U.S. patent application number 11/033687 was filed with the patent office on 2006-07-13 for structure-based designed herbicide resistant products.
Invention is credited to Genichi Kakefuda, Jae-Gyu Kwagh, Karl-Heinz Ott, Gerald W. Stockton.
Application Number | 20060156427 11/033687 |
Document ID | / |
Family ID | 27026922 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060156427 |
Kind Code |
A1 |
Kakefuda; Genichi ; et
al. |
July 13, 2006 |
Structure-based designed herbicide resistant products
Abstract
Disclosed herein are structure-based modelling methods for the
preparation of acetohydroxy acid synthase (AHAS) variants,
including those that exhibit selectively increased resistance to
herbicides such as imidazoline herbicides and AHAS inhibiting
herbicides. The invention encompasses isolated DNAs encoding such
variants, vectors that include the DNAs, and methods for producing
the variant polypeptides and herbicide resistant plants containing
specific AHAS gene mutations. Methods for weed control in crops are
also provided.
Inventors: |
Kakefuda; Genichi; (Yardley,
PA) ; Ott; Karl-Heinz; (Lawrenceville, NJ) ;
Kwagh; Jae-Gyu; (Fairless Hills, PA) ; Stockton;
Gerald W.; (Yardley, PA) |
Correspondence
Address: |
BASF CORPORATION
CARL-BOSCH-STRASSE 38
LUDWIGSHAFEN
D67056
DE
|
Family ID: |
27026922 |
Appl. No.: |
11/033687 |
Filed: |
January 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10407339 |
Apr 4, 2003 |
6855533 |
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11033687 |
Jan 12, 2005 |
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09367512 |
Aug 17, 2000 |
6576455 |
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10407339 |
Apr 4, 2003 |
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PCT/US96/05782 |
Apr 19, 1996 |
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09367512 |
Aug 17, 2000 |
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08455355 |
May 31, 1995 |
5928937 |
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PCT/US96/05782 |
Apr 19, 1996 |
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08426125 |
Apr 20, 1995 |
5853973 |
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08455355 |
May 31, 1995 |
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Current U.S.
Class: |
800/278 ;
703/11 |
Current CPC
Class: |
C12N 9/88 20130101; G16B
15/00 20190201; C12N 15/8278 20130101 |
Class at
Publication: |
800/278 ;
703/011 |
International
Class: |
C12N 15/82 20060101
C12N015/82; G06G 7/48 20060101 G06G007/48; G06G 7/58 20060101
G06G007/58; A01H 1/00 20060101 A01H001/00 |
Claims
1-84. (canceled)
85. A variant plant acetohydroxy acid synthase (AHAS) protein
comprising at least one mutation at an amino acid residue
corresponding to amino acid residue M53 of SEQ ID NO:1, wherein the
mutation is Met53Thr, wherein said variant plant AHAS protein is
more resistant to an herbicide than the wild-type AHAS protein.
86. A variant AHAS protein as defined in claim 85, wherein said
herbicide is selected from the group consisting of an
imidazolinones, sulfonylureas, triazolopyrimidine, sulfomamides,
pyrimidyl-oxy-benzoic acids, sulfamoylureas, sulfonylcarboxamides,
and combinations thereof.
87. A variant AHAS protein as defined in claim 85, wherein said
AHAS protein is derived from Arabidopsis thaliana.
88. A variant AHAS protein as defined in claim 85, wherein said
variant AHAS protein has catalytic activity that is more resistant
to at least one herbicide than is wild type AHAS.
89. A variant AHAS protein as defined in claim 85, wherein said
variant AHAS has more than about 20% of the catalytic activity of
wild-type AHAS.
90. A variant AHAS protein as defined in claim 85, wherein said
variant AHAS is at least 2-fold more resistant to
imidazolinone-based herbicides than to sulfonylurea-based
herbicides.
Description
FIELD OF THE INVENTION
[0001] This invention pertains to structure-based modelling and
design of variants of acetohydroxy acid synthase (AHAS) that are
resistant to imidazolinones and other herbicides, the AHAS
inhibiting herbicides, AHAS variants themselves, DNA encoding these
variants, plants expressing these variants, and methods of weed
management.
BACKGROUND OF THE INVENTION
[0002] Acetohydroxy acid synthase (AHAS) is an enzyme that
catalyzes the initial step in the biosynthesis of isoleucine,
leucine, and valine in bacteria, yeast, and plants. For example,
the mature AHAS from Zea Mays is approximately a 599-amino acid
protein that is localized in the chloroplast (see FIG. 1). The
enzyme utilizes thiamine pyrophosphate (TPP) and flavin adenine
dinucleotide (FAD) as cofactors and pyruvate as a substrate to form
acetolactate. The enzyme also catalyzes the condensation of
pyruvate and 2-ketobutyrate to form acetohydroxybutyrate. AHAS is
also known as acetolactate synthase or acetolactate pyruvate lyase
(carboxylating), and is designated EC 4.1.3.18. The active enzyme
is probably at least a homodimer. Ibdah et al. (Protein Science,
3:479-S, 1994), in an abstract, disclose one model for the active
site of AHAS.
[0003] A variety of herbicides including imidazolinone compounds
such as imazethapyr (PURSUIT.RTM.--American Cyanamid
Company--Wayne, N.J.), sulfonylurea-based compounds such as
sulfometuron methyl (OUST.RTM.--E.I. du Pont de Nemours and
Company-Wilmington, Del.), triazolopyrimidine sulfonamides
(Broadstrike.TM.--Dow Elanco; see Gerwick, et al., Pestic. Sci.
29:357-364, 1990), sulfamoylureas (Rodaway et al., Mechanisms of
Selectively of Ac 322,140 in Paddy Rice, Wheat and Barley,
Proceedings of the Brighton Crop Protection Conference--Weeds,
1993), pyrimidyl-oxy-benzoic acids (STABLE.RTM.--Kumiai Chemical
Industry Company, E.I. du Pont de Nemours and Company; see, The
Pesticide Manual 10th Ed. pp. 688-889, Clive Tomlin, Ed., British
Crop Protection Council, 49 Downing Street, Farmham, Surrey G49
7PH, UNITED KINGDOM), and sulfonylcarboxamides (Alvarado et al.,
U.S. Pat. No. 4,883,914) act by inhibiting AHAS enzymatic activity.
(See, Chaleff et al., Science 224:1443, 1984; LaRossa et al., J.
Biol. Chem. 259:8753, 1984; Ray, Plant Physiol. 7:827, 11984;
Shaner et al., Plant Physiol. 11:545, 1984). These herbicides are
highly effective and environmentally benign. Their use in
agriculture, however, is limited by their lack of selectivity,
since crops as well as undesirable weeds are sensitive to the
phytotoxic effects of these herbicides.
[0004] Bedbrook et al., U.S. Pat. Nos. 5,013,659, 5,141,870, and
5,378,824, disclose several sulfonylurea resistant AHAS variants.
However, these variants were either obtained by mutagenizing
plants, seeds, or cells and selecting for herbicide-resistant
mutants, or were derived from such mutants. This approach is
unpredictable in that it relies (at least initially) on the random
chance introduction of a relevant mutation, rather than a rational
design approach based on a structural model of the target
protein.
[0005] Thus, there is still a need in the art for methods and
compositions that provide selective wide spectrum and/or specific
herbicide resistance in cultivated crops. The present inventors
have discovered that selective herbicide resistant variant forms of
AHAS and plants containing the same can be prepared by
structure-based modelling of AHAS against pyruvate oxidase (POX),
identifying an herbicide binding pocket or pockets on the AHAS
model, and designing specific mutations that alter the affinity of
the herbicide for the binding pocket. These variants and plants are
not inhibited or killed by one or more classes of herbicides and
retain sufficient AHAS enzymatic activity to support crop
growth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is an illustration of a 600 amino acid sequence
corresponding to the approximately 599 amino acid sequence of
acetohydroxy acid synthase (AHAS) from Zea Mays which is given as
an example of a plant AHAS enzyme. The sequence does not include a
transit sequence, and the extra glycine is vestigial from a
thrombin cleavage site. Residues Met53, Arg128, and Phe135 are
shown in bold.
[0007] FIG. 2 is an illustration of the alignment of the sequence
of maize AHAS and pyruvate oxidase (POX) from Lactobacillus
planarum.
[0008] FIG. 3 is a schematic representation of the secondary
structure of an AHAS subunit. Regular secondary structure elements,
.alpha.-helices and .beta.-sheets, are depicted as circles and
ellipses, respectively, and are numbered separately for each of the
three domains within a subunit. Loops and coiled regions are
represented by black lines, with numbers representing the
approximate beginnings and ends of the elements. The locations of
cofactor binding sites and known mutation sites are indicated by
octahedrons and stars, respectively.
[0009] FIG. 4 is an illustration of a computer-generated model of
the active site of maize AHAS with imazethapyr (PURSUIT.RTM.
herbicide) modeled into the binding pocket.
[0010] FIG. 5 is an illustration of the homology among AHAS amino
acid sequences derived from different plant species. pAC 751 is
maize als 2 AHAS isozyme as expressed from the pAC 751 E. Coli
expression vector as in FIG. 1; Maize als 2 is the maize als 2 AHAS
isozyme; Maize als 1 is the maize als 1 AHAS isozyme; Tobac 1 is
the tobacco AHAS SuRA isozyme; Tobac 2 is the tobacco AHAS SuRB
isozyme; Athcsr 12 is the Arabidopsis thaliana Csr 1.2 AHAS gene;
Bnaal 3 is the Brassica napus AHAS III isozyme; and Bnaal 2 is the
Brassica napus AHAS II isozyme.
[0011] pAC 751 and Maize als 2 are identical genes except that
Maize als 2 starts at the beginning of the transit sequence and pAC
751 starts at the putative mature N-terminal site with an
additional glycine at the N-terminal due to the thrombin
recognition sequence in the pGEX-2T expression vector. The
N-terminal glycine is not a natural amino acid at that
position.
[0012] Amino acid sequence alignments of the AHAS proteins were
generated by PILEUP (GCG Package--Genetics Computer Group,
Inc.,--University Research Park--Madison, Wis.). The consensus
sequence was generated by PRETTY GCG Package.
[0013] FIG. 6 is a photographic illustration of an
SDS-polyacrylamide gel stained for protein showing purification of
maize AHAS. The lanes contain (from left to right): A, Molecular
weight markers; B, Crude E. coli cell extract; C,
Glutathione-agarose affinity purified preparation; D, Thrombin
digest of the affinity purified preparation; E, Second pass through
glutathione-agarose column and Sephacryl S-100 gel filtration.
[0014] FIG. 7 is a graphic illustration of the results of in vitro
assays of the enzymatic activity of wild-type and mutant AHAS
proteins in the absence and in the presence of increasing
concentrations of imazethapyr (PURSUIT.sup..smallcircle. herbicide)
The Y axis represents the % of activity of the mutant enzyme,
wherein the 100% value is measured in the absence of inhibitor.
[0015] FIG. 8 is a graphic illustration of the results of in vitro
assays of the enzymatic activity of wild-type and mutant AHAS
proteins in the absence and presence of increasing concentrations
of sulfometuron methyl (OUST.RTM. herbicide). The Y axis represents
the % of activity of the mutant enzyme, wherein the 100% value is
measured in the absence of inhibitor.
[0016] FIG. 9 is a graphic illustration of in vitro assays of the
enzymatic activity of wild-type Arabidopsis AHAS protein and the
Met124Ile mutant Arabidopsis AHAS protein in the absence and
presence of increasing concentrations of imazethapyr (PURSUIT.RTM.
herbicide) and sulfometuron methyl (OUST.RTM. herbicide). The Y
axis represents the % activity of the mutant enzyme, wherein the
100% value is measured in the absence of inhibitor.
[0017] FIG. 10 is a graphic illustration of in vitro assays of the
enzymatic activity of wild-type Arabidopsis AHAS protein and the
Met124His mutant Arabidopsis AHAS protein in the absence and
presence of increasing concentrations of imazethapyr (PURSUIT.RTM.
herbicide) and sulfometuron methyl (OUST.RTM. herbicide). The Y
axis represents the X activity of the mutant enzyme, wherein the
100% value is measured in the absence of inhibitor.
[0018] FIG. 11 is a graphic illustration of in vitro assays of the
enzymatic activity of wild-type Arabidopois AHAS protein and
Arg199Glu mutant Arabidopsis AHAS protein in the absence and
presence of increasing concentrations of imazethapyr (PURSUIT.RTM.
herbicide) and sulfometuron methyl (OUST.RTM. herbicide). The Y
axis represents the % activity of the mutant enzyme, wherein the
100% value is measured in the absence of inhibitor.
[0019] FIG. 12 is a schematic illustration of a DNA vector used for
plant transformation, which contains the nptII gene (encoding
kanamycin resistance) under the control of the 35S promoter and an
AHAS gene (wild type or variant) under the control of the
Arabidopsis AHAS promoter.
[0020] FIG. 13 is a photograph showing the root development of
tobacco plants transformed with the Arabidopsis AHAS gene
containing either the Met124Ile or Arg199Glu mutation and a
non-transformed control. Plants were grown for 18 days after
transfer into medium containing 0.25 .mu.M imazethapyr.
[0021] FIG. 14 is a photograph showing tobacco plants transformed
with the Arabidopsis AHAS gene containing either the Met124Ile, Met
124His, or Arg199Glu mutation and a non-transformed control, which
had been sprayed with twice the field rate (100 g/ha) of
imazethapyr.
[0022] FIG. 15 is a photograph showing the results of germination
tests performed in the presence of the herbicide CL 299,263
(imazamox), which were performed on seeds harvested from primary
tobacco plant transformants that had been transformed with the
Arabidopsis AHAS gene containing either the Met124Ile, Met 124His,
or Arg199Glu mutation.
[0023] The present invention provides a structure-based modelling
method for the production of herbicide resistant AHAS variant
protein. The method includes: [0024] (a) aligning a target AHAS
protein on pyruvate oxidase template or an AHAS modelling
equivalent thereof to derive the three-dimensional structure of the
target AHAS protein; [0025] (b) modelling one or more herbicides
into the three-dimensional structure to localize an herbicide
binding pocket in the target AHAS protein; [0026] (c) selecting as
a target for a mutation, at least one amino acid position in the
target AHAS protein, wherein the mutation alters the affinity of at
least one herbicide for the binding pocket; [0027] (d) mutating DNA
encoding the target AHAS protein to produce a mutated DNA encoding
a variant AHAS containing the mutation, such as, for example, at
least one different amino acid, at the position; and [0028] (e)
expressing the mutated DNA in a first cell, under conditions in
which the variant AHAS containing the mutation, such as, for
example, the different amino acid(s), at the position is
produced.
[0029] The method further may include: [0030] (f) expressing DNA
encoding wild-type AHAS protein parallel in a second cell; [0031]
(g) purifying the wild-type and the variant AHAS proteins from the
cells; [0032] (h) assaying the wild-type and the variant AHAS
proteins for catalytic activity in conversion of pyruvate to
acetolactate or in the condensation of pyruvate and 2-ketobutyrate
to form acetohydroxybutyrate, in the absence and in the presence of
the herbicide; and [0033] (i) repeating steps (c)-(h), wherein the
DNA encoding the AHAS variant of step (e) is used as the
AHAS-encoding DNA in step (c) until a first herbicide resistant
AHAS variant protein is identified having: [0034] (i) in the
absence of the at least one herbicide, [0035] (a) catalytic
activity alone sufficient to maintain the viability of a cell in
which it is expressed; or [0036] (b) catalytic activity in
combination with any herbicide resistant AHAS variant protein also
expressed in the cell, which may be the same as or different than
the first AHAS variant protein, sufficient to maintain the
viability of a cell in which it is expressed; [0037] wherein the
cell requires AHAS activity for viability; and [0038] (ii)
catalytic activity that is more resistant to the at least one
herbicide than is wild-type AHAS.
[0039] An alternate structure-based modelling method for the
production of herbicide resistant AHAS variant protein is also
provided. This method includes: [0040] (a) aligning a target AHAS
protein on a first AHAS template derived from a polypeptide having
the sequence of FIG. 1 or a functional equivalent thereof to derive
the three-dimensional structure of the target AHAS protein; [0041]
(b) modelling one or more herbicides into the three-dimensional
structure to localize an herbicide binding pocket in the target
AHAS protein; [0042] (c) selecting as a target for a mutation, at
least one amino acid position in the target AHAS protein, wherein
the mutation alters the affinity of at least one herbicide for the
binding pocket; [0043] (d) mutating DNA encoding the target AHAS
protein to produce a mutated DNA encoding a variant AHAS containing
the mutation at the position; and [0044] (e) expressing the mutated
DNA in a first cell, under conditions in which the variant AHAS
containing the mutation at the position is produced.
