,
E141, or A160 or a combination thereof. In some embodiments, no
other mutations are introduced into the target nucleic acid. Also
provided as a plant or plant cell resulting from the
above-described method. Such a plant will contain a
non-naturally-occurring nucleic acid sequence encoding the
hypersensitive PYR/PYL polypeptide.
VII. PYR/PYL Fusion Proteins
[0154] In some embodiments, the hypersensitive PYR/PYL polypeptides
described herein are provided as fusion proteins, i.e.,
translational fusions with one or more fusion partner. In some
embodiments, a hypersensitive PYR/PYL polypeptide is fused with a
transcriptional activation or modulation domain. A non-limiting
example of such a domain is VP16 or VP64. The fusion proteins can
further comprise a nuclear localization signal sequence such that
the fusion protein, when translated in a eukaryotic host cell, is
localized to the cell nucleus. Also provided are polynucleotides
encoding such fusion proteins as well as host cells comprising and
expressing such polynucleotides. The polynucleotides in such
instances will be heterologous to the host cell, i.e., will not be
naturally occurring, for example transformed into the cell.
[0155] Such fusion proteins are useful, for example, in controlling
eukaryotic gene expression in the cell when co-expressed with a
sequence-specific DNA binding domain fused with) ABA INSENSITIVE 1
(ABI1) or other proteins having specific binding affinity for
PYR/PYL proteins binding ABA. Exemplary sequence-specific DNA
binding domains include, but are not limited to zinc-finger
proteins, TALENS, transcription factor DNA binding domains, and
RNA-guided DNA-binding domains of inactive Cas9 (dCas9). When both
fusion proteins are co-expressed in the cell in the presence of
ABA, the two fusion proteins will co-localized due to the binding
of ABA1 to the ABA-binding PYR/PYL protein, thereby bringing the
transcriptional activation or modulation domain in proximity to the
target promoter, thereby regulating gene expression. Examples of
systems and their use in gene regulation, are described in, e.g.,
Konermann et al., Nature 500:472-476 (2013) and Liang et al.,
Science Vol. 4 Issue 164 (2011).
RNA Directed Genome Modification
[0156] In one aspect provided herein is a method for introducing a
mutation in situ at a PYR/PYL mutation target site in a plant cell
genome, as described herein. For example, in some embodiments, the
PYR/PYL mutation target site comprises a nucleic acid that encodes
for V89 of PYL-E or E149 of PYL. In certain embodiments the method
comprises introducing into the plant cell: 1) a CRISPR ribonucleic
acid (crRNA) that includes a sequence substantially identical to
SEQ ID NOS: 363, 364, 365, 366, 367 or 369; 2) a transacting
ribonucleic acid (tracRNA); 3) a nuclease (e.g., Cas9); and 4) a
repair nucleic acid that can undergo homologous recombination that
contains the mutation. According to the subject method, the crRNA
and tracRNA directs the nuclease to the PYR/PYL mutation target
site in a plant cell genome. Upon its recruitment, the nuclease
(e.g., Cas9) creates a double strand break at the PYR/PYL mutation
target site. The double strand break at the PYR/PYL mutation target
site facilitates homologous recombination of the repair nucleic
acid containing the mutation with a region of the plant cell genome
that includes the PYR/PYL mutation target site, thereby introducing
the mutation at the PYR/PYL mutation target site.
[0157] Mutations can be introduced into any suitable plant cell
using the subject method. In some embodiments, the plant cell is a
plant embryo. In certain embodiments, the plant cell is a maize
plant cell.
[0158] Each component of the method can be introduced into the
plant cell using any suitable method known in the art. In certain
embodiments, the crRNA and tracRNA are introduced into the cell as
an expression cassette containing a polynucleotide (i.e., DNA)
encoding the crRNA and/or traRNA. In some embodiments, the
expression cassette includes an RNA polymerase promoter operably
linked to the polynucleotide encoding the crRNA and/or traRNA,
thereby allowing transcription of the crRNA and/or traRNA. In some
embodiments, the Cas9 is introduced into the cell as an expression
vector containing a promoter operably linked to a polynucleotide
encoding Cas9. Any suitable promoter can be used, including but not
limtied to, the promoters described herein. In certain embodiments,
the promoter is a ubiquitin-1 promoter (e.g., prUbi-10). DNA
construct (.e.g, the expression cassettes and vectors described
herein) can be introduced directly to plant tissue, for example,
using ballistic methods, such as DNA particle bombardment.
[0159] Each of the crRNA, and the tracRNA, nuclease can be
introduced separately or together as part of one expression vector
into the cell of interest (e.g., a maize plant cell). In certain
embodiments, the crRNA and the tracRNA are fused together to create
a guide ribonucleic acid (gRNA). In some embodiments, the gRNA
includes, from 5' to 3', a crNA linked to a tracRNA. In certain
embodiments the crRNA, tracRNA, and nuclease (e.g., Cas9) are
introduced together as nucleic acid cassettes included in one
expression vector. Each component of the subject method is
discussed in detail below.
Guide RNA
[0160] In one aspect provided, provided herein is a guide RNA
(gRNA) comprising a CRISPR ribonucleic acid (crRNA) and a
transacting RNA (tracRNA).
[0161] The crRNA of the subject gRNA comprises a nucleotide
sequence that is complementary to a sequence in a PYR/PYL mutation
target site and includes a sequence that is substantially identical
to SEQ ID NOS: 363, 364, 365, 366, 367 or 369. In certain
embodiments, the crRNA has a sequence that is substantially
identical to SEQ ID NOS: 363, 364, 365, 366, 367 or 369. The
subject crRNAs provided herein are particularly useful for creating
mutations at a PYR/PYL mutation target site that includes a nucleic
acid encoding for an amino acid corresponding to V89 (SEQ ID NOS:
363, 364, 365, 366, 367) and E149 (SEQ ID NO:369) of PYL-E. As used
herein, a "PYR/PYL mutation target site" refers to a region of a
polynucleotide encoding for a PYR/PYL receptor that includes the
site where a mutation is introduced by the subject method. The
crRNA interacts with the PYR/PYL mutation target site in a
sequence-specific manner by hybridization to a sequence in the
PYR/PYL mutation target site (e.g., the complementary strand of the
PYR/PYL mutation target site) and, together with the tracRNA of the
gRNA, recruits Cas9 endonuclease to the PYR/PYL mutation target
site. Cas9 endonuclease recruited by the gRNA to the PYR/PYL
mutation target site introduces a double strand break in the
PYR/PYL mutation target site. Any of the mutations described herein
can be made in any wildtype PYR/PYL polypeptide. Analogous amino
acid substitutions can be made, for example, in PYR/PYL receptors
other than PYL-E by aligning the PYR/PYL receptor polypeptide
sequence to be mutated with the PYL-E receptor polypeptide
sequence. Analogous amino acid positions in PYR/PYL recetpros are
shown in FIGS. 2 and 3.
[0162] In some embodiments, the PYR/PYL mutation target site has
the sequence of SEQ ID NO: 362, which includes a nucleic acid
encoding for V89 of PYL-E. In some embodiments, the PYR/PYL
mutation target site has the sequence of SEQ ID NO:368, which
includes a nucleic acid encoding for E149 of PYL-E.
[0163] In some embodiments, a crRNA has a length of 10 nucleotides
(nt) to 100 nucleotides (nt). In some embodiments, the crRNA has a
length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 nt and includes a sequence that is substantially identical
to SEQ ID NOS: 362, 363, 364, 365, 366 or 368. In some embodiments,
the crRNA has a length of at least 17 nt. In some embodiments, the
crRNA has a length of 17 nt to 18 nt, 17 nt to 19 nt, 17 nt to 20
nt, 17 nt to 21 nt, 17 nt to 22 nt, 17 nt to 23 nt, 17 nt to 24 nt,
17 nt to 25 nt, 17 nt to 30 nt, 17 nt to 35 nt, 17 nt to 40 nt, 17
nt to 45 nt, 17 nt to 50 nt, 17 nt to 55 nt, 17 nt to 60 nt, 17 nt
to 65 nt, 17 nt to 70 nt, 17 nt to 75 nt, 17 nt to 80 nt, 17 nt to
85 nt, 17 nt to 90 nt, 17 nt to 95 nt, or 17 nt to 100 nt. In some
embodiments, the crRNA has a length of 12 nt to 25 nt, 13 nt to 25
nt, 14 nt to 25 nt, 15 nt to 25 nt, 16 nt to 25 nt, 17 nt to 25 nt,
18 nt to 25 nt, 19 nt to 25 nt, 20 nt to 25 nt, 21 nt to 25 nt, or
22 nt to 25 nt.
[0164] In some embodiments, the crRNA is 17 nt in length, In some
embodiments, the crRNA is 18 nt in length. In some embodiments, the
crRNA is 19 nt in length. In some embodiments, the crRNA is 20 nt
in length. In some embodiments, the crRNA is 21 nt in length. In
some embodiments the crRNA is 22 nt in length. In some embodiments,
the crRNA is 23 nt in length. In some embodiments, the crRNA is 24
nt in length. In some embodiments, the crRNA is 25 nt in
length.
[0165] In some embodiments, the crRNA is at least 17 nt in length,
In some embodiments, the crRNA is at least 18 nt in length. In some
embodiments, the crRNA is at least 19 nt in length. In some
embodiments, the crRNA is at least 20 nt in length. In some
embodiments, the crRNA is at least 21 nt in length. In some
embodiments the crRNA is at least 22 nt in length. In some
embodiments, the crRNA is at least 23 nt in length. In some
embodiments, the crRNA is at least 24 nt in length. In some
embodiments, the crRNA is at least 25 nt in length.
[0166] In some embodiments, the guideRNA (gRNA) includes a
transacting RNA (tracRNA). Transacting RNA of the subject guideRNA
interacts with the crRNA to recruit a nuclease to the site of a
PYR/PYL mutation target site. Upon its recruitment to the PYR/PYL
mutation target site, the nuclease creates a double strand break
(DSB) in the PYR/PYL mutation target site. Any suitable tracRNA
capable of recruiting a Cas9 to a PYR/PYL mutation target site can
be used with the subject gRNA. In some embodiments, the tracRNA is
encoded by a nucleotide having a sequence that is substantially
identical to SEQ ID NO: 370. In certain embodiments of the subject
method, the tracRNA and the crRNA are introduced into the plant
cells separately (e.g., on different expression vectors). In some
embodiments, the tracRNA is linked to the crRNA and introduced into
the plant cell as a guideRNA (gRNA).
[0167] In another aspect, provided herein is a nucleic acid that
includes a polynucleotide encoding any of the subject gRNAs
described herein.
[0168] In another aspect, provided herein is an expression cassette
that includes an RNA polymerase promoter operably linked to any of
the subject gRNAs described herein. Any suitable RNA polymerase
promoter capable of driving transcription of the nucleic acid
encoding the subject gRNA can be used. In some embodiments, the
promoter is an inducible promoter, including, but not limited, to
any of the inducible promoters described herein. In other
embodiments, the promoter is a constitutive promoter, including,
but not limited to any of the constitutive promoters described
herein. In yet other embodiments, the promoter is a tissue-specific
promoter, including, but not limited to, any of the tissue-specific
promoters described herein. In certain embodiments, the RNA
polymerase promoter is an RNA polymerase III (polIII) promoter. In
particular embodiments, the polIII promoter is a U3 promoter or a
U6 promoter. In certain embodiment, the expression cassette has the
sequence of any one of SEQ ID NOS; 371-373.