[0045] This method can further include: [0046] (f) expressing DNA
encoding wild-type AHAS protein in parallel in a second cell;
[0047] (g) purifying the wild-type and the variant AHAS protein
from the cells; [0048] (h) assaying the wild-type and the variant
AHAS protein for catalytic activity in conversion of pyruvate to
acetolactate or in the condensation of pyruvate and 2-ketobutyrate
to form acetohydroxybutyrate, in the absence and in the presence of
the herbicide; and [0049] (i) repeating steps (c)-(h), wherein the
DNA encoding the AHAS variant of step (e) is used as the
AHAS-encoding DNA in step (c) until a first herbicide resistant
AHAS variant protein is identified having: [0050] (i) in the
absence of the at least one herbicide, [0051] (a) catalytic
activity alone sufficient to maintain the viability of a cell in
which it is expressed; or [0052] (b) catalytic activity in
combination with any herbicide resistant AHAS variant protein also
expressed in the cell, which may be the same as or different than
the first AHAS variant protein, sufficient to maintain the
viability of a cell in which it is expressed; [0053] wherein the
cell requires AHAS activity for viability; and [0054] (ii)
catalytic activity that is more resistant to the at least one
herbicide than is wild-type AHAS.
[0055] In another alternate embodiment, the method includes: [0056]
(a) aligning a target AHAS protein on a first AHAS template having
an identified herbicide binding pocket and having the sequence of
FIG. 1 or a functional equivalent thereof to derive the
three-dimensional structure of the target AHAS protein; [0057] (b)
selecting as a target for a mutation, at least one amino acid
position in the target AHAS protein, wherein the mutation alters
the affinity of at least one herbicide for the binding pocket;
[0058] (c) mutating DNA encoding the target AHAS protein to produce
a mutated DNA encoding a variant AHAS containing the mutation at
the position; and [0059] (d) expressing the mutated DNA in a first
cell, under conditions in which the variant AHAS containing the
mutation at the position is produced.
[0060] This method can further include: [0061] (e) expressing DNA
encoding wild-type target AHAS protein in parallel in a second
cell; [0062] (f) purifying the wild-type and the variant AHAS
protein from the cells; [0063] (g) assaying the wild-type and the
variant AHAS protein for catalytic activity in conversion of
pyruvate to acetolactate or in the condensation of pyruvate and
2-ketobutyrate to form acetohydroxybutyrate, in the absence and in
the presence of the herbicide; and [0064] (h) repeating steps
(b)-(g), wherein the DNA encoding the AHAS variant of step (d) is
used as the AHAS-encoding DNA in step (b) until a first herbicide
resistant AHAS variant protein is identified having: [0065] (i) in
the absence of the at least one herbicide, [0066] (a) catalytic
activity alone sufficient to maintain the viability of a cell in
which it is expressed; or [0067] (b) catalytic activity in
combination with any herbicide resistant AHAS variant protein also
expressed in the cell, which may be the same as or different than
the first AHAS variant protein, sufficient to maintain the
viability of a cell in which it is expressed; [0068] wherein the
cell requires AWLS activity for viability; and [0069] (ii)
catalytic activity that is more resistant to the at least one
herbicide than is wild-type AWLS.
[0070] In preferred embodiments of the above methods, the catalytic
activity in the absence of the herbicide is at least about 5% and
most preferably is more than about 20% of the catalytic activity of
the wild-type AHAS. Where the herbicide is an imidazolinone
herbicide, the herbicide resistant AHAS variant protein preferably
has: [0071] (i) catalytic activity in the absence of the herbicide
of more than about 20% of the catalytic activity of the wild-type
AHAS; [0072] (ii) catalytic activity that is relatively more
resistant to the presence of imidazolinone herbicides compared to
wild-type AHAS; and [0073] (iii) catalytic activity that is
relatively more sensitive to the presence of sulfonylurea
herbicides compared to imidazolinone herbicides.
[0074] The present invention further provides isolated DNA encoding
acetohydroxy acid synthase (AHAS) variant proteins, the variant
proteins comprising an AHAS protein modified by:
[0075] (i) substitution of at least one different amino acid
residue at an amino acid residue of the sequence of FIG. 1 selected
from the group consisting of P48, G49, S52, M53, E54, A84, A95,
T96, S97, G98, P99, G100, A101, V125, R127, R128, M129, I130, G131,
T132, D133, F135, Q136, D186, I187, T259, T260, L261, M262, G263,
R276, M277, L278, G279, H281, G282, T283, V284, G300, V301, R302,
F303, D304, R306, V307, T308, G309, K310, I311, E312, A313, F314,
A315, S316, R317, A318, K319, I 320, E329, I330, K332, N333, K334,
Q335, T404, G413, V414, G415, Q416, H417, Q418, M419, W420, A421,
A422, L434, S435, S436, A437, G438, L439, G440, A441, M442, G443,
D467, G468, S469, L471, N473, L477, M479, Q495, H496, L497, G498,
M499, V501, Q502, Q504, D505, R506, Y508, K509, A510, N511, R512,
A513, H514, T515, S524, H572, Q573, E574, H575, V576, L577, P578,
M579, I580, P581, G583, G584, functional equivalents of any of the
foregoing, and any combination of any of the foregoing;
[0076] (ii) deletion of up to 5 amino acid residues preceding, or
up to 5 amino acid residues following at least one amino acid
residue of the sequence of FIG. 1 selected from the group
consisting of P48, G49, S52, M53, E54, A84, A95, T96, S97, G98,
P99, G100, A101, V125, R127, R128, M129, I130, G131, T132, D133,
F135, Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277,
L278, G279, H281, G282, T283, V284, G300, V301, R302, F303, D304,
R306, V307, T308, G309, K310, I311, E312, A313, F314, A315, S316,
R317, A318, K319, I320, E329, I330, K332, N333, K334, Q335, T404,
G413, V414, G415, Q416, H417, Q418, M419, W420, A421, A422, L434,
S435, S436, A437, G438, L439, G440, A441, M442, G443, D467, G468,
S469, L471, N473, L477, M479, Q495, H496, L497, G498, M499, V501,
Q502, Q504, D505, R506, Y508, K509, A510, N511, R512, A513, H514,
T515, S524, H572, Q573, E574, H575, V576, L577, P578, M579, I580,
P581, G583, G584, functional equivalents of any of the foregoing,
and any combination of any of the foregoing;
[0077] (iii) deletion of at least one amino acid residue or a
functional equivalent thereof between Q124 and H150 of the sequence
of FIG. 1;
[0078] (iv) addition of at least one amino acid residue or a
functional equivalent thereof between Q124 and H150 of the sequence
of FIG. 1;
[0079] (v) deletion of at least one amino acid residue or a
functional equivalent thereof between G300 and D324 of the sequence
of FIG. 1;
[0080] (vi) addition of at least one amino acid residue or a
functional equivalent thereof between G300 and D324 of the sequence
of FIG. 1; or
[0081] (vii) any combination of any of the foregoing.
[0082] In this numbering system, residue #2 corresponds to the
putative amino terminus of the mature protein, i.e., after removal
of a chloroplast targeting peptide.
[0083] The above modifications are directed to altering the ability
of an herbicide, and preferably an imidazolinone-based herbicide,
to inhibit the enzymatic activity of the protein. In a preferred
embodiment, the isolated DNA encodes an herbicide-resistant variant
of AHAS. Also provided are DNA vectors comprising DNA encoding
these AHAS variants, variant AHAS proteins themselves, and cells,
grown either in vivo or in cell culture, that express the AHAS
variants or comprise these vectors.
[0084] In another aspect, the present invention provides a method
for conferring herbicide resistance on a cell or cells and
particularly a plant cell or cells such as, for example, a seed. An
AHAS gene, preferably the Arabidopsis thaliana AHAS gene, is
mutated to alter the ability of an herbicide to inhibit the
enzymatic activity of the AHAS. The mutant gene is cloned into a
compatible expression vector, and the gene is transformed into an
herbicide-sensitive cell under conditions in which it is expressed
at sufficient levels to confer herbicide resistance on the
cell.
[0085] Also contemplated are methods for weed control, wherein a
crop containing an herbicide resistant AHAS gene according to the
present invention is cultivated and treated with a weed-controlling
effective amount of the herbicide.
[0086] Also disclosed is a structure-based modelling method for the
preparation of a first herbicide which inhibits AHAS activity. The
method comprises: [0087] (a) aligning a target AHAS protein on
pyruvate oxidase template or an AHAS modelling functional
equivalent thereof to derive the three-dimensional structure of the
target AHAS protein; [0088] (b) modelling a second herbicide having
AHAS inhibiting activity into the three-dimensional structure to
derive the location, structure, or a combination thereof of an
herbicide binding pocket in the target AHAS protein; and [0089] (c)
designing a non-peptidic first herbicide which will interact with,
and preferably will bind to, an AHAS activity inhibiting effective
portion of the binding pocket, wherein the first herbicide inhibits
the AHAS activity sufficiently to destroy the viability of a cell
which requires AHAS activity for viability.
[0090] An alternative structure-based modelling method for the
production of a first herbicide which inhibits AHAS activity, is
also enclosed. The method comprises: [0091] (a) aligning a target
AHAS protein on a first AHAS template derived from a polypeptide
having the sequence of FIG. 1 or a functional equivalent thereof,
to derive the three-dimensional structure of the target AHAS
protein; [0092] (b) modelling a second herbicide having AHAS
inhibiting activity into the three-dimensional structure to derive
the location, structure, or a combination thereof of an herbicide
binding pocket in the target AHAS protein; and [0093] (c) designing
a non-peptidic first herbicide which will interact with, and
preferably will bind to, an AHAS activity inhibiting effective
portion of the binding pocket, wherein the first herbicide inhibits
the AHAS activity sufficiently to destroy the viability of a cell
which requires AHAS activity for viability.
[0094] Preferably in each method, the first herbicide contains at
least one functional group that interacts with a functional group
of the binding pocket.
DETAILED DESCRIPTION OF THE INVENTION
[0095] The present invention encompasses the rational design or
structure-based molecular modelling of modified versions of the
enzyme AHAS and AHAS inhibiting herbicides. These modified enzymes
(AHAS variant proteins) are resistant to the action of herbicides.
The present invention also encompasses DNAs that encode these
variants, vectors that include these DNAs, the AHAS variant
proteins, and cells that express these variants. Additionally
provided are methods for producing herbicide resistance in plants
by expressing these variants and methods of weed control. The DNA
and the AHAS variants of the present invention were discovered in
studies that were based on molecular modelling of the structure of
AHAS.
Rational Structure-Based Design of AHAS Variants and AHAS
Inhibiting Herbicides
[0096] Herbicide-resistant variants of AHAS according to the
present invention are useful in conferring herbicide resistance in
plants and can be designed with the POX model, AHAS model, or
functional equivalents thereof, such as, for example,
transketolases, carboligases, pyruvate decarboxylase, proteins that
bind FAD and/or TPP as a cofactor, or any proteins which have
structural features similar to POX and/or AHAS; with an AHAS model
such as a model having the sequence of FIG. 1; or with a functional
equivalent of the sequence of FIG. 1 including a variant modeled
from a previous model. Proteins that can be used include any
proteins having less than a root mean square deviation of less than
3.5 angstroms in their C.alpha. carbons relative to any of the
above-listed molecules. AHAS directed herbicides can be similarly
modelled from these templates. A functional equivalent of an AHAS
amino acid sequence is a sequence having substantial, i.e., 60-70%,
homology, particularly in conserved regions such as, for example, a
putative binding pocket. The degree of homology can be determined
by simple alignment based on programs known in the art, such as,
for example, GAP and PILEUP by GCG. Homology means identical amino
acids or conservative substitutions. A functional equivalent of a
particular amino acid residue in the AHAS protein of FIG. 1 is an
amino acid residue of another AHAS protein which when aligned with
the sequence of FIG. 1 by programs known in the art, such as, for
example, GAP and PILEUP by GCG, is in the same position as the
amino acid residue of FIG. 1.
[0097] Rational design steps typically include: (1) alignment of a
target AHAS protein with a POX backbone or structure or an AHAS
backbone or structure; (2) optionally, and if the AHAS backbone has
an identified herbicide binding pocket, modelling one or more
herbicides into the three-dimensional structure to localize an
herbicide binding pocket in the target protein; (3) selection of a
mutation based upon the model; (4) site-directed mutagenesis; and
(5) expression and purification of the variants. Additional steps
can include (6) assaying of enzymatic properties and (7) evaluation
of suitable variants by comparison to the properties of the
wild-type AHAS. Each step is discussed separately below.
[0098] 1. Molecular Modelling
[0099] Molecular modelling (and particularly protein homology
modelling) techniques can provide an understanding of the structure
and activity of a given protein. The structural model of a protein
can be determined directly from experimental data such as x-ray
crystallography, indirectly by homology modelling or the like, or
combinations thereof (See White, et al., Annu. Rev. Biophys.
Biomol. Struct., 23:349, 1994). Elucidation of the
three-dimensional structure of AHAS provides a basis for the
development of a rational scheme for mutation of particular amino
acid residues within AHAS that confer herbicide resistance on the
polypeptide.
[0100] Molecular modelling of the structure of Zea mays AHAS, using
as a template the known X-ray crystal structure of related pyruvate
oxidase (POX) from Lactobacillus plantarum, provides a
three-dimensional model of AHAS structure that is useful for the
design of herbicide-resistant AHAS variants or AHAS inhibiting
herbicides. This modelling procedure takes advantage of the fact
that AHAS and POX share a number of biochemical characteristics and
may be derived from a common ancestral gene (Chang et al., J.
Bacteriol. 170:3937, 1988).
[0101] Because of the high degree of cross-species homology in AHAS
the modelled AHAS described herein or functional equivalents
thereof can also be used as templates for AHAS variant protein
design.
[0102] Derivation of one model using interactive molecular graphics
and alignments is described in detail below. The three-dimensional
AHAS structure that results from this procedure predicts the
approximate spatial organization of the active site of the enzyme
and of the binding site or pocket of inhibitors such as herbicides
including, but not limited to, imidazolinone herbicides. The model
is then refined and re-interpreted based on biochemical studies
which are also described below.
[0103] Protein homology modelling requires the alignment of the
primary sequence of the protein understudy with a second protein
whose crystal structure is known. Pyruvate oxidase (POX) was chosen
for AHAS homology modelling because POX and AHAS share a number of
biochemical characteristics. For example, both AHAS and POX share
aspects of enzymatic reaction mechanisms, as well as cofactor and
metal requirements. In both enzymes thiamine pyrophosphate (TPP),
flavin adenine dinucleotide (FAD), and a divalent cation are
required for enzymatic activity. FAD mediates a redox reaction
during catalysis in POX but presumably has only a structural
function in AHAS, which is possibly a vestigial remnant from the
evolution of AHAS from POX. Both enzymes utilize pyruvate as a
substrate and form hydroxyethyl thiamine pyrophosphate as a stable
reaction intermediate (Schloss, J. V. et al. In Biosynthesis of
branched chain amino acids, Barak, Z. J. M., Chipman, D. M.,
Schloss, J. V. (eds) VCH Publishers, Weinheim, Germany, 1990).
[0104] Additionally, AHAS activity is present in chimeric POX-AHAS
proteins consisting of the N-terminal half of POX and the
C-terminal half of AHAS, and there is a small degree of AHAS
activity exhibited by POX itself. AHAS and POX also exhibit similar
properties in solution (Risse, B. et al, Protein Sci. 1: 1699 and
1710, 1992; Singh, B. K., & Schmitt, G. K. (1989), FEBS
Letters, 258: 113; Singh, B. K. et al. (1989) In: Prospects for
Amino Acid Biosynthesis Inhibitors in Crop Protection and
Pharmaceutical Chemistry, (Lopping, L. G., et al., eds., BCPC
Monograph p. 87). With increasing protein concentration, both POX
and AHAS undergo stepwise transitions from monomers to dimers and
tetramers. Increases in FAD concentration also induce higher orders
of subunit assembly. The tetrameric form of both proteins is most
stable to heat and chemical denaturation.