Expression Vectors Including gRNA and Cas9 Nuclease
[0169] In another aspect, provided herein is an expression vector
that includes one or more of the guide RNA (gRNA) expression
cassettes provided herein and an expression cassette including a
promoter operably linked to a polynucleotide encoding a
CRISPR-associated endonuclease 9 (Cas9). In some embodiments, the
promoter operably linked to the Cas9 is a ubiquitin-1 promoter
(prUbi-10). In some embodiments, the expression includes an
expression cassette containing a polynucleotide encoding a gRNA
having a crRNA that is substantially identical to SEQ ID NOS: 363,
364, 365, 366, 367 (see, e.g., FIG. 5). In some embodiments, the
expression vector includes an expression cassette containing a
polynucleotide encoding a gRNA having a crRNA that is substantially
identical to SEQ ID NOS: 369 (see, e.g., FIG. 6). In some
embodiments, the expression vector includes a first expression
cassette containing a polynucleotide encoding a gRNA having a crRNA
that is substantially identical to SEQ ID NOS: 363, 364, 365, 366,
367; a second expression cassette containing a polynucleotide
encoding a gRNA having a crRNA that is substantially identical to
SEQ ID NO: 369; and a third expression cassette including a
promoter operably linked to a polynucleotide encoding a
CRISPR-associated endonuclease 9 (Cas9) (see, e.g., FIG. 7).
Methods of Producing PYR/PYL Variant Plants Using RNA Directed
Genome Modification
[0170] Expression vectors disclosed herein are useful, for example,
for introducing a mutation in a plant in situ at a genomic PYR/PYL
mutation target site. Thus, in another aspect, provided herein is a
method for of producing a plant having a mutation at a genomic
PYR/PYL mutation target site. In some embodiments, the method
includes introducing into plant cells an expression vector encoding
for a gRNA and Cas9 as disclosed herein and at least one repair
nucleic acid comprising the mutation of interest. According to the
subject method, the crRNA and tracRNA directs the nuclease to the
PYR/PYL mutation target site in a plant cell genome. Upon its
recruitment, the nuclease (e.g., Cas9) creates a double strand
break at the PYR/PYL mutation target site. The double strand break
at the PYR/PYL mutation target site facilitates homologous
recombination of the repair nucleic acid containing the mutation of
interest with a region of the plant cell genome that includes the
PYR/PYL mutation target site, thereby introducing the mutation at
the PYR/PYL mutation target site. In certain embodiments, the
repair nucleic acid has a sequence that is substantially identical
to any one of the sequence of SEQ ID NOS:374 to 378. In other
embodiments, the repair nucleic acid has a sequence that is
complementary to a sequence that is substantially identical to any
one of the sequences of SEQ ID NOS:374 to 386. In specific
embodiments, the repair nucleic acid has a sequence that is
substantially identical to SEQ ID NO:376. In other embodiments, the
repair nucleic acid has a sequence that is complementary to a
sequence that is substantially identical to SEQ ID NO:376. In
another embodiment, the repair nucleic acid has a sequence that is
substantially identical to SEQ ID NO:378. In other embodiments, the
repair nucleic acid has a sequence that is complementary to a
sequence that is substantially identical to SEQ ID NO:378. In yet
another embodiment of the method, two repair nucleic acids are
introduced, where the repair nucleic acids have sequences that are
substantially identical to SEQ ID NO:376 and SEQ ID NO:378. In
another embodiment of the method, two repair nucleic acids are
introduced, where the repair nucleic acids have sequences are
complementary to sequences that are substantially identical to SEQ
ID NO:376 and SEQ ID NO:378.
[0171] In certain embodiments, the method further includes the step
of selecting plant cells having the mutation. Selection for
mutation can be performed by any useful technique known in the art,
including, but not limited PCR amplification followed by
sequencing, capillary electrophoresis and Nuclease Serveyer assay.
In some embodiments, the method is for the production of a maize
plant.
[0172] In yet another aspect, provided herein is a kit for
producing a plant having a mutation in a PYR/PYL nucleic acid as
described herein. In some embodiments, the kit includes any one of
the subject expression vectors disclosed herein and at least one
repair nucleic acid, wherein the repair nucleic acid comprises a
PYL-E mutation and is capable of introducing the PYL-E mutation in
situ in a plant cell genome by homologous recombination upon a Cas9
cleavage event. In certain embodiments, the kit includes a repair
nucleic acid that has a sequence that is substantially identical to
SEQ ID NOS:374 to 386.
EXAMPLES
[0173] The following examples are offered to illustrate, but not to
limit the claimed invention.
[0174] The affinity of a receptor for a target ligand is typically
determined by non-covalent interactions between ligand-binding
residues and the ligand. Mutations in such residues can have
negative, positive or neutral effects on the strength of the
receptor-ligand interaction. The affinity of a receptor-ligand
interaction is intrinsically correlated with the concentration of
ligand required to elicit biological effects, with high affinity
ligands requiring lower concentrations relative to low affinity
ligands. A mutant receptor with increased affinity for a ligand can
in some cases elicit greater biological effect relative to a wild
type receptor, when both are activated under identical conditions
by the same concentration of activating ligand. Thus, mutations
that make a receptor hypersensitive to a ligand can be useful for
engineering organisms that elicit stronger responses to the ligand
relative to wild type. Furthermore, ABA hypersensitive plants
possess enhanced ABA responses and improved drought tolerance
(Wang, Y., et al. Plant J. 43, 413-424 (2005)). Based on these
considerations, we set out to systematically establish specific ABA
receptor mutations that increase ABA responsiveness. This was done
by testing a collection of PYR1 variants with all possible single
amino acid substitution mutations in ligand binding residues. Thus
we conducted site-saturated mutagenesis of ABA-contacting residues,
which we define as those that are within 5A or ABA or
ABA-contacting water residues in available X-ray coordinates. This
collection of mutants was constructed previously, as described in
PCT Application No. PCT/US2012/043121 and Mosquna et al., Proc Natl
Acad Sci USA 108: 20838-20843 (2011). This collection of mutants
was made by mutagenizing a previously described pBD GAL-PYR1
template (Park, S.-Y., et al. Science 324, 1068-1071 (2011)). In
response to ABA, this particular plasmid encodes a fusion protein
that binds to a co-expressed GAL4 activation domain-HAB1 fusion
protein, encoded by the plasmid pACT-HAB1. This binding
reconstitutes a functional GAL4 transcriptional activator and
subsequent transcription of a .beta.-galactosidase reporter gene,
which in turn enables colorimetric based detection of agonist
promoted receptor-PP2C interaction when lysed cells are exposed to
the substrate X-gal. The mutant clones were individually
transformed into S. cerevisiae strain Y190 containing pACT-HAB1.
Yeast transformants were selected for the presence of plasmids on
synthetic dextrose (SD) agar plates lacking Leu and Trp (SD-LT) and
examined for PP2C interactions by using X-gal staining to monitor
.beta.-gal reporter gene expression levels. Individual clones were
arrayed into 96 well plates and then spotted onto SD-LT lawn (i.e.
one-well) plates containing 0, 0.5 or 5.0 .mu.M (+)-ABA. Each assay
plate contained 95 mutant clones and one wild type PYR1 positive
control clone. The spotted cells were cultured at 30.degree. C. for
48 hours after which they were lysed by chloroform and stained with
an X-gal solution, as previously described (Park, S.-Y., et al.
(2009) Science 324, 1068-1071). Positive were defined as those
mutants that displayed staining on 0.5 .mu.M (+)-ABA but no
staining on plates lacking (+)-ABA. After this initial screening
exercise, all positives clones were retested on plates containing
0. 0.25, 0.5 and 1 .mu.M (+)-ABA and stained for galactosidase
activity as described above. Mutant clones showing detectable
staining on 0.5 .mu.M (+)-ABA or lower were scored as
hypersensitive mutants. FIG. 1 depicts results of PYR1 mutant-HAB1
interactions as assayed in a yeast two-hybrid assay under different
ABA concentrations, with darker spots indicating increased
interaction. This data is also summarized below:
TABLE-US-00003 Minimal conc. For ABA Mutant SEQ ID NO: Residue WT
AA Mutant response (.mu.M) WT 1 1 F61L 124 61 F L 0.25 F61M 125 61
F M 0.25 V81I 126 81 V I 0.25 V81Y 127 81 V Y 0.25 I110C 128 110 I
C 0.25 I110S 129 110 I S 0.5 E141C 130 141 E C 0.5 E141I 131 141 E
I 0.25 E141L 132 141 E L 0.25 E141M 133 141 E M 0.25 E141N 134 141
E N 0.5 E141T 135 141 E T 0.5 E141V 136 141 E V 0.5 E141W 137 141 E
W 0.5 E141Y 138 141 E Y 0.25 A160C 139 160 A C 0.25 A160I 140 160 A
I 0.25 A160V 141 160 A V 0.25
[0175] Highly Hypersensitive ABA Receptors Constructed by
Combinatorial Mutagenesis
[0176] Additive or synergistic interactions between the single
hypersensitive mutations identified can increase a receptor's
sensitivity to ABA. To identify potentially beneficial
combinations, we used combinatorial mutagenesis to construct
receptors that contain combinations of subsets of the best single
mutants identified and then screened these to identify receptors
with increased sensitivity. Mutagenic primers complementary to the
appropriate regions of PYR1 coding sequence were designed to enable
the following mutations to be incorporated into a PYR1 template
DNA: F61L, F61M, V81I, V81Y, I110C, I110S, E141I, E141L, E141M,
E141Y, A160C, A160I, A160V. Equimolar concentrations of these
primers were combined with a mixture of wild type primers for each
target site (4 mol percent relative to the mutant primer pool) and
the primer mix utilized with the QuickChange Lightning Multi
Site-Directed PCR Mutagenesis kit (Agilent, USA) using the pBD-PYR1
template DNA. The use of wild type primers in the reaction mixtures
enabled, in principle, all double, triple, quadruple and pentuple
mutant combinations to be synthesized in the mutagenesis reaction.
The reaction products were transformed into competent E. coli cells
to yield a pool of .about.10,000 clones, which was then used to
prepare plasmid DNA for the mutant library. The pool of mutant
plasmids was subsequently introduced into the previously described
pAD-HAB1 MAV99 reporter strain (Peterson, F. C., et al. (2010)
Nature Structural & Molecular Biology 17, 1109-1113). In this
reporter strain, a GAL4 promoter drives expression of a URA3
reporter gene in a genetic background where the endogenous URA3
gene is disrupted, which enables positive selections using uracil
deficient media. Thus, mutant clones that encode receptors that can
interact with HAB1 can be positively selected using this system.
The transformed yeast cells containing the mutant receptor library
were next plated onto growth medium lacking uracil and containing
50 nM ABA, a concentration of ABA that is too low to enable growth
of control strains. 26 colonies with uracil-independent growth were
identified, which were isolated and re-tested on medium lacking ABA
to eliminate cones enabling ligand-independent (i.e. constitutive)
interactions of receptor with HAB1. Plasmids from yeast cells
containing non-constitutive receptors were isolated and sequenced,
which revealed that the following 4 highly hypersensitive
combination mutants had been isolated:
PYR1F61L, A160C,
PYR1F61M, A160V,
PYR1F61M, I110S, A160V,
PYR1F61L, V81I, I110C, A160V.
[0177] The plasmids for each of these mutants and their
corresponding single mutations were transformed into the previously
described yeast reporter strain, Y190 pAD-HAB1 (Park, S.-Y., et al.
(2009) Science 324, 1068-1071). The transformed yeast cells were
grown on selective media containing a range of ABA concentrations
and cells lysed and stained to reveal .beta.-galactosidase
activity, as shown in FIG. 4.