[0105] Furthermore, the crystal structure of POX from Lactobacillus
planarum had been solved by Muller et al., Science 259:965, 1993.
The present inventors found that based in part upon the degree of
physical, biochemical, and genetic homology between AHAS and POX,
the X-ray crystal structure of POX could be used as a structural
starting point for homology modelling of the AHAS structure.
[0106] AHAS and L. plantarum POX sequences were not similar enough
for a completely computerized alignment, however. Overall, only
about 20% of the amino acids are identical, while about 50% of the
residues are of similar class (i.e. acidic, basic, aromatic, and
the like). However, if the sequences are compared with respect to
hydrophilic and hydrophobic residue classifications, over 500 of
the 600 amino acids match. Secondary structure predictions for AHAS
(Holley et al., Proc. Natl. Acad. Sci. USA 86:152, 1989) revealed a
strong similarity to the actual secondary structure of POX. For
nearly 70% of the residues, the predicted AHAS secondary structure
matches that of POX.
[0107] POX monomers consist of three domains, all having a central,
parallel .beta.-sheet with crossovers consisting of .alpha.-helices
and long loops. (Muller et al., Science 259:965, 1993). The
topology of the sheets differs between the domains, i.e. in the
first and third domains, the strands are assembled to the
.beta.-sheet in the sequence 2-1-3-4-6-5, while in the .beta.-sheet
of the second domain, the sequence reads 3-2-1-4-5-6.
[0108] Computer generated alignments were based on secondary
structure prediction and sequence homology. The conventional
pair-wise sequence alignment method described by Needleman and
Wunch, J. Mol. Biol, 48: 443, 1970, was used. Two sequences were
aligned to maximize the alignment score. The alignment score
(homology score) is the sum of the scores for all pairs of aligned
residues, plus an optional penalty for the introduction of gaps
into the alignment. The score for the alignment of a pair of
residues is a tabulated integer value. The homology scoring system
is based on observing the frequency of divergence between a given
pair of residues. (M O Dayhoff, R H Schwartz & B C Orcutt
"Atlas of Protein Sequence and Structure" vol. 5 suppl. 3 pp.
345-362, 1978).
[0109] The alignments were further refined by repositioning gaps so
as to conserve continuous regular secondary structures. Amino acid
substitutions generated by evaluation of likely alignment schemes
were compared by means of interactive molecular graphics.
Alignments with the most conservative substitutions with respect to
the particular functionality of the amino acids within a given site
were chosen. The final alignment of both POX and AHAS is displayed
in FIG. 2. Conserved clusters of residues were identified, in
particular for the TPP binding site and for parts of the FAD
binding site. The alignment revealed a high similarity between AHAS
and POX for the first domain, for most parts of the second domain,
and for about half of the third domain. Most of the regions that
aligned poorly and may fold differently in POX and in AHAS were
expected to be at the surface of the protein and were not involved
in cofactor or inhibitor binding. The prediction of mutation sites
is not substantially affected by small shifts in the alignment.
[0110] Most TPP binding residues are highly conserved between POX
and AHAS (e.g. P48-G49-G50). In some cases, residues that were
close to TPP differ between POX and AHAS but remain within a region
that is highly conserved (for example, residues 90-110). On the
other hand, the FAD binding site appeared to be less conserved.
Although some FAD binding residues were strongly conserved (for
example, D325-I326-D327-P328), others clearly differed between AWLS
and POX (for example, residues in the loop from positions 278 to
285 are not homologous. A detailed analysis revealed that, at least
for some of the less-conserved contact sites, the interactions were
mediated by the polypeptide backbone rather than by the side
chains. Hence, conservation was only required for the polypeptide
fold and was not required for the amino acid sequence (for example,
the backbone of residues 258-263 binds the ribitol chain of FAD).
One half of the adenine and the isoalloxazine binding sites clearly
differ.
[0111] After aligning the primary structure, a homology model was
built by transposition of AHAS amino acid sequences to the POX
template structure. Missing coordinates were built stepwise using
templates of amino acid residues to complete undefined side chains.
Data bank searches and energy minimization of small parts of the
molecule were used to complete the conformations of undefined loop
regions. The cofactors TPP and FAD were modeled into their binding
pockets. This model was then subjected to a complete, 5000 cycle
energy minimization. All computer modelling was performed in an
IRIS Indigo Elan R4000 Workstation from Silicon Graphics Co.
Interactive molecular modelling and energy-minimization were
performed using Quanta/CHARMm 4.0 from Molecular Simulations Inc.
During this step, the conformation was stable, indicating that no
strongly disfavored interactions, such as, for example, close van
der Waals contacts, had occurred. The results are shown
schematically in FIG. 3.
[0112] Characteristics of Predicted AHAS Structure
[0113] Inspection of the modelled AHAS structure described above
revealed that most of the protein folds with a backbone that is
energetically reasonable, with most hydrophilic side chains
accessible to the solvent. The surface of the .beta.-sheets are
smooth and accommodate the cross-over regions that are attached to
them.
[0114] A model for dimeric AHAS was generated by duplicating the
coordinates of the energy minimized monomeric AHAS and
superimposing the two copies on two POX subunits using pairs of
C.alpha. coordinates as defined in the alignment scheme. The
polypeptide chain of AHAS folds into three similarly folded domains
composed of a six-stranded parallel .beta.-sheet core surrounded by
long "loops" and .alpha.-helices. Two subunits are assembled such
that the first domain of one subunit is in close proximity to the
cofactor-binding domains 2 and 3 of the other subunit. A
solvent-filled space remains between the subunits at this site.
This pocket, which is defined by the confluence of the three
domains, is the proposed entry site for the substrate. It is also
proposed to be the binding site for herbicides.
[0115] The inner surface of the binding pocket is outlined by the
cofactors. The thiazol of TPP is positioned at the bottom of the
pocket. Domain 3 contributes to the inner surface of the pocket
with a short .alpha.-helix that points its axis towards the
pyrophosphate of TPP, compensating the phosphate charges with its
dipolar moment. This critical helix, which starts with G498, a
"turn" residue in close contact with TPP, and which ends at F507,
contains three known mutation sites for sulfonylurea resistance:
V500, W503, and F507 (See, U.S. Pat. Nos. 5,013,659; 5,141,870; and
5,378,824). In domain 1, the loop defined as P48-S52 (between
.beta.-strand 2 and .alpha.-helix 2) faces W503, a mutation in
which confers resistance to imidazolinones. Residues Y47 to G50 are
also in contact with TPP. This loop is adjacent to P184-Q189,
another turn, which connects the last strand of the .beta.-sheet of
domain 1 with a .beta.-strand that connects with domain 2. Within
the pocket, near its entrance, is a long region of domain 1 that
interacts with a complementary stretch of domain 2. Residues
125-129 and 133-137 of domain 1 and residues 304-313 of domain 2
are at the surface of the pocket. A turn consisting of T96-G100 is
between loop 125-129 and TPP. A further stretch of domain 3 and two
regions of domain 2 that line the binding pocket are at the
opposite corner of the pocket. Residues 572, 575, 582, and 583 of
domain 3 define the pocket surface on one side. The remaining part
of the interior of the pocket's surface is defined by FAD and by a
loop, L278-G282, that contacts the isoalloxazine ring of FAD.
[0116] The structural models of the AHAS protein can also be used
for the rational design of herbicides or AHAS inhibitors.
[0117] 2. Modelling of Herbicides into Binding Sites
[0118] Imazethapyr, the active imidazolinone in PURSUIT.RTM., was
positioned into its proposed binding site using interactive
molecular graphics (FIG. 4) and the software described above (FIG.
4). K185 was chosen as an "anchor" to interact with the charge of
the carboxyl group. The imidazolinone's NH--CO unit was placed to
form hydrogen bonds to G50 and A50. This positioned the methyl
substitute of imazethapyr close to V500 on the backbone of the
small .alpha.-helix. The isopropyl group is possibly bound by
hydrophobic residues of the amino acids in the region of residues
125-135 that contribute to the inner surface of the pocket. The
pyridine ring is most probably "sandwiched" between A134 or F135,
F507 and W503. W503 also interacts with the imidazolinone ring
system.
[0119] In a similar fashion, the sulfonylurea herbicides were
modelled into a site that partially overlapped the described
imidazolinone binding site. Overlap of sulfonylurea and
imidazolinone binding sites was consistent with competition binding
experiments and with established mutant data, which show that the
same mutation in maize, W503L, can confer resistance to both
herbicides. In these models, most of the known mutation sites that
confer sulfonylurea herbicide resistance, i.e. G50, A51, K185,
V500, W503, F507, are in close contact to the bound herbicides.
P126 and A51 are required for keeping the K185 side chain in place
by generating a hydrophobic pore. S582, a site for specific
imidazolinone resistance, is distant from the binding region and is
located in the region where the homology is so poor that a change
in the fold is expected. The FAD binding site apparently has low
homology between AHAS and POX in this region; S582 is a residue
that confers resistance in maize, and that S582 and its adjacent
residues are in close contact to the active site pocket. It is
proposed that FAD and the loop region encompassing residues 278 to
285 move slightly away from the third domain, (downward in FIG. 4)
and that a loop that contains S582 folds into the space between the
helix at positions 499 to 507 and the loop at positions 278 to 285.
D305, another known resistance site, is close to FAD and modulates
the interaction between domains 1 and 2. M280 may either be
involved in positioning of the helix at positions 498 to 507 or
directly in inhibitor binding. M280 and D305 could also be directly
involved in inhibitor binding if domains 1 and 2 move slightly
closer to each other.
[0120] 3. Selection of Mutations
[0121] Specific amino acid residues are pinpointed as sites for the
introduction of mutations into the primary sequence of AHAS. These
amino acids are selected based upon their position in that if that
amino acid residue position is modified, there will be a resultant
alteration (i.e. decline) in the affinity of an herbicide for the
binding pocket. It is not necessary that the mutation position
reside in the binding pocket as amino acid residues outside the
pocket itself can alter the pocket charge or configuration. The
selection of target sites for mutation is achieved using molecular
models as described above. For example according to the model
above, arginine at position 128 (designated R128 in FIG. 1 using
the single-letter code for amino acids) is located near the
entrance to the substrate- and herbicide-binding pocket and has a
large degree of conformational freedom that may allow it to
participate in transport of charged herbicides into the binding
pocket. Therefore, this residue is substituted by alanine to remove
both its charge and its long hydrophobic side chain. (The resulting
mutation is designated R128A).
[0122] The mutations may comprise simple substitutions, which
replace the wild-type sequence with any other amino acid.
Alternatively, the mutations may comprise deletions or additions of
one or more amino acids, preferably up to 5, at a given site. The
added sequence may comprise an amino acid sequence known to exist
in another protein, or may comprise a completely synthetic
sequence. Furthermore, more than one mutation and/or more than one
type of mutation may be introduced into a single polypeptide.
[0123] 4. Site-Directed Mutagenesis
[0124] The DNA encoding AHAS can be manipulated so as to introduce
the desired mutations. Mutagenesis is carried out using methods
that are standard in the art, as described in, for example,
Higuchi, R., Recombinant PCR, In M. A. Innis, et al., eds, PCR
Protocols: A Guide to Methods and Applications, Academic Press, pp.
177-183, 1990.
[0125] 5. Expression and Purification of Variants
[0126] The mutated or variant AHAS sequence is cloned into a DNA
expression vector (Bee, e.g., Example 3) and is expressed in a
suitable cell such as, for example, E. coli. Preferably, the DNA
encoding AHAS is linked to a transcription regulatory element, and
the variant AHAS is expressed as part of a fusion protein, for
example, glutathione-S-transferase, to facilitate purification (see
Example 3 below). The variant AHAS is then purified using affinity
chromatography or any other suitable method known in the art.
"Purification" of an AHAS polypeptide refers to the isolation of
the AHAS polypeptide in a form that allows its enzymatic activity
to be measured without interference by other components of the cell
in which the polypeptide is expressed.
[0127] 6. Assaying of Enzymatic Properties
[0128] The purified variant AHAS may be assayed for one or more of
the following three properties: [0129] (a) specific or catalytic
activity for conversion of pyruvate to acetolactate (expressed as
units/mg pure AHAS, wherein a unit of activity is defined as 1
.mu.mole acetolactate produced/hour), or for condensation of
pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (expressed
as units/mg pure AHAS, wherein a unit of activity is defined as 1
.mu.mole acetohydroxybutyrate produced/hr.; [0130] (b) level of
inhibition by herbicide, such as, for example, imidazolinone
(expressed as IC.sub.50, the concentration at which 50% of the
activity of the enzyme is inhibited); and [0131] (c) selectivity of
resistance to the selected herbicide vs. other herbicides. The
selectivity index is defined as the fold resistance of the mutant
to imidazolinones relative to the wild-type enzyme, divided by the
fold resistance of the same mutant to other herbicides also
relative to the wild-type). Fold resistance to an herbicide
relative to the wild-type enzyme is expressed as the IC.sub.50 of
variant, divided by the IC.sub.50 of the wild type. The selectivity
index (S.I.) is thus represented by the following equation: S . I .
= IC 50 .times. .times. of .times. .times. variant .times. .times.
for .times. .times. herb . A / IC 50 .times. .times. of .times.
.times. wild .times. .times. type .times. .times. for .times.
.times. herb . A IC 50 .times. .times. of .times. .times. variant
.times. .times. for .times. .times. herb . B / IC 50 .times.
.times. of .times. .times. wild .times. .times. type .times.
.times. for .times. .times. herb . B . ##EQU1##
[0132] S.I. IC.sub.50 of variant for herb.A/IC.sub.50 of wild type
for herb.A herb.B. IC.sub.50 of variant for herb.B/IC.sub.50 of
wild type for
[0133] Suitable assay systems for making these determinations
include, but are not limited to, those described in detail in
Example 4 below.
[0134] 7.a. Evaluation of Suitable Variants
[0135] The enzymatic properties of variant AHAS polypeptides are
compared to the wild-type AHAS. Preferably, a given mutation
results in an AHAS variant polypeptide that retains in vitro
enzymatic activity towards pyruvate or pyruvate and 2-ketobutyrate,
i.e., the conversion of pyruvate to acetolactate or in the
condensation of pyruvate and 2-ketobutyrate to form
acetohydroxybutyrate (and thus is expected to be biologically
active in vivo), while exhibiting catalytic activity that is
relatively more resistant to the selected herbicide(s) than is
wild-type AHAS. Preferably, the variant AHAS exhibits: [0136] (i)
in the absence of the at least one herbicide, [0137] (a) catalytic
activity alone sufficient to maintain the viability of a cell in
which it is expressed; or [0138] (b) catalytic activity in
combination with any herbicide resistant AHAS variant protein also
expressed in the cell, which may be the same as or different than
the first AHAS variant protein, sufficient to maintain the
viability of a cell in which it is expressed; [0139] wherein the
cell requires AHAS activity for viability; and [0140] (ii)
catalytic activity that is more resistant to the at least one
herbicide than is wild type AHAS;
[0141] and that is relatively more resistant to the herbicide(s)
than is wild-type AHAS.
[0142] Therefore, any one specific AHAS variant protein need not
have the total catalytic activity necessary to maintain the
viability of the cell, but must have some catalytic activity in an
amount, alone or in combination with the catalytic activity of
additional copies of the same AHAS variant and/or the catalytic
activity of other AHAS variant protein(s), sufficient to maintain
the viability of a cell that requires AHAS activity for viability.
For example, catalytic activity may be increased to minimum
acceptable levels by introducing multiple copies of a variant
encoding gene into the cell or by introducing the gene which
further includes a relatively strong promoter to enhance the
production of the variant.
[0143] More resistant means that the catalytic activity of the
variant is diminished by the herbicide(s), if at all, to a lesser
degree than wild-type AHAS catalytic activity is diminished by the
herbicide(s). Preferred more resistant variant AHAS retains
sufficient catalytic to maintain the viability of a cell, plant, or
organism wherein at the same concentration of the same
herbicide(s), wild-type AHAS would not retain sufficient catalytic
activity to maintain the viability of the cell, plant, or
organism.
[0144] Preferably the catalytic activity in the absence of
herbicide(s) is at least about 5% and, most preferably, is more
than about 20% of the catalytic activity of the wild-type AHAS in
the absence of herbicide(s). Most preferred AHAS variants are more
resistant to imidazolinone herbicides than to other herbicides such
as sulfonylurea-based herbicides, though in some applications
selectivity is neither needed nor preferred.
[0145] In the case of imidazolinone-resistant variant AHAS, it is
preferred that the AHAS variant protein has
[0146] (i) catalytic activity in the absence of said herbicide of
more than about 20% of the catalytic activity of said wild-type
AHAS;
[0147] (ii) catalytic activity that is relatively more resistant to
presence of imidazolinone herbicides compared to wild type AHAS;
and
[0148] (iii) catalytic activity that is relatively more sensitive
to the presence of sulfonylurea herbicides compared to
imidazolinone herbicides. Most preferred herbicide-resistant AHAS
variants exhibit a minimum specific activity of about 20 units/mg,
minimal or no inhibition by imidazolinone, and a selectivity index
ranging from about 1.3 to about 3000 relative to other
herbicides.
[0149] Without wishing to be bound by theory, it is believed that
systematic and iterative application of this method to wild type or
other target AHAS protein will result in the production of AHAS
variants having the desired properties of high enzymatic activity
as explained above and resistance to one or more classes of
herbicides. For example, mutation of a wild-type AHAS sequence at a
particular position to a given amino acid may result in a mutant
that exhibits a high degree of herbicide resistance but a
significant loss of enzymatic activity towards pyruvate or pyruvate
and 2-ketobutyrate. In a second application of the above method,
the starting or target AHAS polypeptide would then be this variant
(in place of the wild-type AHAS). Rational design then involves
substituting other amino acids at the originally mutated position
and/or adding or deleting amino acids at selected points or ranges
in the expectation of retaining herbicide resistance but also
maintaining a higher level of enzymatic activity.
[0150] The structure-based rational design of herbicide resistant
AHAS proteins offers many advantages over conventional approaches
that rely on random mutagenesis and selection. For example, when
substitution of a particular amino acid with another requires
substitution of more than one nucleotide within the codon, the
likelihood of this occurring randomly is so low as to be
impractical. By contrast, even double or triple changes in
nucleotide sequence within a codon can be easily implemented when
suggested by a rational design approach. For example, one
rationally designed mutation to confer selective imidazolinone
resistance requires a change from arginine to glutamate. Arginine
is encoded by CGT, CGC, CGA, CGG, AGA, AGG, while glutamate is
encoded by GAA and GAG. Since none of the arginine codons begins
with GA, this mutation would require a double substitution of
adjacent nucleotides which would occur so rarely using random
mutagenesis as to be unpredictable and unrepeatable with any
certainty of success. Although mutation frequency can be increased
during random mutagenesis, alterations in nucleotide sequence would
have an equal probability of occurring throughout the AHAS gene, in
the absence of prior site-direction of the mutations. This
increases the chance of obtaining an irrelevant mutation that
interferes with enzymatic activity. Similarly, it would be rare,
using random mutagenesis, to find a multiple amino acid
substitution, deletion, or substitution/deletion mutation that
confers herbicide resistance while maintaining catalytic activity.
Deletion mutations that confer herbicide resistance would also be
unlikely using a random mutagenesis approach. Deletions would need
to be limited to small regions and would have to occur in triplets
so as to retain the AHAS reading frame in order to retain enzymatic
activity.
[0151] However, with a rational structure-based approach, double
amino acid substitution and/or deletion mutations are relatively
easily achieved and precisely targeted. Furthermore, different
mutagens used in random mutagenesis create specific types of
mutations. For example, sodium azide creates point substitution
mutations in plants, while radiation tends to create deletions.
Accordingly, two mutagenesis protocols would have to be employed to
obtain a multiple combination substitution/deletion.
[0152] Finally, the present structure-based method for rational
design of herbicide-resistant AHAS variants allows for iterative
improvement of herbicide resistance mutations, a step that is not
facilitated by random mutagenesis. Identification of a mutation
site for herbicide resistance by random mutagenesis may offer
little, if any, predictive value for guiding further improvements
in the characteristics of the mutant. The present structure-based
approach, on the other hand, allows improvements to be implemented
based on the position, environment, and function of the amino acid
position in the structural model.
[0153] The iterative improvement method also allows the independent
manipulation of three important properties of AHAS: level of
resistance, selectivity of resistance, and catalytic efficiency.
For example, compensatory mutations can be designed in a predictive
manner. If a particular mutation has a deleterious effect on the
activity of an enzyme, a second compensatory mutation may be used
to restore activity. For example, a change in the net charge within
a domain when a charged residue is introduced or lost due to a
mutation can be compensated by introducing a second mutation.
Prediction of the position and type of residue(s) to introduce,
delete, or substitute at the second site in order to restore
enzymatic activity requires a knowledge of structure-function
relationships derived from a model such as that described
herein.
[0154] 7.b. Design of Non-Peptide Herbicides or AHAS Inhibitors
[0155] A chemical entity that alters and may fit into the active
site of the target protein or bind in any position where it could
inhibit activity may be designed by methods known in the art, such
as, for example, computer design programs that assist in the design
of compounds that specifically interact with a receptor site.
[0156] An example of such a program is LUDI (Biosym
Technologies--San Diego, Calif.) (see also, Lam, et al., Science
263:380, 1994; Thompson, et al., J. Med. Chem., 37:3100, 1994).
[0157] The binding pocket and particularly the amino acid residues
that have been identified as being involved as inhibitor binding
can be used as anchor points for inhibitor design.
[0158] The design of site-specific herbicides is advantageous in
the control of weed species that may spontaneously develop
herbicide resistance in the field, particularly due to mutations in
the AHAS gene.
Herbicide-Resistant AHAS Variants: DNA, Vectors, and
Polypeptides
[0159] The present invention also encompasses isolated DNA
molecules encoding variant herbicide-resistant AHAS polypeptides.
Genes encoding AHAS polypeptides according to the present invention
may be derived from any species and preferably a plant species, and
mutations conferring herbicide resistance may be introduced at
equivalent positions within any of these AHAS genes. The
equivalence of a given codon position in different AHAS genes is a
function of both the conservation of primary amino acid sequence
and its protein and the retention of similar three-dimensional
structure. For example, FIG. 5 illustrates the high degree of
sequence homology between AHAS polypeptides derived from different
plant species. These AHAS polypeptides exhibit at least about 60 to
about 70% overall homology. Without wishing to be bound by theory,
it is believed that in regions of the polypeptide having a highly
conserved sequence, the polypeptide chain conformation will also be
preserved. Thus, it is possible to use an AHAS-encoding sequence
from one species for molecular modelling, to introduce mutations
predictively into an AHAS gene from a second species for initial
testing and iterative improvement, and finally, to introduce the
optimized mutations into AHAS derived from yet a third plant
species for expression in a transgenic plant.
[0160] In one series of embodiment, these AHAS DNAs encode variants
of an AHAS polypeptide and preferably of the maize AHAS polypeptide
of FIG. 1 in which the polypeptide is modified by substitution at
or deletion preceding or following one or more of FIG. 1 amino acid
residues P48, G49, S52, M53, E54, A84, A95, T96, S97, G98, P99,
G100, A101, V125, R127, R128, M129, I130, G131, T132, D133, F135,
Q136, D186, I187, T259, T260, L261, M262, G263, R276, M277, L278,
G279, H281, G282, T283, V284, G300, V301, R302, F303, D304, R306,
V307, T308, G309, K310, I311, E312, A313, F314, A315, S316, R317,
A318, K319, I320, E329, 1330, K332, N333, K334, Q335, T404, G413,
V414, G415, Q416, H417, Q418, M419, W420, A421, A422, L434, S435,
S436, A437, G438, L439, G440, A441, M442, G443, D467, G468, S469,
L471, N473, L477, M479, Q495, H496, L497, G498, M499, V501, Q502,
Q504, D505S, R506, Y508, K509, A510, N511, R512, A513, H514, T515,
S524, H572, Q573, E574, H575, V576, L577, P578, M579, I580, P581,
G583, G584, functional equivalents of any of the foregoing;
insertions or deletions between FIG. 1 Q124 and H150 or functional
equivalents thereof; insertions or deletions between FIG. 1 G300
and D324 or functional equivalents thereof; and any combination of
any of the foregoing thereof.
[0161] The mutations, whether introduced into the polypeptide of
FIG. 1 or at equivalent positions in another plant AHAS gene, may
comprise alterations in DNA sequence that result in a simple
substitution of any one or more other amino acids or deletions of
up to 5 amino acid residues proceeding or up to 5 amino acids
residues following any of the residence listed above. Suitable
amino acid substituents include, but are not limited to, naturally
occurring amino acids.
[0162] Alternatively, the mutations may comprise alterations in DNA
sequence such that one or more amino acids are added or deleted in
frame at the above positions. Preferably, additions comprise about
3 to about 30 nucleotides, and deletions comprise about 3 to about
30 nucleotides. Furthermore, a single mutant polypeptide may
contain more than one similar or different mutation.
[0163] The present invention encompasses DNA and corresponding RNA
sequences, as well as sense and antisense sequences. Nucleic acid
sequences encoding AHAS polypeptides may be flanked by natural AHAS
regulatory sequences, or may be associated with heterologous
sequences, including promoters, enhancers, response elements,
signal sequences, polyadenylation sequences, introns, 5'- and
3'-noncoding regions, and the like. Furthermore, the nucleic acids
can be modified to alter stability, solubility, binding affinity
and specificity. For example, variant AHAS-encoding sequences can
be selectively methylated. The nucleic acid sequences of the
present invention may also be modified with a label capable of
providing a detectable signal, either directly or indirectly.
Exemplary labels include radioisotopes, fluorescent molecules,
biotin, and the like.
[0164] The invention also provides vectors comprising nucleic acids
encoding AHAS variants. A large number of vectors, including
plasmid and fungal vectors, have been described for expression in a
variety of eukaryotic and prokaryotic hosts. Advantageously,
vectors may also include a promotor operably linked to the AHAS
encoding portion. The encoded AHAS may be expressed by using any
suitable vectors and host cells, using methods disclosed or cited
herein or otherwise known to those skilled in the relevant art.
Examples of suitable vectors include without limitation pBIN-based
vectors, pBluescript vectors, and pGEM vectors.
[0165] The present invention also encompasses both variant
herbicide-resistant AHAS polypeptides or peptide fragments thereof.
As explained above, the variant AHAS polypeptides may be derived
from the maize polypeptide shown in FIG. 1 or from any plant or
microbial AHAS polypeptide, preferably plant AHAS polypeptide. The
polypeptides may be further modified by, for example,
phosphorylation, sulfation, acylation, glycosylation, or other
protein modifications. The polypeptides may be isolated from
plants, or from heterologous organisms or cells (including, but not
limited to, bacteria, yeast, insect, plant, and mammalian cells)
into which the gene encoding a variant AHAS polypeptide has been
introduced and expressed. Furthermore, AHAS polypeptides may be
modified with a label capable of providing a detectable signal,
either directly or indirectly, including radioisotopes, fluorescent
compounds, and the like.
Chemical-Resistant Plants and Plants Containing Variant AHAS
Genes
[0166] The present invention encompasses transgenic cells,
including, but not limited to seeds, organisms, and plants into
which genes encoding herbicide-resistant AHAS variants have been
introduced. Non-limiting examples of suitable recipient plants are
listed in Table 1 below: TABLE-US-00001 TABLE 1 RECIPIENT PLANTS
COMMON NAME FAMILY LATIN NAME Maize Gramineae Zea mays Maize, Dent
Gramineae Zea mays dentiformis Maize, Flint Gramineae Zea mays
vulgaris Maize, Pop Gramineae Zea mays microsperma Maize, Soft
Gramineae Zea mays amylacea Maize, Sweet Gramineae Zea mays
amyleasaccharata Maize, Sweet Gramineae Zea mays saccharate Maize,
Waxy Gramineae Zea mays ceratina Wheat, Dinkel Pooideae Triticum
spelta Wheat, Durum Pooideae Triticum durum Wheat, English Pooideae
Triticum turgidum Wheat, Large Spelt Pooideae Triticum spelta
Wheat, Polish Pooideae Triticum polonium Wheat, Poulard Pooideae
Triticum turgidum Wheat, Singlegrained Pooideae Triticum monococcum
Wheat, Small Spelt Pooideae Triticum monococcum Wheat, Soft
Pooideae Triticum aestivum Rice Gramineae Oryza sativa Rice,
American Wild Gramineae Zizania aguatica Rice, Australian Gramineae
Oryza australiensis Rice, Indian Gramineae Zizania aguatica Rice,
Red Gramineae Oryza glaberrima Rice, Tuscarora Gramineae Zizania
aguatica Rice, West African Gramineae Oryza glaberrima Barley
Pooideae Hordeum vulgare Barley, Abyssinian Pooideae Hordeum
irregulare Intermediate, also Irregular Barley, Ancestral Pooideae
Hordeum spontaneum Tworow Barley. Beardless Pooideae Hordeum
trifurcatum Barley, Egyptian Pooideae Hordeum trifurcatum Barley,
fourrowed Pooideae Hordeum vulgare polystichon Barley, sixrowed
Pooideae Hordeum vulgare hexastichon Barley, Tworowed Pooideae
Hordeum distichon Cotton, Abroma Dicotyledoneae Abroma augusta
Cotton, American Malvaceae Gossypium hirsutum Upland Cotton,
Asiatic Malvaceae Gossypium arboreum Tree, also Indian Tree Cotton,
Brazilian, Malvaceae Gossypium barbadense also, Kidney, and,
brasiliense Pernambuco Cotton, Levant Malvaceae Gossypium herbaceum
Cotton, Long Silk, Malvaceae Gossypium barbadense also Long Staple,
Sea Island Cotton, Mexican, Malvaceae Gossypium hirsutum also Short
Staple Soybean, Soya Leguminosae Glycine max Sugar beet
Chenopodiaceae Beta vulgaris altissima Sugar cane Woody-plant
Arenga pinnata Tomato Solanaceae Lycopersicon esculentum Tomato,
Cherry Solanaceae Lycopersicon esculentum cerasiforme Tomato,
Common Solanaceae Lycopersicon esculentum commune Tomato, Currant
Solanaceae Lycopersicon pimpinellifolium Tomato, Husk Solanaceae
Physalis ixocarpa Tomato, Hyenas Solanaceae Solanum incanum Tomato,
Pear Solanaceae Lycopersicon esculentum pyriforme Tomato, Tree
Solanaceae Cyphomandra betacea Potato Solanaceae Solanum tuberosum
Potato, Spanish, Convolvulaceae Ipomoea batatas Sweet potato Rye,
Common Pooideae Secale cereale Rye, Mountain Pooideae Secale
montanum Pepper, Bell Solanaceae Capsicum annuum grossum Pepper,
Bird, also Solanaceae Capsicum annuum minimum Cayenne, Guinea
Pepper, Bonnet Solanaceae Capsicum sinense Pepper, Bullnose,
Solanaceae Capsicum annuum grossum also Sweet Pepper, Cherry
Solanaceae Capsicum annuum cerasiforme Pepper, Cluster, Solanaceae
Capsicum annuum also Red Cluster fasciculatum Pepper, Cone
Solanaceae Capsicum annuum conoides Pepper, Goat, also Solanaceae
Capsicum frutescens Spur Pepper, Long Solanaceae Capsicum
frutescens longum Pepper, Oranamental Solanaceae Capsicum annuum
Red, also Wrinkled abbreviatum Pepper, Tabasco Red Solanaceae
Capsicum annuum conoides Lettuce, Garden Compositae Lactuca sativa
Lettuce, Asparagus, Compositae Lactuca sativa also Celery
asparagina Lettuce, Blue Compositae Lactuca perennis Lettuce, Blue,
also Compositae Lactuca pulchella Chicory Lettuce, Cabbage,
Compositae Lactuca sativa capitata also Head Lettuce, Cos, also
Compositae Lactuca sativa longifolia Longleaf, Romaine Lettuce,
Crinkle, Compositae Lactuca sativa crispa also Curled, Cutting,
Leaf Celery Umbelliferae Apium graveolens dulce Celery, Blanching,
Umbelliferae Apium graveolens dulce also Garden Celery, Root, also
Umbelliferae Apium graveolens Turniprooted rapaceum Eggplant,
Garden Solanaceae Solanum melongena Sorghum Sorghum All crop
species Alfalfa Leguminosae Medicago sativum Carrot Umbelliferae
Daucus carota sativa Bean, Climbing Leguminosae Phaseolus vulgaris
vulgaris Bean, Sprouts Leguminosae Phaseolus aureus Bean, Brazilian
Leguminosae Canavalia ensiformis Broad Bean, Broad Leguminosae
Vicia faba Bean, Common, also Leguminosae Phaseolus vulgaris
French, White, Kidney Bean, Egyptian Leguminosae Dolichos lablab
Bean, Long, also Leguminosae Vigna sesquipedalis Yardlong Bean,
Winged Leguminosae Psophocarpus tetragonolobus Oat, also Common,
Avena Sativa Side, Tree Oat, Black, also Avena Strigosa Bristle,
Lopsided Oat, Bristle Avena Pea, also Garden, Leguminosae Pisum,
sativum sativum Green, Shelling Pea, Blackeyed Leguminosae Vigna
sinensis Pea, Edible Podded Leguminosae Pisum sativum axiphium Pea,
Grey Leguminosae Pisum sativum speciosum Pea, Winged Leguminosae
Tetragonolobus purpureus Pea, Wrinkled Leguminosae Pisum sativum
medullare Sunflower Compositae Helianthus annuus Squash, Autumn,
Dicotyledoneae Cucurbita maxima Winter Squash, Bush, also
Dicotyledoneae Cucurbita pepo melopepo Summer Squash, Turban
Dicotyledoneae Cucurbita maxima turbaniformis Cucumber
Dicotyledoneae Cucumis sativus Cucumber, African, Momordica
charantia also Bitter Cucumber, Squirting, Ecballium elaterium also
Wild Cucumber, Wild Cucumis anguria Poplar, California Woody-Plant
Populus trichocarpa Poplar, European Populus nigra Black Poplar,
Gray Populus canescens Poplar, Lombardy Populus italica Poplar,
Silverleaf, Populus alba also White Poplar, Western Populus
trichocarpa Balsam Tobacco Solanaceae Nicotiana Arabidopsis
Cruciferae Arabidopsis thaliana Thaliana Turfgrass Lolium Turfgrass
Agrostis Other families of turfgrass Clover Leguminosae
[0167] Expression of the variant AHAS polypeptides in transgenic
plants confers a high level of resistance to herbicides including,
but not limited to, imidazolinone herbicides such as, for example,
imazethapyr (PURSUIT.RTM.), allowing the use of these herbicides
during cultivation of the transgenic plants.