[0178] Increased Affinity of a Mutant Hypersensitive Receptor
[0179] The ligand sensitivity of PYR1 and HAB1 yeast two hybrid
strains generally correlates with receptor affinity. To examine if
this was the case for the hypersensitive mutations identified by
our functional screens, we conducted isothermal titration
calorimetry (ITC) to measure the heat produced by a mutant
receptor-ABA binding reaction and infer the ligand binding
dissociation constant (Kd). The affinity of wild type PYR1 has been
previously measured using ITC and estimated to be 97.+-.36 .mu.M
(Dupeux et al. 2010). The PYR1-A160V mutant receptor was expressed
in E. coli BL21(DE3) as a fusion to the small ubiquitin like
protein SUMO, using the vector pSUMO (LifeSensors, USA), which
improves the solubility of proteins in E. coli and contains an
NH2-terminal hexa-histidine tag that facilitates purification using
immobilized metal affinity chromatography (IMAC). PYR1 A160V was
cloned into pSUMO by using PCR product generated from a
pBD-PYR1(A160V) yeast two hybrid construct as template, and
sequence validated. A short flexible linker and tobacco etch virus
(TEV) protease cleavage site (sequence
NH2-GGGSQFGSGGGGGSGSENLYFQS-COOH; SEQ ID NO:411) was incorporated
in between the SUMO tag and the receptor to enable cleavage of the
recombinant protein by TEV protease, which yields PYR1(A160V) plus
an NH2-terminal QS appendage. Recombinant SUMO-TEV-PYR1 (A160V)
protein was produced in E. coli and purified by immobilized metal
affinity chromatography as previously described (Okamoto et al.,
Proceedings of the National Academy of Sciences of the United
States of America 110, no. 29 (2013): 12132-12137). The purified
fusion protein was digested with recombinant TEV protease according
to established protocols, and the cleaved protein subsequently
separated from both the SUMO tag and uncleaved protein by passing
the cleavage reaction over an IMAC column, which does not retain
the cleaved PYR1(A160V) product. The cleaved protein was purified
by gel filtration using a Superdex column (GE Healthcare, USA) and
concentrated by centrifugal concentration using Amicon filters
(EMD, USA), as previously described (Dupeux et al, The EMBO Journal
30, no. 20 (2011): 4171-4184). The concentrated protein was
utilized for ITC experiments, using a TA instruments Nano ITC Low
Volume instrument, repeatedly injecting 2.5 .mu.L of a 600 .mu.M
(+)-ABA solution into a reaction cell containing 60 .mu.M
PYR1(A160V) every 300 seconds for 200 minutes. Both the ABA and
protein were dissolved in a buffer containing PBS, 1 mM
2-mercaptoethanol and 0.012% DMSO. The thermograms generated were
processed using the instrument's software to a normalized fit
single binding site model, which yielded a Kd of 1.5 .mu.M and a
binding stoichiometry of 1.068. These data demonstrate that the
A160V mutation possesses increased ABA affinity relative to wild
type PYR1, consistent with the increased sensitivity indicated by
yeast two hybrid assays.
Targeted Genome Modification
[0180] Non-transgenic plants harboring induced mutations in
specific genes can be obtained in multiple ways. Chemical
mutagenesis of an organism can be used to create random genome-wide
mutations and populations of mutagenized individuals can be scanned
using high-throughput mutation detection methods to identify
individuals harboring specific mutations in genes of interest. For
example, TILLING (Targeting Induced Local Lesions in Genomes)
enables an investigator to identify non-naturally occurring
induced-mutations in a gene by using PCR to amplify a gene of
interest from 1000s of mutagenized individuals and use
hetero-duplex specific nucleases, such as celery nuclease CEL1, to
identify plants harboring a mutation in the PCR amplified region
(McCallum, C. M., et al. (2000). Nat. Biotechnol. 18, 455-457).
Many technologies are available for polymorphism identification in
addition to endonucleases, including direct sequencing of PCR
products obtained from mutagenized individuals.
[0181] To identify maize plants containing ABA receptors with
increased sensitivity an EMS mutagenized population is created and
from this population all ABA receptor genes are PCR amplified from
1000s of mutagenized plants. The amplified products are scanned for
polymorphisms using TILLING methodology and polymorphic fragments
identified are sequenced to define the specific mutations present.
From this, individuals harboring mutations corresponding to the
polymorphisms described in this application are identified.
[0182] The most likely mutants to be obtained using this strategy
are those that can be encoded by a single nucleotide substitution,
which can be established by examining the codon table. For example,
receptors with mutations homologous to F61L or F61M in PYR1 can be
obtained in receptor homologs by screening for different single
nucleotide substitutions depending on the gene sequence, such as
UUU->CUU, or UUC->CUC. The same is true for A160V
(GCN->GUN), V81I (GUU->AUU, GUC->AUC, GUA->AUA), V81Y
(GUU->UUU, GUC->UUC). In principle, any single mutation can
be isolated by chemical mutagenesis TILLING, but in practice the
subset of changes that can arise by a single nucleotide
substitution are most likely to be obtained. The examples provided
above are representative, not exhaustive, and other single
nucleotide substitutions enabling desired mutations, such as E141V
and I110S, are also possible.
[0183] Other mutation induction systems can be used to target
mutations in specific genes, such as genome editing methods, which
have the advantages of increasing the frequency of single and
multiple mutations at a defined target site (Lozano-Juste, J., and
Cutler, S. R. (2014) Trends in Plant Science 19, 284-287). The
sequence-specific introduction of a double stranded DNA break (DSB)
in a genome leads to the recruitment of DNA repair factors at the
breakage site, which then repair lesion by either the error-prone
non-homologous end joining (NHEJ) or homologous recombination (HR)
pathways. NHEJ repairs the breaks, but is imprecise and often
creates diverse mutations at and around the DSB. In cells in which
the HR machinery repairs the DSB, sequences with homology flanking
the DSB, including exogenously supplied sequences, can be
incorporated at the region of the DSB. DSBs can therefore be
leveraged by geneticists to increase the frequency of mutations at
defined sites, however intrinsic differences between the relative
roles of HR and NHEJ can affect the mutation types at a targets
locus. A number of technologies have been developed to create DSBs
at specific sites including synthetic zinc finger nucleases (ZFNs),
transcription activator-like endonucleases (TALENs) and most
recently the clustered regularly interspaced short palindromic
repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. This
system is based on a bacterial immune system against invading
bacteriophages in which a complex of 2 small RNAs, the CRISPR-RNA
(crRNA) and the transactivating crRNA (tracrRNA) directs a nuclease
(Cas9) to a specific DNA sequence complementary to the crRNA. Using
any of these systems, an investigator can create DSBs at
pre-determined sites in cells expressing the genome editing
constructs. In order for homologous recombination to occur, a DNA
cassette homologous to the targeted site must be provided,
preferably at a high concentration so that HR is favored or NHEJ.
Multiple strategies are conceivable for realizing this, including
template delivery using agrobacterium mediated transformation or
particle bombardment of DNA templates, and one recently described
method uses a modified viral genome to provide the double stranded
DNA template. For example, Baltes et al. 2014 (Baltes, N. J., et
al. (2014) Plant Cell 26, 151-163) recently demonstrated that an
engineered geminivirus that was introduced into plant cells using
Agrobacterium mediated transformation could be engineered to
produce DNA recombination templates in cells where a ZFN was
co-expressed.
[0184] In some aspects, once DSBs have been created using any
number of technologies, such sites can be exploited to facilitate
isolation of targeted genetic changes by either homologous
recombination of nucleotide substitutions, deletions or insertions.
For example, ABA receptor genes can be targeted using genome
editing technologies and progeny plants of the mutagenized plants
be screened using the methods outlined above to identify mutations
at sites that increase ABA sensitivity. Delivery of genome editing
constructs into organisms can involve both unstable transient
expression constructs or stable integration of constructs into
genomes delivered by Agrobacterium mediated transformation. In the
latter case, stable transgenic plants can be used to express
genome-editing constructs in plants to increase mutation
frequencies at the target site. Once the desired mutants are
isolated through polymorphism scans (analogous to those used in
TILLING), individuals can be back crossed wild type lines to
segregate away transgenic insertion events.
[0185] Conceptually, these methods are analogous to TILLING and the
methods for identifying defined mutations would be similar, however
because of the targeted nature of these methods, the frequency of
mutations at defined sites will be higher and mutations involving
changes of more than a single base pair can be identified more
readily.
Targeted Amino Acid Modification of Maize ABA Receptors Mediated by
CRISPR-Cas9
1. Maize ABA Receptor (ZmPYL) Target Gene Modification
[0186] In vivo modification of plant ABA receptors is described
using maize ZmPYL genes as an example. Maize genome contains more
than 10 ABA receptors (ZmPYL-A to ZmPYL-P) that share several
highly conserved amino acids with AtPYL1. To obtain ABA
hypersensitive mutations in the endogenous ZmPYL-E (GRMZM2G165567)
gene, two amino acids (V89 and E149) are chosen as targets for
sequence-specific mutagenesis into desired mutant amino acids,
alanine and leucine (V89A and E149L), respectively, using
homology-directed gene repair mediated by sequence-specific
nucleases and donor DNA template. Currently, there are several
technology platforms for making sequence-specific nucleases,
including for example zinc-finger nuclease, TALE nuclease,
CRISPR-Cas9 and engineered meganuclease (Puchta and Fauser (2014),
Synthetic nucleases for genome engineering in plants: prospects for
a bright future. Plant Journal 78:727-741; Chen and Gao (2014)
Targeted genome modification technologies and their applications in
crop improvements. Plant Cell Rep. 33:575-583), any of which can be
used to produce a plant comprising an in situ hypersensitive
mutation in a genomic coding sequence PYR/PYL polypeptide as
described herein.
[0187] The following examples describe the use of CRISPR-Cas9
system for making targeted gene modification in endogenous ZmPYL-E
gene. CRISPR-Cas9-mediated gene modification requires these
components: Cas9 nuclease, crRNA (CRISPR RNA) recognizing the
mutagenesis target, tracRNA (transactivating RNA) and repair donor
DNA template molecule. For easiness of use, crRNA and tracRNA are
fused and delivered as a single guide RNA molecule (gRNA or sgRNA)
[Sander and Joung (2014) CRISPR-Cas systems for editing, regulating
and targeting genomes. 32:347-355].
2. Optimization of Cas9 and its Expression in Maize Cells
[0188] In order to achieve good expression in maize cells, Type II
Cas9 gene from Streptococcus pyogenes SF370 is optimized with
maize-preferred codons. Nuclear localization signal is also
incorporated into the C-terminus of Cas9 to improve its targeting
to nucleus. Below is the optimized Cas9 sequence (cBCas9Nu-01, aka,
cCas9-01). To express Cas9 in maize cells, the maize-optimized Cas9
gene (cBCas9Nu-01) is placed under the control of maize ubiquitin-1
promoter (prUbi1-10) and is followed by a terminator sequences
(tNOS) (FIG. 5).