[0168] Methods for the introduction of foreign genes into plants
are known in the art. Non-limiting examples of such methods include
Agrobacterium infection, particle bombardment, polyethylene glycol
(PEG) treatment of protoplasts, electroporation of protoplasts,
microinjection, macroinjection, tiller injection, pollen tube
pathway, dry seed imbibition, laser perforation, and
electrophoresis. These methods are described in, for example, B.
Jenes et al., and S. W. Ritchie et al. In Transgenic Plants, Vol.
1, Engineering and Utilization, ed. S.-D. Kung, R. Wu, Academic
Press, Inc., Harcourt Brace Jovanovich 1993; and L. Mannonen et
al., Critical Reviews in Biotechnology, 14:287-310, 1994.
[0169] In a preferred embodiment, the DNA encoding a variant AHAS
is cloned into a DNA vector containing an antibiotic resistance
marker gene, and the recombinant AHAS DNA-containing plasmid is
introduced into Agrobacterium tumefaciens containing a Ti plasmid.
This "binary vector system" is described in, for example, U.S. Pat.
No. 4,490,838, and in An et al., Plant Mol. Biol. Manual A3:1-19
(1988). The transformed Agrobacterium is then co-cultivated with
leaf disks from the recipient plant to allow infection and
transformation of plant cells. Transformed plant cells are then
cultivated in regeneration medium, which promotes the formation of
shoots, first in the presence of the appropriate antibiotic to
select for transformed cells, then in the presence of herbicide. In
plant cells successfully transformed with DNA encoding
herbicide-resistant AHAS, shoot formation occurs even in the
presence of levels of herbicide that inhibit shoot formation from
non-transformed cells. After confirming the presence of variant
AHAS DNA using, for example, polymerase chain reaction (PCR)
analysis, transformed plants are tested for their ability to
withstand herbicide spraying and for their capabilities for seed
germination and root initiation and proliferation in the presence
of herbicide.
Other Applications
[0170] The methods and compositions of the present invention can be
used in the structure-based rational design of herbicide-resistant
ALAS variants, which can be incorporated into plants to confer
selective herbicide resistance on the plants. Intermediate variants
of ALAS (for example, variants that exhibit sub-optimal specific
activity but high resistance and selectivity, or the converse) are
useful as templates for the design of second-generation ALAS
variants that retain adequate specific activity and high resistance
and selectivity.
[0171] Herbicide resistant AHAS genes can be transformed into crop
species in single or multiple copies to confer herbicide
resistance. Genetic engineering of crop species with reduced
sensitivity to herbicides can:
[0172] (1) Increase the spectrum and flexibility of application of
specific effective and environmentally benign herbicides such as
imidazolinone herbicides;
[0173] (2) Enhance the commercial value of these herbicides;
[0174] (3) Reduce weed pressure in crop fields by effective use of
herbicides on herbicide resistant crop species and a corresponding
increase in harvest yields;
[0175] (4) Increase sales of seed for herbicide-resistant
plants;
[0176] (5) Increase resistance to crop damage from carry-over of
herbicides applied in a previous planting;
[0177] (6) Decrease susceptibility to changes in herbicide
characteristics due to adverse climate conditions; and
[0178] (7) Increase tolerance to unevenly or mis-applied
herbicides.
[0179] For example, transgenic AHAS variant protein containing
plants can be cultivated. The crop can be treated with a weed
controlling effective amount of the herbicide to which the AHAS
variant transgenic plant is resistant, resulting in weed control in
the crop without detrimentally affecting the cultivated crop.
[0180] The DNA vectors described above that encode
herbicide-resistant AHAS variants can be further utilized so that
expression of the AHAS variant provides a selectable marker for
transformation of cells by the vector. The intended recipient cells
may be in culture or in situ, and the AHAS variant genes may be
used alone or in combination with other selectable markers. The
only requirement is that the recipient cell is sensitive to the
cytotoxic effects of the cognate herbicide. This embodiment takes
advantage of the relative low cost and lack of toxicity of, for
example, imidazolinone-based herbicides, and may be applied in any
system that requires DNA-mediated transformation.
EXEMPLIFICATION WITH RESPECT TO PREFERRED EMBODIMENTS
[0181] The following examples are intended to illustrate the
present invention without limitation.
Example 1
Design of Herbicide-Resistant AHAS Variants
[0182] Residues located close to the proposed herbicide binding
site of the model described in detail above were selected for
mutagenesis in order to design an active AHAS polypeptide with
decreased herbicide binding capacity. Each site at the surface of
the pocket was considered in terms of potential interactions with
other residues in the pocket, as well as with cofactors and
herbicides. For example, addition of positively charged residue(s)
is expected to interfere with the charge distribution within the
binding site, resulting in a loss in affinity of binding of a
negatively-charged herbicide.
[0183] Three residues were identified as most useful targets for
mutagenesis:
[0184] (1) F135 was believed to interact with both the
isoalloxazine ring of FAD and with the aromatic group of the
herbicides. In accordance with the strategy of introducing more
charged residues into the binding pocket, this residue was changed
to arginine.
[0185] (2) M53 contacts helix 498-507. This helix contains known
herbicide resistance mutation sites and is also implicated in TPP
binding. Furthermore, substitution of glutamic acid at position 53
was believed to favor an interaction with K185, reducing the
affinity of K185 for the carboxylate group of imazethapyr.
[0186] (3) R128 is located near the entrance to the pocket, where
it was believed to be involved in the initial transport of charged
herbicides into the binding pocket. This residue was changed to
alanine to remove both its charge and its long hydrophobic side
chain.
Example 2
Site-Directed Mutagenesis of AHAS to Produce Herbicide-Resistant
Variants
[0187] The Arabidopsis AHAS gene was inserted in-frame to the 3'
end of the coding region of the glutathione S-transferase gene in
the pGEX-2T vector (Pharmacia). Construction of the vector in this
manner maintained the six amino acid thrombin recognition sequence
at the junction of the expressed glutathione-S-transferase
(GST)/AHAS fusion protein. Thrombin digestion of the expressed
fusion protein results in an AHAS protein with an N-terminal
starting position at the end of the transit peptide at a putative
transit peptide processing site, with a residual N-terminal glycine
derived from the thrombin recognition site. The final amino
terminus of the cleaved AHAS protein consists of
Gly-Ser-Ser-Ile-Ser. Site-directed mutations were introduced into
the AHAS gene in this vector.
[0188] Site-directed mutations were constructed according to the
PCR method of Higuchi (Recombinant PCR. In M A Innis, et al. PCR
Protocols: A Guide to Methods and Applications, Academic Press, San
Diego, pp. 177-183, 1990). Two PCR products, each of which overlap
the mutation site, were amplified. The primers in the overlap
region contained the mutation. The overlapping PCR amplified
fragments were combined, denatured, and allowed to re-anneal
together, producing two possible heteroduplex products with
recessed 3'-ends. The recessed 3'-ends were extended by Taq DNA
polymerase to produce a fragment that was the sum of the two
overlapping PCR products containing the desired mutation. A
subsequent re-amplification of this fragment with only the two
"outside" primers resulted in the enrichment of the full-length
product. The product containing the mutation was then re-introduced
into the Arabidopsis AHAS gene in the pGEX-2T vector.
Example 3
Expression and Purification of AHAS Variants
[0189] A. Methods
[0190] E. coli (DH5.alpha.) cells transformed with the pGEX-2T
vector containing either the maize wild type AHAS gene (vector
designation pAC751), the Arabidopsis Ser653Asn mutant, or the
Arabidopsis Ile401Phe mutant were grown overnight in LB broth
containing 50 .mu.g/mL ampicillin. The overnight culture of E. coli
was diluted 1:10 in 1 L LB, 50 .mu.g/mL ampicillin, and 0.1% v/v
antifoam A. The culture was incubated at 37.degree. C. with shaking
until the OD.sub.600 reached approximately 0.8.
Isopropylthiogalactose (IPTG) was added to a final concentration of
1 mM and the culture was incubated for 3 more hours.
[0191] Cells were harvested by centrifugation at 8,670.times.g for
10 minutes in a JA-10 rotor and resuspended in 1/100th of the
original culture volume in MTPBS (16 mM Na.sub.2HPO.sub.4, 4 mM
NaPO.sub.4, 150 mM NaCl, pH 7.3). Triton X-100 and lysozyme were
added to a final concentration of 1% v/v and 100 .mu.g/mL,
respectively. Cells were incubated at 30.degree. C. for 15 minutes
cooled to 4.degree. C. on ice, and were lysed by sonication for 10
seconds at level 7 with a Branson Sonifier Cell Disrupter equipped
with a microtip probe. The cell free extract was centrifuged at
35,000.times.g for 10 min. at 4.degree. C. The supernatant was
decanted and the centrifugation step was repeated.
[0192] Purification of expressed fusion proteins was performed as
modified from Smith and Johnson (Gene 67:31-40, 1988). The
supernatant was warmed to room temperature and was passed through a
2 mL column of glutathione-agarose beads (sulfur linkage, Sigma)
equilibrated in MTPBS. The column was subsequently washed with
MTPBS at room temperature until the A.sub.200 of eluant matched
that of MTPBS. The fusion protein was then eluted using a solution
containing 5 mM reduced glutathione in 50 mM Tris HCL, pH 8.0. The
eluted fusion protein was treated with approximately 30 NIH units
of thrombin and dialyzed against 50 mM citrate pH 6.5 and 150 mM
NaCl.
[0193] The fusion protein was digested overnight at room
temperature. Digested samples were dialyzed against MTPBS and
passed twice through a glutathione-agarose column equilibrated in
MTPBS to remove the released glutathione transferase protein. The
protein fraction that did not bind to the column was collected and
was concentrated by ultrafiltration on a YM10 filter (Amicon). The
concentrated sample was loaded onto a 1.5.times.95 cm Sephacryl
S-100 gel filtration column equilibrated in gel filtration buffer
(50 mM HEPES, 150 mM NaCl, pH 7.0). Two mL fractions were collected
at a flow rate of 0.14 mL/min. Enzyme stability was tested by
storage of the enzyme at 4.degree. C. in gel filtration buffer with
the addition of 0.02% sodium azide and in the presence or absence
of 2 mM thiamine pyrophosphate and 100 .mu.M flavin adenine
dinucleotide (FAD).
[0194] B. Results
[0195] E. coli transformed with the pAC751 plasmid containing the
wide-type AHAS gene fused downstream and in-frame with the GST gene
expressed a 91 kD protein when induced with IPTG. The 91 kD protein
exhibited the predicted molecular mass of a GST/AHAS fusion protein
(the sum of 26 kD and 65 kD, respectively). When the cell free
extract of DH5.alpha./pAC751 was passed through a
glutathione-agarose affinity gel, washed, and eluted with free
glutathione it yielded a preparation enriched in the 91 kD protein
(FIG. 6, lane C). The six amino acid thrombin recognition site
engineered in the junction of GST and AHAS was successfully cleaved
by thrombin (FIG. 6, lane D). The cleaved fusion protein
preparation consisted of the expected 26 kD GST protein and the 65
kD maize AWLS protein. Maize ALAS was purified to homogeneity by a
second pass through the glutathione-agarose column to affinity
subtract GST and subjected to a final Sephacryl S-100 gel
filtration step to eliminated thrombin (FIG. 6, lane E). The 65 kD
protein is recognized on western blots by a monoclonal antibody
raised against a maize AHAS peptide.
[0196] Purified wild type maize AHAS was analyzed by electrospray
mass spectrometry and was determined to have a molecular mass of
64,996 daltons (data not shown). The predicted mass, as calculated
from the deduced amino acid sequence of the gene inserted into the
pGEX-2T vector, is 65,058. The 0.096% discrepancy between the
empirically determined and predicted mass was within tuning
variability of the mass spectrometer. The close proximity of the
two mass determinations suggests that there were no misincorporated
nucleotides during construction of the expression vector, nor any
post-translational modifications to the protein that would cause
gross changes in molecular mass. Moreover, the lack of spurious
peaks in the preparation of purified enzyme indicated that the
sample was free of contamination.
Example 4
Enzymatic Properties of AHAS Variants
[0197] The enzymatic properties of wild-type and variant AHAS
produced in E. coli were measured by a modification of the method
of Singh et al. (Anal. Biochem 171:173-179, 1988) as follows:
[0198] A reaction mixture containing 1.times.AHAS assay buffer (50
mM HEPES pH 7.0, 100 mM pyruvate, 10 mm MgCl.sub.2, 1 mM thiamine
pyrophosphate (TPP), and 50 .mu.M flavin adenine dinucleotide
(FAD)) was obtained either by dilution of enzyme in 2.times. assay
buffer or by addition of concentrated enzyme to 1.times.AHAS assay
buffer. All assays containing imazethapyr and associated controls
contained a final concentration of 5% DMSO due to addition of
imazethapyr to assay mixtures as a 50% DMSO solution. Assays were
performed in a final volume of 250 .mu.L at 37.degree. C. in
microtiter plates. After allowing the reaction to proceed for 60
minutes, acetolactate accumulation was measured colorimetrically as
described by Singh et al., Anal. Biochem 171:173-179, 1988.
[0199] Maize AHAS expressed and purified from pAC751 as described
in Example 3 above is active in the conversion of pyruvate to
acetolactate. Full AHAS activity is dependent on the presence of
the cofactors FAD and TPP in the assay medium. No activity was
detected when only FAD was added to the assay medium. The activity
of the purified enzyme with TPP only, or with no cofactors, was
less than 1% of the activity detected in the presence of both TPP
and FAD. Normally, AHAS present in crude plant extracts is very
labile, particularly in the absence of substrate and cofactors. In
contrast, the purified AHAS from the bacterial expression system
showed no loss in catalytic activity when stored for one month at
4.degree. C. in 50 mM HEPES pH 7.0, 150 mM NaCl, 0.02% NaN.sub.3 in
the presence or absence of FAD and TPP. Furthermore, no degradation
products were visible from these stored preparations when resolved
in SDS-PAGE gels.