3. gRNA Design and Expression 3.1 gRNAs for Mediating V89A
Modification: Structure and its Expression
[0189] For targeted V89A modification, crRNAs of at least 17
nucleotides (nt) long are designed against the maize ZmPYL-E
genomic target sequence (5'-CGCGA CGTCA ACGTC AAGAC-3') (SEQ ID
NO:362) preceding the 5'-CGG-3' PAM (protospacer adjacent motif)
sequence for Cas9-mediated target recognition. For example, gRNAs
of 17-nt (5'-GA CGUCA ACGUC AAGAC-3') (SEQ ID NO:363), 18-nt
(5'-CGA CGUCA ACGUC AAGAC-3') (SEQ ID NO:364), 19-nt (5'-GCGA CGUCA
ACGUC AAGAC-3') (SEQ ID NO:365), 20-nt (5'-CGCGA CGUCA ACGUC
AAGAC-3') (SEQ ID NO:366) or 21-nt (5'-G CGCGA CGUCA ACGUC
AAGAC-3') (SEQ ID NO:367) can be used to guide Cas9 cleavage of the
ZmPYL-E target. crRNA is co-delivered with tracRNA and Cas9 protein
or mRNA to mediate target site cleavage. Preferably, crRNA molecule
is fused with tracRNA molecule covalently into a single guide RNA
(gRNA). gRNAs can be synthesized chemically or produced by in vitro
transcription. In vitro produced gRNAs can be used directly for
physical delivery such as biolistic bombardment with Cas9 RNA or
protein to mediate target cleavage and homology-directed target
modification if repair donor oligonucleotide is co-delivered. More
preferably, gRNA is produced in planta from DNA expression cassette
comprising RNA polymerase III (PolIII) promoter such as plant U3
and U6 promoters such as rice U3 and U6 promoters (prOsU3 and
prOsU6). For prOsU3, the transcription start begins with nucleotide
A, whereas for prOsU6, the transcription starts with nucleotide G
(Shan et al. (2013) Nature Biotechnology 31: 686-688; Xie and Yang
(2013) Molecular Plant 6:1975-1983). For example, to produce gRNA
targeting the endogenous ZmPYKL-E sequence (5'-CGCGA CGTCA ACGTC
AAGAC-3') (SEQ ID NO:362), 19-nt DNA oligonucleotides (5'-GCGA
CGTCA ACGTC AAGAC-3') (SEQ ID NO:365) or 21-nt oligonucleotides
(5'-G CGCGA CGTCA ACGTC AAGAC-3') (SEQ ID NO:367) is fused to the
DNA sequences encoding tracRNA scaffold ((5'-GTTTT AGAGC TAGAA
ATAGC AAGTT AAAAT AAGGC TAGTC CGTTA TCAAC TTGAA AAAGT GGCAC CGAGT
CGGTG C-3') (SEQ ID NO: 370) and PolIII termination sequences
(5'-GTTTT AGAGC TAGAA ATAGC AAGTT AAAAT AAGGC TAGTC CGTTA TCAAC
TTGAA AAAGT GGCAC CGAGT CGGTG CTTTT TTTTT-3'(SEQ ID NO:413), Mali
et al. (2013) Science 339:823-826) and placed under the control of
rice polymerase III promoter U3 (prOsU3) or U6 (prOsU6). Below is
the sequence (SEQ ID NO:371) of the expression cassette comprising
of prOsU3 and coding sequences for the gRNA comprising the 19-nt
V89A crRNA (underlined) fused with tracRNA. This sequence is cloned
into biolistic transformation vector along with the Cas9 expression
cassette to form vector pZmPYLE-V89A (FIG. 5).
TABLE-US-00004 (SEQ ID NO: 371) 5'-GGGAT CTTTA AACAT ACGAA CAGAT
CACTT AAAGT TCTTC TGAAG CAACT TAAAG TTATC AGGCA TGCAT GGATC TTGGA
GGAAT CAGAT GTGCA GTCAG GGACC ATAGC ACAGG ACAGG CGTCT TCTAC TGGTG
CTACC AGCAA ATGCT GGAAG CCGGG AACAC TGGGT ACGTT GGAAA CCACG TGATG
TGGAG TAAGA TAAAC TGTAG GAGAA AAGCA TTTCG TAGTG GGCCA TGAAG CCTTT
CAGGA CATGT ATTGC AGTAT GGGCC GGCCC ATTAC GCAAT TGGAC GACAA CAAAG
ACTAG TATTA GTACC ACCTC GGCTA TCCAC ATAGA TCAAA GCTGG TTTAA AAGAG
TTGTG CAGAT GATCC GTGGC AGCGA CGTCA ACGTC AAGAC GTTTT AGAGC TAGAA
ATAGC AAGTT AAAAT AAGGC TAGTC CGTTA TCAAC TTGAA AAAGT GGCAC CGAGT
CGGTG CTTTT TTTTT-3'
[0190] The sequence example below (SEQ ID NO:372) describes the
expression cassette comprising of prOsU6 promoter and coding
sequences for a gRNA comprising the 21-nt V89A crRNA (underlined)
and tracRNA.
TABLE-US-00005 (SEQ ID NO: 372) 5'-TTTGT GAAAG TTGAA TTACG GCATA
GCCGA AGGAA TAACA GAATC GTTTC ACACT TTCGT AACAA AGGTC TTCTT ATCAT
GTTTC AGACG ATGGA GGCAA GGCTG ATCAA AGTGA TCAAG CACAT AAACG CATTT
TTTTA CCATG TTTCA CTCCA TAAGC GTCTG AGATT ATCAC AAGTC ACGTC TAGTA
GTTTG ATGGT ACACT AGTGA CAATC AGTTC GTGCA GACAG AGCTC ATACT TGACT
ACTTG AGCGA TTACA GGCGA AAGTG TGAAA CGCAT GTGAT GTGGG CTGGG AGGAG
GAGAA TATAT ACTAA TGGGC CGTAT CCTGA TTTGG GCTGC GTCGG AAGGT GCAGC
CCACG CGCGC CGTAC CGCGC GGGTG GCGCT GCTAC CCACT TTAGT CCGTT GGATG
GGGAT CCGAT GGTTT GCGCG GTGGC GTTGC GGGGG ATGTT TAGTA CCACA TCGGA
AACCG AAAGA CGATG GAACC AGCTT ATAAA CCCGC GCGCT GTAGT CAGCT TGCGC
GACGT CAACG TCAAG ACGTT TTAGA GCTAG AAATA GCAAG TTAAA ATAAG GCTAG
TCCGT TATCA ACTTG AAAAA GTGGC ACCGA GTCGG TGCTTTT TTTTT-3'
3.2 gRNA for Mediating E149L Modification: Structure and its
Expression
[0191] For targeted E149L modification of the maize ZmPYL-E, the
two underlined bases in the maize genomic target sequence (5'-GCACC
CTGGT GATCG AGTCG TTCGT GGTCG-3') (SEQ ID NO:368) needs to be
converted into CT to form mutant sequence (5'-GCACC CTGGT GATCC
TGTCG TTCGT GGTCG-3' (SEQ ID NO:412)). In order to achieve that, an
expression cassette for a sequence coding for the 20-nt guide RNA
(5'-CCTGG TGATC CTGTC GTTCG-3', xZmPYLE-E149L) (SEQ ID NO:369),
tracRNA scaffold and PolIII termination sequences (5'-GTTTT AGAGC
TAGAA ATAGC AAGTT AAAAT AAGGC TAGTC CGTTA TCAAC TTGAA AAAGT GGCAC
CGAGT CGGTG CTTTT TTTTT-3' (SEQ ID NO:413), Mali et al. (2013)
Science 339:823-826) was placed under the control of rice
polymerase III promoter U6 (prOsU6) as shown in FIG. 6. prOsU6
promoter initiates transcription after nucleotide G. In FIG. 6, the
prOsU6-E149L gRNA expression cassette has the following sequences
(SEQ ID NO:373) with the 20 bp targeting guide sequence
(xZmPYLE-E149L or xZmPYLe, SEQ ID NO: 369) underlined.
TABLE-US-00006 (SEQ ID NO: 373) 5'-TTTGT GAAAG TTGAA TTACG GCATA
GCCGA AGGAA TAACA GAATC GTTTC ACACT TTCGT AACAA AGGTC TTCTT ATCAT
GTTTC AGACG ATGGA GGCAA GGCTG ATCAA AGTGA TCAAG CACAT AAACG CATTT
TTTTA CCATG TTTCA CTCCA TAAGC GTCTG AGATT ATCAC AAGTC ACGTC TAGTA
GTTTG ATGGT ACACT AGTGA CAATC AGTTC GTGCA GACAG AGCTC ATACT TGACT
ACTTG AGCGA TTACA GGCGA AAGTG TGAAA CGCAT GTGAT GTGGG CTGGG AGGAG
GAGAA TATAT ACTAA TGGGC CGTAT CCTGA TTTGG GCTGC GTCGG AAGGT GCAGC
CCACG CGCGC CGTAC CGCGC GGGTG GCGCT GCTAC CCACT TTAGT CCGTT GGATG
GGGAT CCGAT GGTTT GCGCG GTGGC GTTGC GGGGG ATGTT TAGTA CCACA TCGGA
AACCG AAAGA CGATG GAACC AGCTT ATAAA CCCGC GCGCT GTAGT CAGCT TGCCT
GGTGA TCGAG TCGTT CGGTT TTAGA GCTAG AAATA GCAAG TTAAA ATAAG GCTAG
TCCGT TATCA ACTTG AAAAA GTGGC ACCGA GTCGG TGCTT TTTTT TT-3'
[0192] Alternatively, the guide RNA can also be expressed from a
different polymerase III promoter like rice U3 promoter (prOsU3)
which initiates transcription after nucleotide A. The prOsU3-E149L
gRNA expression cassette has the following sequences (SEQ ID
NO:374) with the 20 bp targeting guide sequence (xZmPYLE-E149L or
xZmPYLe, SEQ ID NO: 369) underlined. This prOsU3-E149L gRNA
expression cassette along with PMI selectable marker gene cassette
and prSoUbi4 driven Cas9 gene expression cassette are inserted into
binary vector backbone to form transformation vector 23190 (FIG.
8).
TABLE-US-00007 (SEQ ID NO: 374) 5'-GGGAT CTTTA AACAT ACGAA CAGAT
CACTT AAAGT TCTTC TGAAG CAACT TAAAG TTATC AGGCA TGCAT GGATC TTGGA
GGAAT CAGAT GTGCA GTCAG GGACC ATAGC ACAGG ACAGG CGTCT TCTAC TGGTG
CTACC AGCAA ATGCT GGAAG CCGGG AACAC TGGGT ACGTT GGAAA CCACG TGATG
TGGAG TAAGA TAAAC TGTAG GAGAA AAGCA TTTCG TAGTG GGCCA TGAAG CCTTT
CAGGA CATGT ATTGC AGTAT GGGCC GGCCC ATTAC GCAAT TGGAC GACAA CAAAG
ACTAG TATTA GTACC ACCTC GGCTA TCCAC ATAGA TCAAA GCTGG TTTAA AAGAG
TTGTG CAGAT GATCC GTGGC ACCTG GTGAT CGAGT CGTTC GGTTT TAGAG CTAGA
AATAG CAAGT TAAAA TAAGG CTAGT CCGTT ATCAA CTTGA AAAAG TGGCA CCGAG
TCGGT GCTTT TTTTT T-3'
4. Generation of Mutants with Targeted Genomic Sequence
Modification in ZmPYL-E Gene 4.1 Generation of Targeted Mutation
V89A in ZmPYL-E Gene with Biolistic Bombardment
[0193] For target gene sequence modification mediated by
homology-directed repair, donor DNA molecule needs to be
co-delivered with Cas9 and gRNA. DNA molecule with at least 15
nucleotides flanking the Cas9 cleavage site and containing the
intended mutant nucleotide(s) is used as repair donor. For
modification of the target sequence 5'-CGCGA CGTCA ACGTC AA/GAC-3'
to result in V89A mutation, the single underlined residue T needs
to be converted to C so valine at position 89 (V89, GTC) is changed
to alanine (A89, GCC). Since the intended Cas9 cleavage site
(indicated by /) is 9 nucleotides downstream, preferably, the
repair DNA molecule should contain sequences at least 15-nt
upstream of TCA and 15-nt downstream of the underlined A in AA/GAC
as in this sequence (5'-GCAGCCT GCGCGACGCC AACGTCAA/GA CCGGCCTGCC
GGC-3') (SEQ ID NO:375). More preferably, the repair DNA molecule
should contain sequences with at least 20-nt upstream of TCA and at
least 20-nt downstream of the underlined A in AA/GAC as outlined in
this sequence (5'-GG TCGGCAGCCT GCGCGACGCC AACGTCAA/GA CCGGCCTGCC
GGCGACGA-3') (SEQ ID NO:376). More preferably, the repair DNA
molecule should contain sequences with more than 30-nt upstream of
TCA and more than 30-nt downstream of the underlined A in AA/GAC as
outlined in this sequence (5'-AC CAGCTC GAGG TCGGCAGCCT GCGCGACGCC
AACGTCAA/GA CCGGCCTGCC GGCGACGACC AGAACCGA-3') (SEQ ID NO:377).