[0200] The specific activities of wild-type AHAS and the M124E,
R199A, and F206R variants are shown in Table 2 below. As determined
from the alignment in FIG. 5, the M124E mutation in Arabidopsis
AHAS is the equivalent of the maize M53E mutation, the R199A
mutation in Arabidopsis is the equivalent of the maize R128A
mutation, and the F206R mutation in Arabidopsis is the equivalent
of the maize F135R mutation. The mutations designed in the maize
AHAS structural model were used to identify the equivalent amino
acid in the dicot Arabidopsis AHAS gene and were incorporated and
tested in the Arabidopsis AHAS gene. This translation and
incorporation of rationally designed herbicide mutations into the
dicot Arabidopsis AHAS gene can facilitate evaluation of herbicide
resistance in plants of a dicot species. TABLE-US-00002 TABLE 2
SPECIFIC ACTIVITY % Catalytic Activity as Specific Activity
Compared to Wild Type Wild-Type 99.8 100 Met124Glu 9.15 9.16
Arg199Ala 86.3 86.5 Phe206Arg 5.07 5.1
[0201] The R199A mutation maintains a high level of catalytic
activity (Table 2) while exhibiting a significant level of
resistance to imazethapyr (FIG. 7). Notably, this variant retains
complete sensitivity to sulfonylureas (FIG. 8). Thus, this variant
fulfills the criteria of high specific activity and selective
herbicide resistance. By contrast, the M124E substitution resulted
in almost complete resistance to imazethapyr (FIG. 7) but also
exhibited severely reduced catalytic activity (Table 2). Relative
to imidazolinone resistance, this variant exhibits greater
sensitivity to sulfonylurea (FIG. 8), suggesting that this residue
is a good candidate for creating a mutation that confers selective
resistance. Substitution of an amino acid other than glutamic acid
may help to maintain catalytic activity. The F206R substitution
yielded similar results to those observed with M124E variant, but
lacked selectivity in resistance.
Example 5
Iterative Improvement of AHAS Herbicide-Resistant Variant Using a
Rational Design Approach
[0202] Changing residue 124 in AHAS from Met to Glu as described in
Example 4 above conferred imidazolinone resistance but also reduced
enzymatic activity to 9.2% of the wild type value. The model of the
maize AHAS structure described above suggested that Met53
(equivalent to the Arabidopsis Met124 residue) interacts with a
series of hydrophobic residues on the face of an .alpha.-helix that
is derived from a separate subunit but are in close proximity to
Met53. Thus, the hydrophobic interaction between Met53 and the
residues on the helix may stabilize both subunit/subunit
association and the conformation of the active site. It was
believed that the substitution of the hydrophobic Met residue with
a charged glutamate residue most probably destabilizes the
inter-subunit hydrophobic interaction and results in a loss of
catalytic activity.
[0203] Based on this structure/function analysis, the activity of
the original Arabidopsis Met124Glu (equivalent to maize Met53Glu)
mutant enzyme was then iteratively improved by substituting a more
hydrophobic amino acid (Ile) at this position. The hydrophobic
nature of the Ile side chain resulted in restoration of activity to
wild type levels (specific activity of 102, equivalent to 102% of
the wild-type activity), but the greater bulk of the Ile side chain
was still able to maintain a significant level of imidazolinone
resistance (FIG. 9).
[0204] By comparison, substitution of a histidine residue at this
position resulting in an AHAS variant exhibiting a specific
activity of 42.5, equivalent to 42.6% of the wild-type activity.
This mutant, nonetheless, exhibited a high degree of resistance to
PURSUIT.RTM. (FIG. 10).
Example 6
Iterative Improvement of AHAS Herbicide-Resistant Variant Using a
Rational Design Approach
[0205] Another example of iterative refinement using the methods of
the present invention involves the Arg128Ala variant. The
structural model of maize AHAS suggested that the Arg128 residue,
which resides at the lip of the herbicide binding pocket,
contributes to channeling charged substrates and herbicides into
the herbicide binding pocket and into the active site. The Arg 128
residue is distant from the TPP moiety, which binds the initial
pyruvate molecule in the reaction mechanism of AHAS, explaining why
the substitution of Arabidopsis AHAS Arg199 (the equivalent to
maize Arg128) to alanine had little effect on the catalytic
activity of the enzyme. The structural model further indicated that
a more radical change could be made at this position to raise the
level of resistance while maintaining high levels of catalytic
activity. On this basis, an iterative improvement of the mutation
was made to substitute the positively charge arginine residue with
a negatively charged glutamate residue. The enzyme thus mutated had
improved levels of resistance to PURSUIT.RTM. while maintaining
high levels of activity (specific activity of 114, equivalent to
114% of the wild-type activity) (FIG. 11).
Example 7
Interchangeability of AHAS Derived From Different Species in
Structure-Based Rational Design of Herbicide-Resistant Variants
[0206] A structural model of the three-dimensional structure of
AHAS is built with a monocot AHAS sequence such as that derived
from maize, as described above. To introduce mutations into AHAS
derived from a dicot species such as Arabidopsis, the sequences of
AHAS derived from the monocot and dicot species are aligned using
the GAP and PILEUP programs (Genetics Computer Group, 575 Sequence
Drive, Madison, Wis. 53711). Equivalent positions are determined
from the computer-generated alignment. The mutations are then
introduced into the dicot AHAS gene as described above. Following
expression of the mutant AHAS protein in E. coli and assessment of
its biochemical properties (i.e., specific activity and resistance
to herbicides), the mutant gene is introduced into a dicot plant by
plant transformation methods as described above.
Example 8
Production of Herbicide-Resistant Plants by Transformation with
Rationally Designed AHAS Gene
DNA Constructs:
[0207] Rationally designed AHAS variant genes contained within E.
coli expression vectors were used as a source of DNA restriction
fragments to replace the equivalent restriction fragment in a
Arabidopsis AHAS gene. This gene is present in a 5.5 kb genomic DNA
fragment which also contains the Arabidopsis AHAS promoter, the
Arabidopsis AHAS termination sequence and 5'- and 3'-flanking DNA.
After DNA sequencing through the mutation sites was performed to
confirm the presence of the proper mutation, the entire 5.5 kb
fragment from each plasmid was inserted into a pBIN based plant
transformation vector (Mogen, Leiden, Netherlands). The plant
transformation vector also contains the neomycin phosphotransferase
II (nptII) kanamycin resistance gene driven by the 35S cauliflower
mosaic virus promoter. The final vector construct is displayed in
FIG. 12. Vectors containing Arabidopsis AHAS genes with Met124Ile,
Met124His, and Arg199Glu mutations (corresponding to Met53Ile,
Met53His, and Arg128Glu mutations in the maize AHAS sequence as
shown in FIG. 1) were labeled pJK002, pJK003, and pJT004,
respectively.
[0208] Each of these vectors was transformed into Agrobacterium
tumefaciens strain LBA4404 (R&D Life Technologies,
Gaithersburg, Md.) using the transformation method described in An
et al., Plant Mol. Biol. Manual A3:1-19 (1988).
Plant Transformation:
[0209] Leaf disc transformation of Nicotiana tabacum cv. Wisconsin
38 was performed as described by Horsch et al. (Science, 227:
1229-1231, 1985) with slight modifications. Leaf discs were cut
from plants grown under sterile conditions and co-cultivated
upsidedown in Murashige Skoog media (Sigma Chemical Co., St. Louis,
Mo.) for 2-3 days at 25.degree. C. in darkness with Agrobacterium
tumefaciens strains containing plasmids pJK002, pJK003, or pJK004.
The discs were blotted dry and transferred to regeneration
Murashige Skoog medium with B5 vitamins containing 1 mg/L
benzyladenine and 0.1 mg/l 1-Napthyl Acetic Acid, 100 mg/L
kanamycin, and 500 mg/L cefotaxime (all obtained from Sigma).
[0210] Initially, transformants were selected by kanamycin
resistance conferred by the nptII gene present in the
transformation vector. Shoots derived from the leaf discs were
excised and placed on fresh Murashige Skoog hormone free media
containing cefotaxime and kanamycin.
In Vivo Herbicide Resistance
[0211] Kanamycin-resistant tobacco shoots were transferred to
medium containing a 0.25 .mu.M imazethapyr. At this concentration
of the imidazolinone herbicide, non-transformed tobacco shoots
(containing endogenous wild-type AHAS) were not able to initiate
root formation. By contrast, root initiation and growth were
observed from tobacco shoots transformed with each of the mutant
AHAS genes. Roots developed from shoots transformed with the
Met124Ile and Arg199Glu mutant genes along with wild type are shown
in FIG. 1. Furthermore, plants transformed with the Met124Ile or
Arg199Glu mutant genes were resistant to spraying with twice the
field rate (100 g/ha) of imazethapyr (FIG. 13). The patterns of
root growth in transformed vs. non-transformed plants in the
presence of herbicide, as well as the behavior after herbicide
spraying suggest that expression of the rationally designed
herbicide resistance genes confers herbicide resistance in
vivo.
Detection of the Rationally Designed Genes in Herbicide Resistant
Tobacco
[0212] Genomic DNA was isolated from the AHAS-transformed tobacco
plants, and the presence of the Arabidopsis AHAS variant genes was
verified by PCR analysis. Differences between the nucleotide
sequences of the Arabidopsis AHAS gene and the two tobacco AHAS
genes were exploited to design PCR primers that amplify only the
Arabidopsis gene in a tobacco genomic DNA background. The
rationally designed herbicide resistance genes were detected, as
shown by amplification of a DNA fragment of the proper size, in a
majority of the herbicide resistant plants. No PCR signal was seen
from non-transformed tobacco plants.
Segregation of Transformed AHAS Genes:
[0213] To monitor segregation of rationally designed AHAS genes in
transformed plants, germination tests were performed. Seeds were
placed in hormone-free Murashige-Skoog medium containing up to 2.5
.mu.M PURSUIT.RTM. and 100 .mu.M kanamycin. The seedlings that
resulted were visually scored for resistance or susceptibility to
the herbicide.
[0214] Since tobacco plants are diploid, it would be expected that
the progeny of self-pollinated plants should segregate 3:1
resistant:susceptible, reflecting the existence of 1 seedling
homozygous for the resistant AHAS gene, 2 seedlings heterozygous
for the resistant AHAS gene, and 1 seedling lacking a resistant
AHAS gene.
[0215] The results indicate that resistant AHAS genes are
segregating in the expected 3:1 ratio, supporting the conclusion
that herbicide resistance is conferred by a single, dominant copy
of a rationally designed AHAS gene.
[0216] These results indicate that rational design of
herbicide-resistant AHAS genes can be used to produce plants that
exhibit herbicide resistant growth in vivo.
Example 9
Production of Plants Cross-Resistant to Different Herbicides by
Transformation with Rationally Designed AHAS Genes
[0217] Tobacco plants transformed with rationally designed AHAS
genes as described in Example 8 above were also tested for
cross-resistance to another herbicide, CL 299,263 (also known as
imazamox). Germination tests were performed on seeds harvested from
the primary transformants containing the Met124Ile, Met124His, and
Arg199Glu Arabidopsis AHAS variant genes, in the absence or
presence of 2.5 .mu.M CL 299,263 (FIG. 15). This concentration of
the herbicide causes severe stunting and bleaching of wild-type
tobacco plants. Tobacco plants transformed with the Met124His AHAS
gene showed the greatest level of resistance (FIG. 15). Arg199Glu
transformants showed an intermediate level of resistance, while
Met124Ile showed little resistance (FIG. 15).
[0218] All patents, applications, articles, publications, and test
methods mentioned above are hereby incorporated by reference.
[0219] Many variations of the present invention will suggest
themselves to those skilled in the art in light of the above
detailed description. Such obvious variations are within the full
intended scope of the appended claims.
Sequence CWU 1
1
9 1 599 PRT Zea Mays 1 Gly Ser Ala Ala Ser Pro Ala Met Pro Met Ala
Pro Pro Ala Thr Pro 1 5 10 15 Leu Arg Pro Trp Gly Pro Thr Asp Pro
Arg Lys Gly Ala Asp Ile Leu 20 25 30 Val Glu Ser Leu Glu Arg Cys