Most preferably, the repair DNA molecule should contain sequences
with more than 50-nt upstream of TCA and more than 50-nt downstream
of the underlined A in AA/GAC as indicated in this sequence (5'-GA
ACTGCGTCGT GCGCGGGGAC CAGCTC GAGG TCGGCAGCCT GCGCGACGCC AACGTCAA/GA
CCGGCCTGCC GGCGACGACC AGAACCGAGC GCCTCGAGCA GCTCGACGA-3') (SEQ ID
NO:378). It should be noted that oligonucletoides with sequences
corresponding to the opposite strand of SEQ ID NO:375 to SEQ ID
NO:378 can also be used for mediating targeted V89A mutation.
[0194] To generate plants carrying V89A mutation, the above
described repair donor DNA oligonucleotide (5'-AC CAGCTC GAGG
TCGGCAGCCT GCGCGACGCC AACGTCAA/GA CCGGCCTGCC GGCGACGACC
AGAACCGA-3') (SEQ ID NO:377) that comprise sequences 30-nt upstream
of TCA and 30-nt downstream of the underlined A in AA/GAC is
co-precipitated with pZmPYLE-V89A vector (FIG. 1) onto gold
particles and bombarded into immature maize embryos (genotype A188,
HiII or other applicable varieties). Methods for maize immature
embryo bombardment, callus induction tissue regeneration and
rooting methods have been described previously except here no
mannose selection is required (Wright et al., 2001, Efficient
biolistic transformation of maize (Zea mays L.) and wheat (Triticum
aestivum L.) using the phosphomannose isomerase gene, pmi, as the
selectable marker. Plant Cell Reports 20:429-436.). Briefly,
immature embryos are isolated from harvested immature ears at about
9-12 days after pollination and pre-cultured for 3 to 5 days on
osmoticum media. Pre-cultured embryos are then bombarded with DNA
vector ZmPYLE-V89A and the donor oligonucleotide using BioRad
PDS-1000 Biolistic particle delivery system. Bombarded embryos are
then incubated in callus induction media and then moved onto
regeneration media to induce shoot formation. Shoots are then moved
to rooting media. Preferably but not essential, a selectable marker
gene cassette like PMI is also added to the ZmPYLE-V89A vector so
only transformed cells containing an integrated gRNA or Cas9
expression cassette will be selected for regeneration. Samples are
then harvested from regenerated plants for genotyping to identify
plants containing the desired V89A mutation in the ZmPYL-E gene.
Genotyping can be done with one or more of the standard mutation
detection methods such as PCR amplification followed by sequencing,
capillary electrophoresis and Nuclease Surveyer assay.
[0195] ZmPYLE-V89A vector carries the gRNA and Cas9 expression
cassettes can also be delivered into maize cells using other
physical delivery method such as protoplast transformation and
silicon carbide whisker-mediated transformation. The repair donor
DNA molecule can also be delivered into cells in the form of
single- or double-stranded molecule that is present as part of a
recombinant DNA molecule such as restriction fragment or plasmid or
T-DNA or viral replicon for generation of transformed cells using
methodologies known in the art. Alternatively, gRNA and Cas9
expression vectors and repair donor vector can be transformed into
maize cells with Agrobacterium-mediated transformation. It should
be noted that for targeted modification, no integration of Cas9 or
gRNA expression vector is required or even preferred. Therefore,
these vectors can be delivered transiently by biolistic
transformation or Agrobacterium-mediated transformation.
4.2 Generation of Targeted Mutation E149L in ZmPYL-E Gene with
Biolistic and Agrobacterium-Mediated Transformation
[0196] Similar to the above example (Section 4.1) for generating
ZmPYLE-V89A mutation, targeted E149L mutation (Table 1) can be
introduced into ZmPYL-E gene using biolistic bombardment using DNA
vectors carrying Cas9 and gRNA expression cassettes such as these
shown in FIG. 6 and FIG. 8 along with repair donor DNA sequences
containing the desired mutation such as in the form of purified
oligonucleotide with this sequence (ZmPYL-Eb, SEQ ID NO:379,
5'-TGACG GGAGG CCGGG CACCC TGGTG ATCCT GTCGT TCGTA GTCGA TGTGC
CTGAT GGCAA-3', Table 2). Other forms of repair donor
oligonucleotides can be used too. For example, the oligonucleotides
can be longer or in the complimentary strand or contain chemical
modifications (e.g. phosphorothioate or methylphosphonate) to
enhance stability or affinity to the target sequences. Chemically
modified oligonucletoides have been described (Deleavey and Damha,
2012, Chemistry & Biology,
http://dx.doi.org/10.1016/j.chembiol.2012.07.011). To demonstrate
utility of such chemically modified oligonucleotides, experiments
were done using oligonucleotides with sequences from the non-coding
strand and also containing phosphorothioate linkage (Table 2,
ZmPYL-Ec-NT-PM, SEQ ID NO:380, 5'-T*T*C*GT GTTGC CATCA GGCAC ATCGA
CTACG AACGA CAGGA TCACC AGGGT GCCCG GCCTC CCGTC AATG*C* T*C-3', *
denotes the presence of phosphorothioate linkage between
nucleotides).
[0197] Targeted mutation E149L in ZmPYL-E gene can also be
generated with DNA molecules delivered via Agrobacterium.
Agrobacterium-mediated transformation methods have been described
elsewhere (Ishida et al. (1996). High efficiency transformation of
maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat.
Biotechnol. 14, 745-750; Negrotto et al. (2000). Theuse of
phosphomannose-isomerase as a selectable marker to recover
transgenic maize plants (Zea mays L.) via Agrobacterium
transformation. Plant Cell Rep. 19, 798-803.). Briefly, the
prOsU6-E149L (SEQ ID NO:373) or prOsU3-E148L (SEQ ID NO:374) gRNA
expression cassette is cloned into a binary vector carrying PMI
selectable marker cassette and also an expression cassette for Cas9
with maize preferred codons forming transformation vector
pZmPYLE-E149L and 23190 (FIG. 6, FIG. 8, Table 1). These vectors
can be used to deliver Cas9 and gRNA expression cassettes into
maize cells with Agrobacterium-mediated transformation. The repair
donor DNA molecule containing the intended mutant sequences
(5'-TGACG GGAGG CCGGG CACCC TGGTG ATCCT GTCGT TCGTA GTCGA TGTGC
CTGAT GGCAA-3') (SEQ ID NO:379) is co-delivered into cells from a
separate T-DNA molecule. However, it can be also be inserted into
the T-DNA region next to the gRNA and Cas9 expression cassettes in
the binary vector pZmPYLE-E149L or 23190 (FIG. 2). The donor repair
template can also be delivered in the form of viral replicon
derived from another T-DNA (Baltes et al. 2014, DNA replicons for
plant genome engineering. Plant Cell. 26:151-163). PMI marker is
used to select for transgenic plants with integrated Cas9 or gRNA
expression cassette. However, it should be noted that stable
transformation of transformation vectors is not essential or even
preferred for generating desired mutations as long as enough plants
are screened since transient expression of Cas9 and gRNA is
sufficient to result in cleavage of the chromosomal target sequence
to induce DNA repair. However, it should be noted that for targeted
modification, no integration of Cas9 or gRNA expression vector is
required or even preferred.
4.3 Generation of Multiple Amino Acid Modifications in ZmPYL-E Gene
Simultaneously
[0198] It should be noted that more than one target can be modified
at the same time if gRNAs and repair donors for multiple target
sequences are present at the same time. For example, both V89A and
E149L mutations can be obtained by co-bombarding vector
pZmPYLE-V89A-E149L containing expression cassettes for Cas9 and two
gRNAs (FIG. 7) along with both repair donor DNA templates, V89A
oligonucleotide (5'-AC CAGCTC GAGG TCGGCAGCCT GCGCGACGCC
AACGTCAA/GA CCGGCCTGCC GGCGACGACC AGAACCGA-3')(SEQ ID NO:377) and
E149L oligonucleotides ODN-ZmPYL-Eb (5'-TGACG GGAGG CCGGG CACCC
TGGTG ATCCT GTCGT TCGTA GTCGA TGTGC CTGAT GGCAA-3') (SEQ ID NO:379)
or ODN-ZmPYL-Ec-NT-PM (SEQ ID NO:380, 5'-T*T*C*GT GTTGC CATCA GGCAC
ATCGA CTACG AACGA CAGGA TCACC AGGGT GCCCG GCCTC CCGTC AATG*C*
T*C-3', * denotes the presence of phosphorothioate linkage between
nucleotides).
[0199] As described for generating plants with single mutations,
bombarded embryos are then incubated in callus induction media and
then moved onto regeneration media to induce shoot formation.
Shoots are then moved to rooting media. PMI marker can be used to
select for transgenic plants with integrated Cas9 or gRNA
expression cassette. However, it should be noted again that stable
transformation of transformation vectors is not essential or even
preferred for generating desired mutations as long as enough plants
are screened to identify plants with desired mutations since
transient expression of Cas9 and gRNA is sufficient to result in
cleavage of the chromosomal target sequence to induce DNA repair.
Samples are then harvested from regenerated plants for genotyping
to identify plants containing the desired V89A and E149L mutations
in the ZmPYL-E gene. Genotyping can be done with one or more of the
standard mutation detection methods such as PCR amplification
followed by sequencing, capillary electrophoresis and Nuclease
Surveyer assay.
4.4 Generation of Additional Targeted Mutations at V89 Position in
ZmPYL-E Gene
[0200] Alternate site-directed changes can be introduced at the V89
position of the ZmPYL-E to obtain ABA hypersensitive mutations by
using similar method described above for creating V89A mutation
except that repair donor oligonucleotide sequences need to be
changed to introduce the corresponding mutations. For example, V89I
and V89Y mutations can be introduced by using the same gRNAs (SEQ
ID NOS: 363 to 367) to guide Cas9 cleavage of the ZmPYL-E target.
Expressing cassettes for gRNA and Cas9 can be delivered into maize
cells simultaneously by any physical or biological methods such as
biolistic bombardment or Agrobacterium-mediated transformation. For
introduction of V89I mutation, the single underlined residue Gin
the ZmPYL-E genomic target sequence 5'-CGCGA CGTCA ACGTC AA/GAC-3'
needs to be converted to A, so valine at position 89 (V89, GTC) is
changed to isoleucine (I89, ATC). Since the intended Cas9 cleavage
site (indicated by /) is 9 nucleotides downstream, preferably, the
repair DNA molecule should contain sequences at least 15-nt
upstream of GTC and 15-nt downstream of the underlined A in AA/GAC
as in this sequence (5'-GGCAGCCT GCGCGACATC AACGTCAAGA CCGGCCTGCC
GGC-3') (SEQ ID NO:381). More preferably, the repair DNA molecule
should contain sequences with at least 20-nt upstream of ATC and at
least 20-nt downstream of the underlined A in AAGAC as outlined in
this sequence (5'-AGG TCGGCAGCCT GCGCGACATC AACGTCAAGA CCGGCCTGCC
GGCGACGA-3') (SEQ ID NO:382). More preferably, the repair DNA
molecule should contain sequences with more than 30-nt upstream of
ATC and more than 30-nt downstream of the underlined A in AAGAC as
outlined in this sequence (5'-GAC CAGCTC GAGG TCGGCAGCCT GCGCGACATC
AACGTCAAGA CCGGCCTGCC GGCGACGACC AGAACCGA-3') (SEQ ID NO:383). Most
preferably, the repair DNA molecule should contain sequences with
more than 50-nt upstream of ATC and more than 50-nt downstream of
the underlined A in AAGAC as indicated in this sequence (5'-GGA
ACTGCGTCGT GCGCGGGGAC CAGCTC GAGG TCGGCAGCCT GCGCGACATC AACGTCAAGA
CCGGCCTGCC GGCGACGACC AGAACCGAGC GCCTCGAGCA GCTCGACGA-3') (SEQ ID
NO:384).
[0201] For introduction of V89Y mutation, the two underlined
residues GT in the maize genomic target sequence 5'-CGCGA CGTCA
ACGTC AA/GAC-3' need to be converted to TA, so the valine residue
at position 89 (V89, GTC) is changed to tyrosine (Y89, TAC). Since
the intended Cas9 cleavage site (indicated by /) is 9 nucleotides
downstream, preferably, the repair DNA molecule should contain
sequences at least 15-nt upstream of GTC and 15-nt downstream of
the underlined A in AA/GAC as in this sequence (5'-GGCAGCCT
GCGCGACTAC AACGTCAAGA CCGGCCTGCC GGC-3') (SEQ ID NO:385). More
preferably, the repair DNA molecule should contain sequences with
at least 20-nt upstream of TAC and at least 20-nt downstream of the
underlined A in AAGAC as outlined in this sequence (5'-AGG
TCGGCAGCCT GCGCGACTAC AACGTCAAGA CCGGCCTGCC GGCGACGA-3') (SEQ ID
NO:386). More preferably, the repair DNA molecule should contain
sequences with more than 30-nt upstream of TAC and more than 30-nt
downstream of the underlined A in AAGAC as outlined in this
sequence (5'-GAC CAGCTC GAGG TCGGCAGCCT GCGCGACTAC AACGTCAAGA
CCGGCCTGCC GGCGACGACC AGAACCGA-3') (SEQ ID NO:387). Most
preferably, the repair DNA molecule should contain sequences with
more than 50-nt upstream of TAC and more than 50-nt downstream of
the underlined A in AA/GAC as indicated in this sequence (5'-GGA
ACTGCGTCGT GCGCGGGGAC CAGCTC GAGG TCGGCAGCCT GCGCGACTAC AACGTCAAGA
CCGGCCTGCC GGCGACGACC AGAACCGAGC GCCTCGAGCA GCTCGACGA-3') (SEQ ID
NO:388).
[0202] Similarly, double mutants containing V89I (or V89Y) and
E149L (see, e.g., SEQ ID NOS:390, 391 and 392) can be obtained by
transforming maize cells with vectors containing expression
cassettes for Cas9 and two gRNAs along with oligonucleotides to
introduce corresponding mutations (E149L, V89I or V89Y) as
described above in section 4.3.
5. Generation of Targeted E169L Genomic Sequence Modification in
Additional ZmPYL Gene Family Members, ZmPYL-D, ZmPYL-F and
ZmPYL-J
[0203] 5.1 Mutagenesis Targets and gRNA Design
[0204] Similar to examples described above for endogenous ZmPYL-E
gene (Example 4 and Table 1), additional ZmPYL gene family members
were also chosen for targeted genome editing to replace specific
nucleotides so the amino acid residue corresponding to E.sup.169 in
the ABA receptors (ZmPYL-D, ZmPYL-F and ZmPYL-J) is changed to a
hypersenstive form L.sup.169. These intended changes are summarized
in Table 1. These experiments aimed to modify the corresponding
conserved amino acid residue E (glutamic acid) into L (Leucine) in
homologous ZmPYL genes.
TABLE-US-00008 TABLE 1 ZmPYL mutations and gRNA sequences and
transformation vectors Desired Trans- WT maize mutant gRNA target
sequence formation ZmPYL protein protein in transformation vector
vector gene sequence sequence (SEQ. ID. NO. and notes) name ZmPYL-E
LVIE.sup.149SFV LVIL.sup.149SFV 5'-cctgg tgatc gagtc gttcg-3' 23190
(GRMZM2G16 (SEQ. ID. NO: 369; target site 5567_P02) in coding
strand, base replacement 5 bp away from the Cas9 cleavage site)
ZmPYL-D TLVIE.sup.169SFV TLVIL.sup.169SFV 5'-gtcgg ggacg tcgac
gacga-3' 23136 (GRMZM2G04 (SEQ. ID. NO: 393; target site 8733_P02)
in template strand, base replacement 8 bp away from the Cas9
cleavage site) ZmPYL-D LVIE.sup.169SFV LVIL.sup.169SFV 5'-gaggt
catcg acggc cggcc-3' 23189 (GRMZM2G04 (SEQ. ID. NO: 394; target
site 8733_P02) in coding strand, base replacement 19 bp away from
the Cas9 cleavage site) ZmPYL-F LVIE.sup.164SFV LVIL.sup.164SFV
5'-gctcg tgatc gagtc cttcg 22981 (GRMZM2G05 tgg-3' (SEQ. ID. NO:
395; 3882_P01) longer targeting guide sequence (23 bp), target site
in coding strand, base replacement 8 bp away from the Cas9 cleavage
site) ZmPYL-F LVIE.sup.164SFV LVIL.sup.164SFV 5'-gctcg tgatc gagtc
cttcg-3' 23191 (GRMZM2G05 (SEQ. ID. NO: 396; shorter 3882_P01)
targeting guide sequence (20 bp), target site in coding strand,
base replacement 5 bp away from the Cas9 cleavage site) ZmPYL-J
VVLE.sup.148SYV VVLE.sup.148SYV 5'-cgtcg acgac gtagg actcg-3' 23192
(GRMZM2G15 (SEQ. ID. NO: 397; target site 4987_P01) in template
strand, base replacement at the Cas9 cleavage site)
5.2 Constructions of Vectors for Expression of gRNAs Targeting
ZmPYL-D, ZmPYL-F and ZmPYL-J Genes
[0205] Similar to examples described above for constructing 23190
for expressing gRNA for endogenous ZmPYL-E gene (Example 4),
transformation vectors expressing Cas9 and different gRNAs (Table
1) for ZmPYL-D (23136 and 23189), ZmPYL-F (22981 and 23191) and
ZmPYL-J (23192) genes were constructed (FIGS. 9A-9B, 10A-10B and
11). The gRNA targeting sequence for different ZmPYL genes are
listed in Table 1 (SEQ ID NO:393 to 397). In these vectors, the
whole gRNA coding regions [.about.20 nucleotide targeting guides
(SEQ ID NO:393 to 397), tracRNA scaffold and PolIII termination
sequences (5'-GTTTT AGAGC TAGAA ATAGC AAGTT AAAAT AAGGC TAGTC CGTTA
TCAAC TTGAA AAAGT GGCAC CGAGT CGGTG CTTTT TTTTT-3')] were placed
under the control of rice polymerase III U3 promoter (prOsU3).
These vectors also contain a PMI selectable marker gene cassette
for selecting stable transformants. These vectors can be used for
transformation mediated by Agrobacterium-mediated transformation or
used directly for particle bomdbarment.
5.3 Generation of Genome Edited Novel Alleles (Targeted Mutagenesis
and Allele Replacement Mutants) Mediated CRISPR-Cas in ZmPYL-D,
ZmPYL-F and ZmPYL-J Genes
[0206] Novel alleles including targeted mutagenesis and allele
replacement mutants can be generated via CRISPR-Cas system in the
presence of repair donor DNA by Agrobacterium-mediated
transformation or particle bomdbarment as described in Example 4
for ZmPYL-E. Here specific examples are provided for targeted
notations in ZmPYL-D, ZmPYL-F and ZmPYL-J genes using biolistic
co-delivery of transformation vectors (Table 1 and FIG. 9A-B to 11)
and repair donor oligodeoxynucleotides with desired mutations
(Table 2 and Seq ID NO:398 to 410). Oligodeoxynucleotides (ODNs) of
different length, strand (coding and non-coding template) or
modification (with and without phosphorothioate linkage
modification at the ends) (Seq ID NO:397 to 409) were used to
mediate mutagenesis in different ZmPYL genes.
TABLE-US-00009 TABLE 2 Repair donor DNA oligonucleotide sequences
Cas9 and gRNA Oligo- expression deoxynucleotide Length ZmPYL gene
vector(s) (ODN) (nt) Notes Seq. ID. No. ZmPYL-D 23136 ODN-ZmPYL-Dc
75 PAM #1 and target site in template strand, Seq. ID. No. 398 base
replacement 8 bp away from the Cas9 cleavage site ZmPYL-D 23136
ODN-ZmPYL-Dd- 75 PAM#1 and target site in template strand, base
Seq. ID. No. 399 NT replacement 8 bp away from the Cas9 cleavage
site; PAM in donor ODN removed ZmPYL-D 23136 ODN-ZmPYL-Dd- 75 PAM#1
and target site in coding strand, base Seq. ID. No. 400 NT-PM
replacement 8 bp away from the Cas9 cleavage site; PAM in donor ODN
removed; ODN with phosphorothioate linkage modification ZmPYL-D
23189 ODN-ZmPYL-Db 88 PAM #2 and target site in coding strand, base
Seq. ID. No. 401 replacement 19 bp away from the Cas9 cleavage
site; PAM in donor ODN removed; ZmPYL-E 23190 ODN- ZmPYL-Eb 60
Target site in coding strand, base replacement Seq. ID. No. 379 5
bp away from the Cas9 cleavage site; ODN in non-coding strand; PAM
in donor ODN removed ZmPYL-E 23190 ODN-ZmPYL-Ec- 72 Target site in
coding strand, base replacement Seq. ID. No. 380 NT-PM 5 bp away
from the Cas9 cleavage site; ODN with phosphorothioate linkage
modification; PAM in donor ODN removed ZmPYL-F 22981, 23191
ODN-ZmPYL-Fa 60 Target site in coding strand, base replacement Seq.