Gly Val Arg Asp Val Phe Ala Tyr Pro 35 40 45 Gly Gly Ala Ser Met
Glu Ile His Gln Ala Leu Thr Arg Ser Pro Val 50 55 60 Ile Ala Asn
His Leu Phe Arg His Glu Gln Gly Glu Ala Phe Ala Ala 65 70 75 80 Ser
Gly Tyr Ala Arg Ser Ser Gly Arg Val Gly Val Cys Ile Ala Thr 85 90
95 Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Ala Leu Ala Asp Ala Leu
100 105 110 Leu Asp Ser Val Pro Met Val Ala Ile Thr Gly Gln Val Pro
Arg Arg 115 120 125 Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile
Val Glu Val Thr 130 135 140 Arg Ser Ile Thr Lys His Asn Tyr Leu Val
Leu Asp Val Asp Asp Ile 145 150 155 160 Pro Arg Val Val Gln Glu Ala
Phe Phe Leu Ala Ser Ser Gly Arg Pro 165 170 175 Gly Pro Val Leu Val
Asp Ile Pro Lys Asp Ile Gln Gln Gln Met Ala 180 185 190 Val Pro Val
Trp Asp Lys Pro Met Ser Leu Pro Gly Tyr Ile Ala Arg 195 200 205 Leu
Pro Lys Pro Pro Ala Thr Glu Leu Leu Glu Gln Val Leu Arg Leu 210 215
220 Val Gly Glu Ser Arg Arg Pro Val Leu Tyr Val Gly Gly Gly Cys Ala
225 230 235 240 Arg Ser Gly Glu Glu Leu Arg Arg Phe Val Glu Leu Thr
Gly Ile Pro 245 250 255 Val Thr Thr Thr Leu Met Gly Leu Gly Asn Phe
Pro Ser Asp Asp Pro 260 265 270 Leu Ser Leu Arg Met Leu Gly Met His
Gly Thr Val Tyr Ala Asn Tyr 275 280 285 Ala Val Asp Lys Ala Asp Leu
Leu Leu Ala Leu Gly Val Arg Phe Asp 290 295 300 Asp Arg Val Thr Gly
Lys Ile Glu Ala Phe Ala Ser Arg Ala Lys Ile 305 310 315 320 Val His
Val Asp Ile Asp Pro Ala Glu Ile Gly Lys Asn Lys Gln Pro 325 330 335
His Val Ser Ile Cys Ala Asp Val Lys Leu Ala Leu Gln Gly Met Asn 340
345 350 Ala Leu Leu Glu Gly Ser Thr Ser Lys Lys Ser Phe Asp Phe Gly
Ser 355 360 365 Trp Asn Asp Glu Leu Asp Gln Gln Lys Arg Glu Phe Pro
Leu Gly Tyr 370 375 380 Lys Tyr Ser Asn Glu Glu Ile Gln Pro Gln Tyr
Ala Ile Gln Val Leu 385 390 395 400 Asp Glu Leu Thr Lys Gly Glu Ala
Ile Ile Gly Thr Gly Val Gly Gln 405 410 415 His Gln Met Trp Ala Ala
Gln Tyr Tyr Thr Tyr Lys Arg Pro Arg Gln 420 425 430 Trp Leu Ser Ser
Ala Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala 435 440 445 Ala Ala
Gly Ala Ser Val Ala Asn Pro Gly Val Thr Val Val Asp Ile 450 455 460
Asp Gly Asp Gly Ser Phe Leu Met Asn Val Gln Glu Leu Ala Met Ile 465
470 475 480 Arg Ile Glu Asn Leu Pro Val Lys Val Phe Val Leu Asn Asn
Gln His 485 490 495 Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr
Lys Ala Asn Arg 500 505 510 Ala His Thr Tyr Leu Gly Asn Pro Glu Asn
Glu Ser Glu Ile Tyr Pro 515 520 525 Asp Phe Val Thr Ile Ala Lys Gly
Phe Asn Ile Pro Ala Val Arg Val 530 535 540 Thr Lys Lys Asn Glu Val
Arg Ala Ala Ile Lys Lys Met Leu Glu Thr 545 550 555 560 Pro Gly Pro
Tyr Leu Leu Asp Ile Ile Val Pro His Gln Glu His Val 565 570 575 Leu
Pro Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Met Ile Leu Asp 580 585
590 Gly Asp Gly Arg Thr Val Tyr 595 2 585 PRT Lactobacillus
planarum 2 Thr Asn Ile Leu Ala Gly Ala Ala Val Ile Lys Val Leu Glu
Ala Trp 1 5 10 15 Gly Val Asp His Leu Tyr Gly Ile Pro Gly Gly Ser
Ile Asn Ser Ile 20 25 30 Met Asp Ala Leu Ser Ala Glu Arg Asp Arg
Ile His Tyr Ile Gln Val 35 40 45 Arg His Glu Glu Val Gly Ala Met
Ala Ala Ala Ala Asp Ala Lys Leu 50 55 60 Thr Gly Lys Ile Gly Val
Cys Phe Gly Ser Ala Gly Pro Gly Gly Thr 65 70 75 80 His Leu Met Asn
Gly Leu Tyr Asp Ala Arg Glu Asp His Val Pro Val 85 90 95 Leu Ala
Leu Ile Gly Gln Phe Gly Thr Thr Gly Met Asn Met Asp Thr 100 105 110
Phe Gln Glu Met Asn Glu Asn Pro Ile Tyr Ala Asp Val Ala Asp Tyr 115
120 125 Asn Val Thr Ala Val Asn Ala Ala Thr Leu Pro His Val Ile Asp
Glu 130 135 140 Ala Ile Arg Arg Ala Tyr Ala His Gln Gly Val Ala Val
Val Gln Ile 145 150 155 160 Pro Val Asp Leu Pro Trp Gln Gln Ile Ser
Ala Glu Asp Trp Tyr Ala 165 170 175 Ser Ala Asn Asn Tyr Gln Thr Pro
Leu Leu Pro Glu Pro Asp Val Gln 180 185 190 Ala Val Thr Arg Leu Thr
Gln Thr Leu Leu Ala Ala Glu Arg Pro Leu 195 200 205 Ile Tyr Tyr Gly
Ile Gly Ala Arg Lys Ala Gly Lys Glu Leu Glu Gln 210 215 220 Leu Ser
Lys Thr Leu Lys Ile Pro Leu Met Ser Thr Tyr Pro Ala Lys 225 230 235
240 Gly Ile Val Ala Asp Arg Tyr Pro Ala Tyr Leu Gly Ser Ala Asn Arg
245 250 255 Val Ala Gln Lys Pro Ala Asn Glu Ala Leu Ala Gln Ala Asp
Val Val 260 265 270 Leu Phe Val Gly Asn Asn Tyr Pro Phe Ala Glu Val
Ser Lys Ala Phe 275 280 285 Lys Asn Thr Arg Tyr Phe Leu Gln Ile Asp
Ile Asp Pro Ala Lys Leu 290 295 300 Gly Lys Arg His Lys Thr Asp Ile
Ala Val Leu Ala Asp Ala Gln Lys 305 310 315 320 Thr Leu Ala Ala Ile
Leu Ala Gln Val Ser Glu Arg Glu Ser Thr Pro 325 330 335 Trp Trp Gln
Ala Asn Leu Ala Asn Val Lys Asn Trp Arg Ala Tyr Leu 340 345 350 Ala
Ser Leu Glu Asp Lys Gln Glu Gly Pro Leu Gln Ala Tyr Gln Val 355 360
365 Leu Arg Ala Val Asn Lys Ile Ala Glu Pro Asp Ala Ile Tyr Ser Ile
370 375 380 Asp Val Gly Asp Ile Asn Leu Asn Ala Asn Arg His Leu Lys
Leu Thr 385 390 395 400 Pro Ser Asn Arg His Ile Thr Ser Asn Leu Phe
Ala Thr Met Gly Val 405 410 415 Gly Ile Pro Gly Ala Ile Ala Ala Lys
Leu Asn Tyr Pro Glu Arg Gln 420 425 430 Val Phe Asn Leu Ala Gly Asp
Gly Gly Ala Ser Met Thr Met Gln Asp 435 440 445 Leu Val Thr Gln Val
Gln Tyr His Leu Pro Val Ile Asn Val Val Phe 450 455 460 Thr Asn Cys
Gln Tyr Gly Phe Ile Lys Asp Glu Gln Glu Asp Thr Asn 465 470 475 480
Gln Asn Asp Phe Ile Gly Val Glu Phe Asn Asp Ile Asp Phe Ser Lys 485
490 495 Ile Ala Asp Gly Val His Met Gln Ala Phe Arg Val Asn Lys Ile
Glu 500 505 510 Gln Leu Pro Asp Val Phe Glu Gln Ala Lys Ala Ile Ala
Gln His Glu 515 520 525 Pro Val Leu Ile Asp Ala Val Ile Thr Gly Asp
Arg Pro Leu Pro Ala 530 535 540 Glu Lys Leu Arg Leu Asp Ser Ala Met
Ser Ser Ala Ala Asp Ile Glu 545 550 555 560 Ala Phe Lys Gln Arg Tyr
Glu Ala Gln Asp Leu Gln Pro Leu Ser Thr 565 570 575 Tyr Leu Lys Gln
Phe Gly Leu Asp Asp 580 585 3 638 PRT Zea Mays 3 Met Ala Thr Ala
Ala Ala Ala Ser Thr Ala Leu Thr Gly Ala Thr Thr 1 5 10 15 Ala Ala
Pro Lys Ala Arg Arg Arg Ala His Leu Leu Ala Thr Arg Arg 20 25 30
Ala Leu Ala Ala Pro Ile Arg Cys Ser Ala Ala Ser Pro Ala Met Pro 35
40 45 Met Ala Pro Pro Ala Thr Pro Leu Arg Pro Trp Gly Pro Thr Asp
Pro 50 55 60 Arg Lys Gly Ala Asp Ile Leu Val Glu Ser Leu Glu Arg
Cys Gly Val 65 70 75 80 Arg Asp Val Phe Ala Tyr Pro Gly Gly Ala Ser
Met Glu Ile His Gln 85 90 95 Ala Leu Thr Arg Ser Pro Val Ile Ala
Asn His Leu Phe Arg His Glu 100 105 110 Gln Gly Glu Ala Phe Ala Ala
Ser Gly Tyr Ala Arg Ser Ser Gly Arg 115 120 125 Val Gly Val Cys Ile
Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 130 135 140 Ser Ala Leu
Ala Asp Ala Leu Leu Asp Ser Val Pro Met Val Ala Ile 145 150 155 160
Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu 165
170 175 Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr
Leu 180 185 190 Val Leu Asp Val Asp Asp Ile Pro Arg Val Val Gln Glu
Ala Phe Phe 195 200 205 Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu
Val Asp Ile Pro Lys 210 215 220 Asp Ile Gln Gln Gln Met Ala Val Pro
Val Trp Asp Lys Pro Met Ser 225 230 235 240 Leu Pro Gly Tyr Ile Ala
Arg Leu Pro Lys Pro Pro Ala Thr Glu Leu 245 250 255 Leu Glu Gln Val
Leu Arg Leu Val Gly Glu Ser Arg Arg Pro Val Leu 260 265 270 Tyr Val
Gly Gly Gly Cys Ala Ala Ser Gly Glu Glu Leu Arg Arg Phe 275 280 285
Val Glu Leu Thr Gly Ile Pro Val Thr Thr Thr Leu Met Gly Leu Gly 290
295 300 Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu Arg Met Leu Gly Met
His 305 310 315 320 Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ala
Asp Leu Leu Leu 325 330 335 Ala Leu Gly Val Arg Phe Asp Asp Arg Val
Thr Gly Lys Ile Glu Ala 340 345 350 Phe Ala Ser Arg Ala Lys Ile Val
His Val Asp Ile Asp Pro Ala Glu 355 360 365 Ile Gly Lys Asn Lys Gln
Pro His Val Ser Ile Cys Ala Asp Val Lys 370 375 380 Leu Ala Leu Gln
Gly Met Asn Ala Leu Leu Glu Gly Ser Thr Ser Lys 385 390 395 400 Lys
Ser Phe Asp Phe Gly Ser Trp Asn Asp Glu Leu Asp Gln Gln Lys 405 410
415 Arg Glu Phe Pro Leu Gly Tyr Lys Thr Ser Asn Glu Glu Ile Gln Pro
420 425 430 Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr Lys Gly Glu
Ala Ile 435 440 445 Ile Gly Thr Gly Val Gly Gln His Gln Met Trp Ala
Ala Gln Tyr Tyr 450 455 460 Thr Tyr Lys Arg Pro Arg Gln Trp Leu Ser
Ser Ala Gly Leu Gly Ala 465 470 475 480 Met Gly Phe Gly Leu Pro Ala
Ala Ala Gly Ala Ser Val Ala Asn Pro 485 490 495 Gly Val Thr Val Val
Asp Ile Asp Gly Asp Gly Ser Phe Leu Met Asn 500 505 510 Val Gln Glu
Leu Ala Met Ile Arg Ile Glu Asn Leu Pro Val Lys Val 515 520 525 Phe
Val Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp Glu Asp 530 535
540 Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Glu
545 550 555 560 Asn Glu Ser Glu Ile Tyr Pro Asp Phe Val Thr Ile Ala
Lys Gly Phe 565 570 575 Asn Ile Pro Ala Val Arg Val Thr Lys Lys Asn
Glu Val Arg Ala Ala 580 585 590 Ile Lys Lys Met Leu Glu Thr Pro Gly
Pro Tyr Leu Leu Asp Ile Ile 595 600 605 Val Pro His Gln Glu His Val
Leu Pro Met Ile Pro Ser Gly Gly Ala 610 615 620 Phe Lys Asp Met Ile
Leu Asp Gly Asp Gly Arg Thr Val Tyr 625 630 635 4 638 PRT Zea Mays
4 Met Ala Thr Ala Ala Thr Ala Ala Ala Ala Leu Thr Gly Ala Thr Thr 1
5 10 15 Ala Thr Pro Lys Ser Arg Arg Arg Ala His His Leu Ala Thr Arg
Arg 20 25 30 Ala Leu Ala Ala Pro Ile Arg Cys Ser Ala Leu Ser Arg
Ala Thr Pro 35 40 45 Thr Ala Pro Pro Ala Thr Pro Leu Arg Pro Trp
Gly Pro Asn Glu Pro 50 55 60 Arg Lys Gly Ser Asp Ile Leu Val Glu
Ala Leu Glu Arg Cys Gly Val 65 70 75 80 Arg Asp Val Phe Ala Tyr Pro
Gly Gly Ala Ser Met Glu Ile His Gln 85 90 95 Ala Leu Thr Arg Ser
Pro Val Ile Ala Asn His Leu Phe Arg His Glu 100 105 110 Gln Gly Glu
Ala Phe Ala Ala Ser Ala Tyr Ala Arg Ser Ser Gly Arg 115 120 125 Val
Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 130 135
140 Ser Ala Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Met Val Ala Ile
145 150 155 160 Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala
Phe Gln Glu 165 170 175 Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr
Lys His Asn Tyr Leu 180 185 190 Val Leu Asp Val Asp Asp Ile Pro Arg
Val Val Gln Glu Ala Phe Phe 195 200 205 Leu Ala Ser Ser Gly Arg Pro
Gly Pro Val Leu Val Asp Ile Pro Lys 210 215 220 Asp Ile Gln Gln Gln
Met Ala Val Pro Ala Trp Asp Thr Pro Met Ser 225 230 235 240 Leu Pro
Gly Tyr Ile Ala Arg Leu Pro Lys Pro Pro Ala Thr Glu Phe 245 250 255
Leu Glu Gln Val Leu Arg Leu Val Gly Glu Ser Arg Arg Pro Val Leu 260
265 270 Tyr Val Gly Gly Gly Cys Ala Ala Ser Gly Glu Glu Leu Cys Arg
Phe 275 280 285 Val Glu Leu Thr Gly Ile Pro Val Thr Thr Thr Leu Met
Gly Leu Gly 290 295 300 Asn Phe Pro Ser Asp Asp Pro Leu Ser Leu Arg
Met Leu Gly Met His 305 310 315 320 Gly Thr Val Tyr Ala Asn Tyr Ala
Val Asp Lys Ala Asp Leu Leu Leu 325 330 335 Ala Phe Gly Val Arg Phe
Asp Asp Arg Val Thr Gly Lys Ile Glu Ala 340 345 350 Phe Ala Gly Arg
Ala Lys Ile Val His Ile Asp Ile Asp Pro Ala Glu 355 360 365 Ile Gly
Lys Asn Lys Gln Pro His Val Ser Ile Cys Ala Asp Val Lys 370 375 380
Leu Ala Leu Gln Gly Met Asn Thr Leu Leu Glu Gly Ser Thr Ser Lys 385
390 395 400 Lys Ser Phe Asp Phe Gly Ser Trp His Asp Glu Leu Asp Gln
Gln Lys 405 410 415 Arg Glu Phe Pro Leu Gly Tyr Lys Ile Phe Asn Glu
Glu Ile Gln Pro 420 425 430 Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu
Thr Lys Gly Glu Ala Ile 435 440 445 Ile Ala Thr Gly Val Gly Gln His
Gln Met Trp Ala Ala Gln Tyr Tyr 450 455 460 Thr Tyr Lys Arg Pro Arg
Gln Trp Leu Ser Ser Ala Gly Leu Gly Ala 465 470 475 480 Met Gly Phe
Gly Leu Pro Ala Ala Ala Gly Ala Ala Val Ala Asn Pro 485 490 495 Gly
Val Thr Val Val Asp Ile Asp Gly Asp Gly Ser Phe Leu Met Asn 500 505
510 Ile Gln Glu Leu Ala Met Ile Arg Ile Glu Asn Leu Pro Val Lys Val
515 520 525 Phe Val Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp
Glu Asp 530 535 540 Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu
Gly Asn Pro Glu 545 550 555 560 Asn Glu Ser Glu Ile Tyr Pro Asp Phe
Val Ala Ile Ala Lys Gly Phe 565 570 575 Asn Ile Pro Ala Val Arg Val
Thr Lys Lys Ser Glu Val His Ala Ala 580 585 590 Ile Lys Lys Met Leu
Glu Ala Pro Gly Pro Tyr Leu Leu Asp Ile Ile 595 600 605 Val Pro His
Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Ala 610
615 620 Phe Lys Asp Met Ile Leu Asp Gly Asp Gly Arg Thr Val Tyr 625
630 635 5 667 PRT Tobacco 5 Met Ala Ala Ala Ala Pro Ser Pro Ser Ser
Ser Ala Phe Ser Lys Thr 1 5 10 15 Leu Ser Pro Ser Ser Ser Thr Ser
Ser Thr Leu Leu Pro Arg Ser Thr 20 25 30 Phe Pro Phe Pro His His
Pro His Lys Thr Thr Pro Pro Pro Leu His 35 40 45 Leu Thr His Thr
His Ile His Ile His Ser Gln Arg Arg Arg Phe Thr 50 55 60 Ile Ser
Asn Val Ile Ser Thr Asn Gln Lys Val Ser Gln Thr Glu Lys 65 70 75 80
Thr Glu Thr Phe Val Ser Arg Phe Ala Pro Asp Glu Pro Arg Lys Gly 85
90 95 Ser Asp Val Leu Val Glu Ala Leu Glu Arg Glu Gly Val Thr Asp
Val 100 105 110 Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gln
Ala Leu Thr 115 120 125 Arg Ser Ser Ile Ile Arg Asn Val Leu Pro Arg
His Glu Gln Gly Gly 130 135 140 Val Phe Ala Ala Glu Gly Tyr Ala Arg
Ala Thr Gly Phe Pro Gly Val 145 150 155 160 Cys Ile Ala Thr Ser Gly
Pro Gly Ala Thr Asn Leu Val Ser Gly Leu 165 170 175 Ala Asp Ala Leu