ID. No. 402 5 or 8 bp away from the Cas9 cleavage site; PAM in
donor ODN not removed ZmPYL-F 22981, 23191 ODN-ZmPYL-Fb 60 Target
site in coding strand, base replacement Seq. ID. No. 403 5 or 8 bp
away from the Cas9 cleavage site; PAM in donor ODN removed ZmPYL-F
22981, 23191 ODN-ZmPYL-Fc 77 Target site in coding strand, base
replacement Seq. ID. No. 404 5 or 8 bp away from the Cas9 cleavage
site; PAM in donor ODN removed ZmPYL-F 22981, 23191 ODN-ZmPYL-Fd-
77 Target site in coding strand, base replacement Seq. ID. No. 405
NT 5 or 8 bp away from the Cas9 cleavage site; PAM in donor ODN
removed ZmPYL-F 22981, 23191 ODN-ZmPYL-Fd- 77 Target site in coding
strand, base replacement Seq. ID. No. 406 NT-PM 5 or 8 bp away from
the Cas9 cleavage site; PAM in donor ODN removed; ODN with
phosphorothioate linkage modification ZmPYL-J 23192 ODN-ZmPYL-Jc 68
Target site in template strand, base Seq. ID. No. 407 short
replacement at the Cas9 cleavage site; ODN in coding strand
sequence ZmPYL-J 23192 ODN--ZmPYL-Jc- 68 Target site in template
strand, base Seq. ID. No. 408 NT replacement at the Cas9 cleavage
site; ODN in non-coding strand sequence ZmPYL-J 23192
ODN--ZmPYL-Jc- 68 Target site in template strand, base Seq. ID. No.
409 NT-PM replacement at the Cas9 cleavage site; ODN in non-coding
strand sequence and with phosphorothioate linkage modification
ZmPYL-J 23192 ODN--ZmPYL-Jc- 88 Target site in template strand,
base Seq. ID. No. 410 long replacement at the Cas9 cleavage site;
ODN in coding strand sequence
[0207] More specifically, the above described transformation vector
(Table 2) expressing Cas9 and gRNA is mixed with its corresponding
repair donor DNA oligonucleotides (SEQ ID NO:398 to 410) and then
precipitated onto gold particles. The coated gold particles are
then used to bombard immature maize embryos of elite inbred
transformation variety NP2222 (DeFramond A J, et al (2013) Corn
Event 5307. U.S. Pat. No. 8,466,346). Other maize genotype such as
A188 and HiII can be used as bombardment target tissue source.
Methods for maize immature embryo bombardment, callus induction
tissue regeneration and rooting methods have been described
previously except here no mannose selection is required (Wright et
al., 2001, Efficient biolistic transformation of maize (Zea mays
L.) and wheat (Triticum aestivum L.) using the phosphomannose
isomerase gene, pmi, as the selectable marker. Plant Cell Reports
20:429-436). For example, for mutagenesis of ZmPYL-F gene mediated
by CRISP-Cas9, immature embryos are isolated from harvested
immature ears at about 9-12 days after pollination and pre-cultured
for 3 to 5 days on osmoticum media. Pre-cultured embryos are then
bombarded with DNA vector 22981 along with one of the
oligonucleotides [ODN-ZmPYL-Fa, ODN-ZmPYL-Fb, ODN-ZmPYL-Fc,
ODN-ZmPYL-Fd-NT or ODN-ZmPYL-Fd-NT-PM (Seq. ID. NO:402 to 406)]
using BioRad PDS-1000 Biolistic particle delivery system. Bombarded
embryos are then incubated in callus induction media and then moved
onto mannose selection media. Selected calli are moved onto
regeneration media to induce shoot formation. Shoots are then moved
to rooting media. Samples are then harvested from regenerated
plants for genotyping to identify plants containing the desired
genomic sequence mutation that results in E164L amino acid change
in the ZmPYL-F gene. Table 3 lists different experiments for
targeted mutagenesis and allele replacement of different ZmPYL
genes. In some experiments, gRNA and Cas9 expression vector was
co-transformed with ZsGreen fluorescent protein vector 12672 for
assessing gene delivery efficiency. In some other experiments, two
or more gRNA expression vectors were co-delivered with two or more
repair donor oligodexynucleotides to mutate two or ZmPYL genes
simultaneously (Table 3). Table 3 shows that ZmPYL-F in a high
percentage of PMI positive events (transformants) contain mutations
at the intended sequences (5'-GCTCG TGATC GAGTC CTTCG/TGGTG
GACGT-3', / indicated predicted Cas9 cleavage position) targeted by
gRNA-Cas9.
TABLE-US-00010 TABLE 3 Targeted mutagenesis and allele replacement
experiments of different ZmPYL genes Number of Number of Total
Total events with putative ODN(s) used for number of number of
mutation(s) events with ZmPYL Transformation generating targeted
Number of immature PMI positive at the target desired allele target
gene vector(s) mutation (s) experiments embryos events site* change
ZmPYL-D 23136 ODN-ZmPYL-Dc 5 7914 279 132 3 ZmPYL-D 23136,
ODN-ZmPYL-Dc 2 2102 TBD TBD TBD 12672 ZmPYL-D 23189 ODN-ZmPYL-Db 1
1750 TBD TBD TBD ZmPYL-E 23190 ODN-ZmPYL-Eb 2 2238 60** 15 and TBD
1 and TBD ZmPYL-F 22981 ODN-ZmPYL-Fb 6 5460 80 and TBD 13 and TBD
TBD ZmPYL-F 22981, ODN-ZmPYL-Fb 5 7225 346 and 171 and 6 and TBD
with other TBD TBD (22980, 22978, 22982)** ZmPYL-F 22981
ODN-ZmPYL-Fc 1 810 TBD TBD TBD ZmPYL-J 23192 ODN-ZmPYL-Jc short 1
855 TBD TBD TBD ZmPYL-J 23192 ODN-ZmPYL-Jc long 1 1605 TBD TBD TBD
ZmPYL-E, 23190, ODN-ZmPYL-Eb, 1 1785 TBD TBD TBD ZmPYL-F 23191
ODN-ZmPYL-Fc ZmPYL-F, 23191 + ODN-ZmPYL-Jd-S-NT, 1 1970 TBD TBD TBD
ZmPYL-J 23192 ODN-ZmPYL-Fd-NT ZmPYL-F, 23191 + ODN-ZmPYL-Jd-S-NT- 1
1505 TBD TBD TBD ZmPYL-J 23192 PM, ODN-ZmPYL-Fd- NT-PM
[0208] Sequencing of ZmPYL-F target region in selected mutants
confirmed qPCR results. FIG. 13 shows sequence alignment of
targeted mutations in ZmPYL-F mediated by gRNA-Cas9 expressed from
vector 22981. Note: * Event with both monoallelic and/or biallelic
mutations; ** These vectors (22980, 22978 or 22982) carry cassettes
for expression of control gRNAs (including NGG sequence) for
testing specificity of gRNA for ZmPYL genes. # TBD, to be
determined; experiments are in progress and no data is available at
the moment.
5.4 Molecular Characterization of Edited ZmPYL Mutants
[0209] Leaf samples are harvested from regenerated plants in root
vessels for molecular analysis or genotyping to identify plants
containing mutations at the target sequence and also containing
desired sequence mutations that results in desired amino acid
change in the ZmPYL genes. Targeted mutants can be identified using
one of the following methods: (1) PCR amplification of the target
region followed by restriction enzyme digestion and gel
electrophoresis if the mutated sequence contains a restriction site
(Lloyd A et al. 2005. Proc. Natl. Acad. Sci. USA 102:2232-37; Zhang
F, et al. 2010. Proc. Natl. Acad. Sci. USA 107:12028-33). This
method is simple, but requires the presence of suitable restriction
site, thus cannot be used for most targets. (2) PCR amplification
of the target region followed by Sanger sequencing or deep
sequencing (Gross, E. et al. 1999. Hum. Genet. 105, 72-78. Shukla V
K, et al. 2009. Nature 459:437-41. Townsend J A, et al. 2009.
Nature 459:442-45.); Sequencing approach is definitive and
sensitive, but takes longer time and throughput can be limited by
capacity. (3) PCR amplification of the target region followed by
denaturation, annealing and capillary electrophoresis (Li-Sucholeik
X C, et al. 1999. Electrophoresis 20, 1224-1232; Larsen L A, et al.
1999. Hum. Mutat. 13, 318-327) or denaturing high-performance
liquid chromatography to detect base pair changes by heteroduplex
analysis (McCallum C M, et al. 2000. Nature Biotechnology 18,
455-457); these methods are limited by throughput and the
identified mutations need to be further verified by sequencing. (4)
PCR amplification of the target region followed by denaturation,
heteroduplex formation/strand annealing, digestion with
mismatch-specific nuclease (such as CEL1 and T7 endonuclease) and
gel electrophoresis (Oleykowski, C. A. et al. 1998. Nucleic Acids
Res. 26, 4597-4602. Colbert et al. 2001. Plant Physiol.
126:480-484; Lombardo A, et al. 2007. Nat. Biotechnol.
25:1298-306), for example using the commercially available
Surveyer.TM. nuclease assay kit (Transgenomic, Gaithersburg, Md.,
USA; Qiu, P., et al. 2004. BioTechniques 36, 702-707). However, the
gel-based assays are not as sensitive as high-throughput DNA
sequencing and can only detect mutation with frequency of 1% or
more. All of the above 4 approaches of identifying a potential
mutant in a target site are based on the presence of a new signal
in a qualitative fashion, either a new band in a gel or a new peak
in a chromatogram that is different from the wild type reference
sequence.
[0210] We have developed an alternative high throughput assay
method for identification of plants with any site-directed mutation
at the targeted sequences based on qPCR (Syngenta Provisional
Patent Application #9207-137PR, case 80484). The method measures
the reduction of the wild type target site sequence in cells or
tissues that have been treated with a site-directed nuclease in a
quantitative fashion in comparison with a reference sample.
Typically, a Taqman-based assay is used for quantification of the
target sequence copy numbers. For detecting potential events with
desired allele replacement, an additional high throughput end-point
assay is designed and performed. In this end point assay, signals
from two MGB probes are used to determine the presence of WT or
expected mutant allele as shown in FIG. 12. Events with putative
allele replacement are selected based on both Taqman copy number
assay (WT target sequence copy number) and end point assay (mutant
copy number) results. Putative events with putative allele
replacement are further confirmed by DNA sequence analysis of
amplified target locus sequences. Table 4 shows the qPCR and end
point assay results of selected number of regerated maize plants
generated from biolistic transformation experiment of vector 22981
(FIG. 10A-10B) co-delivered with oligonucleotide ODN-ZmPYL-Fb (Seq.
ID.No. 402). As shown in Table 4, transformation vector-specific
assays were performed to determine if there is any transgene
insertion (cCas9-01 and cPMI-09 qPCR assays). qPCR assay (ZmPYL-F
cutting site) was also performed to determine the copy number of
the ZmPYL-F maize genomic target site sequence (5'-GCTCG TGATC
GAGTC CTTCG/TGG-3', SEQ ID NO:394). Finally, an end point assay
(ZmPYL-F E164L) was also used to determine if plants have intended
sequence mutation (from GAG to CTG) resulting in E164L amino acid
residue change. For example, plant MZET151104A015A has a single
copy of transgene insertion (for Cas9 and PMI genes), biallelic
mutations at the target sequence since ZmPYL-F cutting site copy
call is 0 (in WT, the copy call should be 2) and no E164L mutation.
Another plant MZET151104A125A has more than 2 copies of
transformation vector (22981) insertion and only one copy of the
ZmPYL-F cutting site is mutated. But plant MZET151104A125A is
positive for end point assay for detecting ZmPYL-F E164L mutation.
This event is thus a candidate event with ZmPYL-F E164L mutation.
Candidate events MZET151104A125A, MZET151104A174A and
MZET151104A180A in Table 4 are then further confirmed by sequencing
analysis of PCR-amplified ZmPYL-F genome sequences.
TABLE-US-00011 TABLE 4 qPCR and end point assay results of
regenerated maize events Assays for genomic Assays for target
sequence transgene vector ZmPYL-F ZmPYL-F cCas9-01 cPMI-09 cutting
site E164L Assay name (qPCR) (qPCR) (qPCR) (End point) (Type) Copy
Copy Copy Null/Het/ Candidate Plant ID Construct ID number number
number Hom event MZET151104A015A 22981 1 1 0 Null MZET151104A017A
22981 1 1 1 Null MZET151104A019A 22981 >2 >2 0 or 1 Null
MZET151104A021A 22981 1 1 1 Null MZET151104A125A 22981 >2 2 1
Het Yes MZET151104A126A 22981 >2 >2 1 Null MZET151104A132A
22981 1 1 or 2 1 or 2 Null MZET151104A138A 22981 1 1 0 Null
MZET151104A141A 22981 1 or 2 1 0 Null MZET151104A158A 22981 >2 2
1 Null MZET151104A174A 22981 >2 >2 1 Het Yes MZET151104A178A
22981 >2 1 1 Null MZET151104A180A 22981 >2 1 1 Het Yes
MZET151104A186A 22981 >2 >2 0 or 1 Null MZET151104A195A 22981
>2 >2 0 Null MZET151104A201A 22981 0 0 1 Null
5.5 Evaluation of ZmPYL Gene Edited Mutants
[0211] ZmPYL gene edited mutants are tested as described for
transgenic T6PP maize plants (Nuccio et al., 2015, Nature
Biotechnology, doi:10.1038/nbt.3277) with managed stress
environment (MSE) trials. Mutant lines that show improved plant
response to water deficit are further tested in multiple location
agronomic equivalency (Ag Eq) trials with mutant lines grown
alongside control plants and using a checkerboard plot layout.
TABLE-US-00012 PYL-E V89A (SEQ ID NO: 389) Met Val Gly Leu Val Gly
Gly Ser Thr Ala Arg Ala Glu His Val Val 1 5 10 15 Ala Asn Ala Gly
Gly Glu Ala Glu Tyr Val Arg Arg Met His Arg His 20 25 30 Ala Pro
Thr Glu His Gln Cys Thr Ser Thr Leu Val Lys His Ile Lys 35 40 45
Ala Pro Val His Leu Val Trp Glu Leu Val Arg Arg Phe Asp Gln Pro 50
55 60 Gln Arg Tyr Lys Pro Phe Val Arg Asn Cys Val Val Arg Gly Asp
Gln 65 70 75 80 Leu Glu Val Gly Ser Leu Arg Asp Ala Asn Val Lys Thr
Gly Leu Pro 85 90 95 Ala Thr Thr Ser Thr Glu Arg Leu Glu Gln Leu
Asp Asp Asp Leu His 100 105 110 Ile Leu Gly Val Lys Phe Val Gly Gly
Asp His Arg Leu Gln Asn Tyr 115 120 125 Ser Ser Ile Ile Thr Val His
Pro Glu Ser Ile Asp Gly Arg Pro Gly 130 135 140 Thr Leu Val Ile Glu
Ser Phe Val Val Asp Val Pro Asp Gly Asn Thr 145 150 155 160 Lys Asp
Glu Thr Cys Tyr Phe Val Glu Ala Val Ile Lys Cys Asn Leu 165 170 175
Asn Ser Leu Ala Glu Val Ser Glu Gln Leu Ala Val Glu Ser Pro Thr 180
185 190 Ser Leu Ile Asp Gln 195 PYL-E V89A E149L (SEQ ID NO: 390)
Met Val Gly Leu Val Gly Gly Ser Thr Ala Arg Ala Glu His Val Val 1 5
10 15 Ala Asn Ala Gly Gly Glu Ala Glu Tyr Val Arg Arg Met His Arg
His 20 25 30 Ala Pro Thr Glu His Gln Cys Thr Ser Thr Leu Val Lys
His Ile Lys 35 40 45 Ala Pro Val His Leu Val Trp Glu Leu Val Arg
Arg Phe Asp Gln Pro 50 55 60 Gln Arg Tyr Lys Pro Phe Val Arg Asn
Cys Val Val Arg Gly Asp Gln 65 70 75 80 Leu Glu Val Gly Ser Leu Arg
Asp Ala Asn Val Lys Thr Gly Leu Pro 85 90 95 Ala Thr Thr Ser Thr
Glu Arg Leu Glu Gln Leu Asp Asp Asp Leu His 100 105 110 Ile Leu Gly
Val Lys Phe Val Gly Gly Asp His Arg Leu Gln Asn Tyr 115 120 125 Ser
Ser Ile Ile Thr Val His Pro Glu Ser Ile Asp Gly Arg Pro Gly 130 135
140 Thr Leu Val Ile Leu Ser Phe Val Val Asp Val Pro Asp Gly Asn Thr
145 150 155 160 Lys Asp Glu Thr Cys Tyr Phe Val Glu Ala Val Ile Lys
Cys Asn Leu 165 170 175 Asn Ser Leu Ala Glu Val Ser Glu Gln Leu Ala
Val Glu Ser Pro Thr 180 185 190 Ser Leu Ile Asp Gln 195 PYL-E V89I
E149L (SEQ ID NO: 391) Met Val Gly Leu Val Gly Gly Ser Thr Ala Arg
Ala Glu His Val Val 1 5 10 15 Ala Asn Ala Gly Gly Glu Ala Glu Tyr
Val Arg Arg Met His Arg His 20 25 30 Ala Pro Thr Glu His Gln Cys
Thr Ser Thr Leu Val Lys His Ile Lys 35 40 45 Ala Pro Val His Leu
Val Trp Glu Leu Val Arg Arg Phe Asp Gln Pro 50 55 60 Gln Arg Tyr
Lys Pro Phe Val Arg Asn Cys Val Val Arg Gly Asp Gln 65 70 75 80 Leu
Glu Val Gly Ser Leu Arg Asp Ile Asn Val Lys Thr Gly Leu Pro 85 90
95 Ala Thr Thr Ser Thr Glu Arg Leu Glu Gln Leu Asp Asp Asp Leu His
100 105 110 Ile Leu Gly Val Lys Phe Val Gly Gly Asp His Arg Leu Gln
Asn Tyr 115 120 125 Ser Ser Ile Ile Thr Val His Pro Glu Ser Ile Asp
Gly Arg Pro Gly 130 135 140 Thr Leu Val Ile Leu Ser Phe Val Val Asp
Val Pro Asp Gly Asn Thr 145 150 155 160 Lys Asp Glu Thr Cys Tyr Phe
Val Glu Ala Val Ile Lys Cys Asn Leu 165 170 175 Asn Ser Leu Ala Glu
Val Ser Glu Gln Leu Ala Val Glu Ser Pro Thr 180 185 190 Ser Leu Ile
Asp Gln 195 PYL-E V89Y E149L (SEQ ID NO: 392) Met Val Gly Leu Val
Gly Gly Ser Thr Ala Arg Ala Glu His Val Val 1 5 10 15 Ala Asn Ala
Gly Gly Glu Ala Glu Tyr Val Arg Arg Met His Arg His 20 25 30 Ala
Pro Thr Glu His Gln Cys Thr Ser Thr Leu Val Lys His Ile Lys 35 40
45 Ala Pro Val His Leu Val Trp Glu Leu Val Arg Arg Phe Asp Gln Pro
50 55 60 Gln Arg Tyr Lys Pro Phe Val Arg Asn Cys Val Val Arg Gly
Asp Gln 65 70 75 80 Leu Glu Val Gly Ser Leu Arg Asp Tyr Asn Val Lys
Thr Gly Leu Pro 85 90 95 Ala Thr Thr Ser Thr Glu Arg Leu Glu Gln
Leu Asp Asp Asp Leu His 100 105 110 Ile Leu Gly Val Lys Phe Val Gly
Gly Asp His Arg Leu Gln Asn Tyr 115 120 125 Ser Ser Ile Ile Thr Val
His Pro Glu Ser Ile Asp Gly Arg Pro Gly 130 135 140 Thr Leu Val Ile
Leu Ser Phe Val Val Asp Val Pro Asp Gly Asn Thr 145 150 155 160 Lys
Asp Glu Thr Cys Tyr Phe Val Glu Ala Val Ile Lys Cys Asn Leu 165 170
175 Asn Ser Leu Ala Glu Val Ser Glu Gln Leu Ala Val Glu Ser Pro Thr
180 185 190 Ser Leu Ile Asp Gln 195 ODN-ZmPYL-Dc (SEQ ID NO: 398)
AGGTC ATCGA CGGCC GGCCA GGGAC GCTCG TCATC CTGTC ATTCG TCGTC GACGT
CCCCG ACGGC AACAC CAAGG ODN-ZmPYL-Dd-NT (SEQ ID NO: 399) CCTTG
GTGTT GCCGT CGGGG ACGTC GACGA CGAAT GACAG GATGA CGAGC GTCCC TGGCC
GGCCG TCGAT GACCT ODN-ZmPYL-Dd-NT-PM (SEQ ID NO: 400) C*C*T*T GGTGT
TGCCG TCGGG GACGT CGACG ACGAA TGACA GGATG ACGAG CGTCC CTGGC CGGCC
GTCGA TGA*C*C *T *denotes phosphorothioate modification
ODN-ZmPYL-Db (SEQ ID NO: 401) CCATC CTCAC CGTCC ACCCG GAGGT CATCG
ACGGC CGACC AGGGA CGCTC GTCAT CCTGT CCTTC GTCGT CGACG TCCCC GACGG
CAA ODN-ZmPYL-Fa (SEQ ID NO: 402) CGACG GCCGA CCGGG GACGC TCGTG
ATCCT GTCCT TCGTG GTGGA CGTCC CCGAC GGCAA ODN-ZmPYL-Fb (SEQ ID NO:
403) CGACG GCCGA CCGGG GACGC TCGTG ATCCT GTCCT TCGTA GTGGA CGTCC
CCGAC GGCAA ODN-ZmPYL-Fc (SEQ ID NO: 404) AGGTC ATCGA CGGCC GACCG
GGGAC GCTCG TGATC CTGTC CTTCG TAGTG GACGT CCCCG ACGGC AACAC CAAGG
AC ODN-ZmPYL-Fd-NT (SEQ ID NO: 405) GTCCT TGGTG TTGCC GTCGG GGACG
TCCAC TACGA AGGAC AGGAT CACGA GCGTC CCCGG TCGGC CGTCG ATGAC CT
ODN-ZmPYL-Fd-NT-PM (SEQ ID NO: 406) G*T*C*CT TGGTG TTGCC GTCGG
GGACG TCCAC TACGA AGGAC AGGAT CACGA GCGTC CCCGG TCGGC CGTCG ATGA*C*
C*T *denotes phosphorothioate modification ODN-ZmPYL-Jc short (SEQ
ID NO: 407) CACCG AGTTC CAGCC GGGCC CCTAC TGCGT CGTGC TCCTG TCCTA
CGTCG TCGAC GTCCC CGACG GCA ODN-ZmPYL-Jc short-NT (SEQ ID NO: 408)
TGCCG TCGGG GACGT CGACG ACGTA GGACA GGAGC ACGAC GCAGT AGGGG CCCGG
CTGGA ACTCG GTG ODN-ZmPYL-Jc short-NT-PM (SEQ ID NO: 409) T*G*C*CG
TCGGG GACGT CGACG ACGTA GGACA GGAGC ACGAC GCAGT AGGGG CCCGG CTGGA
ACTCG *G*T*G *denotes phosphorothioate modification ODN-ZmPYL-Jc
long (SEQ ID NO: 410) TCACC TCCGT CACCG AGTTC CAGCC GGGCC CCTAC
TGCGT CGTGC TCCTG TCCTA CGTCG TCGAC GTCCC CGACG GCAAC ACCGA GGA
[0212] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
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=US20160194653A1).
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=US20160194653A1).
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