Leu Asp Ser Val Pro Ile Val Ala Ile Thr Gly Gln 180 185 190 Val Pro
Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile 195 200 205
Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met Asp 210
215 220 Val Glu Asp Ile Pro Arg Val Val Arg Glu Ala Phe Phe Leu Ala
Arg 225 230 235 240 Ser Gly Arg Pro Gly Pro Ile Leu Ile Asp Val Pro
Lys Asp Ile Gln 245 250 255 Gln Gln Leu Val Ile Pro Asp Trp Asp Gln
Pro Met Arg Leu Pro Gly 260 265 270 Tyr Met Ser Arg Leu Pro Lys Leu
Pro Asn Glu Met Leu Leu Glu Gln 275 280 285 Ile Val Arg Leu Ile Ser
Glu Ser Lys Lys Pro Val Leu Tyr Val Gly 290 295 300 Gly Gly Cys Ser
Gln Ser Ser Glu Asp Leu Arg Arg Phe Val Glu Leu 305 310 315 320 Thr
Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ala Phe Pro 325 330
335 Thr Gly Asp Glu Leu Ser Leu Ser Met Leu Gly Met His Gly Thr Val
340 345 350 Tyr Ala Asn Tyr Ala Val Asp Ser Ser Asp Leu Leu Leu Ala
Phe Gly 355 360 365 Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu
Ala Phe Ala Ser 370 375 380 Arg Ala Lys Ile Val His Ile Asp Ile Asp
Ser Ala Glu Ile Gly Lys 385 390 395 400 Asn Lys Gln Pro His Val Ser
Ile Cys Ala Asp Ile Lys Leu Ala Leu 405 410 415 Gln Gly Leu Asn Ser
Ile Leu Glu Ser Lys Glu Gly Lys Leu Lys Leu 420 425 430 Asp Phe Ser
Ala Trp Arg Gln Glu Leu Thr Glu Gln Lys Val Lys His 435 440 445 Pro
Leu Asn Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala 450 455
460 Ile Gln Val Leu Asp Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr
465 470 475 480 Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Tyr Tyr
Lys Tyr Arg 485 490 495 Lys Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu
Gly Ala Met Gly Phe 500 505 510 Gly Leu Pro Ala Ala Ile Gly Ala Ala
Val Gly Arg Pro Asp Glu Val 515 520 525 Val Val Asp Ile Asp Gly Asp
Gly Ser Phe Ile Met Asn Val Gln Glu 530 535 540 Leu Ala Thr Ile Lys
Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu 545 550 555 560 Asn Asn
Gln His Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr 565 570 575
Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Asn Glu Ala 580
585 590 Glu Ile Phe Pro Asn Met Leu Lys Phe Ala Glu Ala Cys Gly Val
Pro 595 600 605 Ala Ala Arg Val Thr His Arg Asp Asp Leu Arg Ala Ala
Ile Gln Lys 610 615 620 Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp
Val Ile Val Pro His 625 630 635 640 Gln Glu His Val Leu Pro Met Ile
Pro Ser Gly Gly Ala Phe Lys Asp 645 650 655 Val Ile Thr Glu Gly Asp
Gly Arg Ser Ser Tyr 660 665 6 664 PRT Tobacco 6 Met Ala Ala Ala Ala
Ala Ala Pro Ser Pro Ser Phe Ser Lys Thr Leu 1 5 10 15 Ser Ser Ser
Ser Ser Lys Ser Ser Thr Leu Leu Pro Arg Ser Thr Phe 20 25 30 Pro
Phe Pro His His Pro His Lys Thr Thr Pro Pro Pro Leu His Leu 35 40
45 Thr Pro Thr His Ile His Ser Gln Arg Arg Arg Phe Thr Ile Ser Asn
50 55 60 Val Ile Ser Thr Thr Gln Lys Val Ser Glu Thr Gln Lys Ala
Glu Thr 65 70 75 80 Phe Val Ser Arg Phe Ala Pro Asp Glu Pro Arg Lys
Gly Ser Asp Val 85 90 95 Leu Val Glu Ala Leu Glu Arg Glu Gly Val
Thr Asp Val Phe Ala Tyr 100 105 110 Pro Gly Gly Ala Ser Met Glu Ile
His Gln Ala Leu Thr Arg Ser Ser 115 120 125 Ile Ile Arg Asn Val Leu
Pro Arg His Glu Gln Gly Gly Val Phe Ala 130 135 140 Ala Glu Gly Tyr
Ala Arg Ala Thr Gly Phe Pro Gly Val Cys Ile Ala 145 150 155 160 Thr
Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala 165 170
175 Leu Leu Asp Ser Val Pro Ile Val Ala Ile Thr Gly Gln Val Pro Arg
180 185 190 Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val
Glu Val 195 200 205 Thr Arg Ser Ile Thr Lys His Asn Tyr Leu Val Met
Asp Val Glu Asp 210 215 220 Ile Pro Arg Val Val Arg Glu Ala Phe Phe
Leu Ala Arg Ser Gly Arg 225 230 235 240 Pro Gly Pro Val Leu Ile Asp
Val Pro Lys Asp Ile Gln Gln Gln Leu 245 250 255 Val Ile Pro Asp Trp
Asp Gln Pro Met Arg Leu Pro Gly Tyr Met Ser 260 265 270 Arg Leu Pro
Lys Leu Pro Asn Glu Met Leu Leu Glu Gln Ile Val Arg 275 280 285 Leu
Ile Ser Glu Ser Lys Lys Pro Val Leu Tyr Val Gly Gly Gly Cys 290 295
300 Ser Gln Ser Ser Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly Ile
305 310 315 320 Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ala Phe Pro
Thr Gly Asp 325 330 335 Glu Leu Ser Leu Ser Met Leu Gly Met His Gly
Thr Val Tyr Ala Asn 340 345 350 Tyr Ala Val Asp Ser Ser Asp Leu Leu
Leu Ala Phe Gly Val Arg Phe 355 360 365 Asp Asp Arg Val Thr Gly Lys
Leu Glu Ala Phe Ala Ser Arg Ala Lys 370 375 380 Ile Val His Ile Asp
Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys Gln 385 390 395 400 Pro His
Val Ser Ile Cys Ala Asp Ile Lys Leu Ala Leu Gln Gly Leu 405 410 415
Asn Ser Ile Leu Glu Ser Lys Glu Gly Lys Leu Lys Leu Asp Phe Ser 420
425 430 Ala Trp Arg Gln Glu Leu Thr Val Gln Lys Val Lys Tyr Pro Leu
Asn 435 440 445 Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln Tyr Ala
Ile Gln Val 450 455 460 Leu Asp Glu Leu Thr Asn Gly Ser Ala Ile Ile
Ser Thr Gly Val Gly 465 470 475 480 Gln His Gln Met Trp Ala Ala Gln
Tyr Tyr Lys Tyr Arg Lys Pro Arg 485 490 495 Gln Trp Leu Thr Ser Gly
Gly Leu Gly Ala Met Gly Phe Gly Leu Pro 500 505 510 Ala Ala Ile Gly
Ala Ala Val Gly Arg Pro Asp Glu Val Val Val Asp 515 520 525 Ile Asp
Gly Asp Gly Ser Phe Ile Met Asn Val Gln Glu Leu Ala Thr 530 535 540
Ile Lys Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu Asn Asn Gln 545
550 555 560 His Leu Gly Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys
Ala Asn 565 570 575 Arg Ala His Thr Tyr Leu Gly Asn Pro Ser Asn Glu
Ala Glu Ile Phe 580 585 590 Pro Asn Met Leu Lys Phe Ala Glu Ala Cys
Gly Val Pro Ala Ala Arg 595 600 605 Val Thr His Arg Asp Asp Leu Arg
Ala Ala Ile Gln Lys Met Leu Asp 610 615 620 Thr Pro Gly Pro Tyr Leu
Leu Asp Val Ile Val Pro His Gln Glu His 625 630 635 640 Val Leu Pro
Met Ile Pro Ser Gly Gly Ala Phe Lys Asp Val Ile Thr 645 650 655 Glu
Gly Asp Gly Arg Ser Ser Tyr 660 7 671 PRT Arabidopsis thaliana 7
Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser Ser Ser Ile Ser Phe 1 5
10 15 Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu Pro Ile
Ser 20 25 30 Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys Ser
Ser Ser Ser 35 40 45 Ser Arg Arg Arg Gly Ile Lys Ser Ser Ser Pro
Ser Ser Ile Ser Ala 50 55 60 Val Leu Asn Thr Thr Thr Asn Val Thr
Thr Thr Pro Ser Pro Thr Lys 65 70 75 80 Pro Thr Lys Pro Glu Thr Phe
Ile Ser Arg Phe Ala Pro Asp Gln Pro 85 90 95 Arg Lys Gly Ala Asp
Ile Leu Val Glu Ala Leu Glu Arg Gln Gly Val 100 105 110 Glu Thr Val
Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His Gln 115 120 125 Ala
Leu Thr Arg Ser Ser Ser Ile Arg Asn Val Leu Pro Arg His Glu 130 135
140 Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser Gly Lys
145 150 155 160 Pro Gly Ile Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr
Asn Leu Val 165 170 175 Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val
Pro Leu Val Ala Ile 180 185 190 Thr Gly Gln Val Pro Arg Arg Met Ile
Gly Thr Asp Ala Phe Gln Glu 195 200 205 Thr Pro Ile Val Glu Val Thr
Arg Ser Ile Thr Lys His Asn Tyr Leu 210 215 220 Val Met Asp Val Glu
Asp Ile Pro Arg Ile Ile Glu Glu Ala Phe Phe 225 230 235 240 Leu Ala
Thr Ser Gly Arg Pro Gly Pro Val Leu Val Asp Val Pro Lys 245 250 255
Asp Ile Gln Gln Gln Leu Ala Ile Pro Asn Trp Glu Gln Ala Met Arg 260
265 270 Leu Pro Gly Tyr Met Ser Arg Met Pro Lys Pro Pro Glu Asp Ser
His 275 280 285 Leu Glu Gln Ile Val Arg Leu Ile Ser Glu Ser Lys Lys
Pro Val Leu 290 295 300 Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Asp
Glu Leu Gly Arg Phe 305 310 315 320 Val Glu Leu Thr Gly Ile Pro Val
Ala Ser Thr Leu Met Gly Leu Gly 325 330 335 Ser Tyr Pro Cys Asp Asp
Glu Leu Ser Leu His Met Leu Gly Met His 340 345 350 Gly Thr Val Tyr
Ala Asn Tyr Ala Val Glu His Ser Asp Leu Leu Leu 355 360 365 Ala Phe
Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala 370 375 380
Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu 385
390 395 400 Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Gly Asp
Val Lys 405 410 415 Leu Ala Leu Gln Gly Met Asn Lys Val Leu Glu Asn
Arg Ala Glu Glu 420 425 430 Leu Lys Leu Asp Phe Gly Val Trp Arg Asn
Glu Leu Asn Val Gln Lys 435 440 445 Gln Lys Phe Pro Leu Ser Phe Lys
Thr Phe Gly Glu Ala Ile Pro Pro 450 455 460 Gln Tyr Ala Ile Lys Val
Leu Asp Glu Leu Thr Asp Gly Lys Ala Ile 465 470 475 480 Ile Ser Thr
Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr 485 490 495 Asn
Tyr Lys Lys Pro Arg Arg Gln Trp Leu Ser Ser Gly Gly Leu Gly 500 505
510 Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn
515 520 525 Pro Asp Ala Ile Val Val Asp Ile Asp Gly Asp Gly Ser Phe
Ile Met 530 535 540 Asn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn
Leu Pro Val Lys 545 550 555 560 Val Leu Leu Leu Asn Asn Gln His Leu
Gly Met Val Met Gln Trp Glu 565 570 575 Asp Arg Phe Tyr Lys Ala Asn
Arg Ala His Thr Phe Leu Gly Asp Pro 580 585 590 Ala Gln Glu Asp Glu
Ile Phe Pro Asn Met Leu Leu Phe Ala Ala Ala 595 600 605 Cys Gly Ile
Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu Arg Glu 610 615 620 Ala
Ile Gln Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val 625 630
635 640 Ile Cys Pro His Gln Glu His Val Leu Pro Met Ile Pro Asn Gly
Gly 645 650 655 Thr Phe Asn Asp Val Ile Thr Glu Gly Asp Gly Arg Ile
Lys Tyr 660 665 670 8 652 PRT Brassica napus 8 Met Ala Ala Ala Thr
Ser Ser Ser Pro Ile Ser Leu Thr Ala Lys Pro 1 5 10 15 Ser Ser Lys
Ser Pro Leu Pro Ile Ser Arg Phe Ser Leu Pro Phe Ser 20 25 30 Leu
Thr Pro Gln Lys Pro Ser Ser Arg Leu His Arg Pro Leu Ala Ile 35 40
45 Ser Ala Val Leu Asn Ser Pro Val Asn Val Ala Pro Glu Lys Thr Asp
50 55 60 Lys Ile Lys Thr Phe Ile Ser Arg Tyr Ala Pro Asp Glu Pro
Arg Lys 65 70 75 80 Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gln
Gly Val Glu Thr 85 90 95 Val Phe Ala Tyr Pro Gly Gly Ala Ser Met
Glu Ile His Gln Ala Leu 100 105 110 Thr Arg Ser Ser Thr Ile Arg Asn
Val Leu Pro Arg His Glu Gln Gly 115 120 125 Gly Val Phe Ala Ala Glu
Gly Tyr Ala Arg Ser Ser Gly Lys Pro Gly 130 135 140 Ile Cys Ile Ala
Thr Ser Gly Pro Gly Ala Thr Asn Leu Val Ser Gly 145 150 155 160 Leu
Ala Asp Ala Met Leu Asp Ser Val Pro Leu Val Ala Ile Thr Gly 165 170
175 Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu Thr Pro
180 185 190 Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu
Val Met 195 200 205 Asp Val Asp Asp Ile Pro Arg Ile Val Gln Glu Ala
Phe Phe Leu Ala 210 215 220 Thr Ser Gly Arg Pro Gly Pro Val Leu Val
Asp Val Pro Lys Asp Ile 225 230 235 240 Gln Gln Gln Leu Ala Ile Pro
Asn Trp Asp Gln Pro Met Arg Leu Pro 245 250 255 Gly Tyr Met Ser Arg
Leu Pro Gln Pro Pro Glu Val Ser Gln Leu Gly 260 265 270 Gln Ile Val
Arg Leu Ile Ser Glu Ser Lys Arg Pro Val Leu Tyr Val 275 280 285 Gly
Gly Gly Ser Leu Asn Ser Ser Glu Glu Leu Gly Arg Phe Val Glu 290 295
300 Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser Tyr
305 310 315 320 Pro Cys Asn Asp Glu Leu Ser Leu Gln Met Leu Gly Met
His Gly Thr 325 330 335 Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp
Leu Leu Leu Ala Phe 340 345 350 Gly Val Arg Phe Asp Asp Arg Val Thr
Gly Lys Leu Glu Ala Phe Ala 355 360 365 Ser Arg Ala Lys Ile Val His
Ile Asp Ile Asp Ser Ala Glu Ile Gly 370 375 380 Lys Asn Lys Thr Pro
His Val Ser Val Cys Gly Asp Val Lys Leu Ala 385 390 395 400 Leu Gln
Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu Leu Lys 405 410 415
Leu
Asp Phe Gly Val Trp Arg Ser Glu Leu Ser Glu Gln Lys Gln Lys 420 425
430 Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro Gln Tyr
435 440 445 Ala Ile Gln Val Leu Asp Glu Leu Thr Gln Gly Lys Ala Ile
Ile Ser 450 455 460 Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln
Phe Tyr Lys Tyr 465 470 475 480 Arg Lys Pro Arg Gln Trp Leu Ser Ser
Ser Gly Leu Gly Ala Met Gly 485 490 495 Phe Gly Leu Pro Ala Ala Ile
Gly Ala Ser Val Ala Asn Pro Asp Ala 500 505 510 Ile Val Val Asp Ile
Asp Gly Asp Gly Ser Phe Ile Met Asn Val Gln 515 520 525 Glu Leu Ala
Thr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Leu Leu 530 535 540 Leu
Asn Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp Arg Phe 545 550
555 560 Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asp Pro Ala Arg
Glu 565 570 575 Asn Glu Ile Phe Pro Asn Met Leu Gln Phe Ala Gly Ala
Cys Gly Ile 580 585 590 Pro Ala Ala Arg Val Thr Lys Lys Glu Glu Leu
Arg Glu Ala Ile Gln 595 600 605 Thr Met Leu Asp Thr Pro Gly Pro Tyr
Leu Leu Asp Val Ile Cys Pro 610 615 620 His Gln Glu His Val Leu Pro
Met Ile Pro Ser Gly Gly Thr Phe Lys 625 630 635 640 Asp Val Ile Thr
Glu Gly Asp Gly Arg Thr Lys Tyr 645 650 9 637 PRT Brassica napus 9
Met Ala Ser Phe Ser Phe Phe Gly Thr Ile Pro Ser Ser Pro Thr Lys 1 5
10 15 Ala Ser Val Phe Ser Leu Pro Val Ser Val Thr Thr Leu Pro Ser
Phe 20 25 30 Pro Arg Arg Arg Ala Thr Arg Val Ser Val Ser Ala Asn
Ser Lys Lys 35 40 45 Asp Gln Asp Arg Thr Ala Ser Arg Arg Glu Asn
Pro Ser Thr Phe Ser 50 55 60 Ser Lys Tyr Ala Pro Asn Val Pro Arg
Ser Gly Ala Asp Ile Leu Val 65 70 75 80 Glu Ala Leu Glu Arg Gln Gly
Val Asp Val Val Phe Ala Tyr Pro Gly 85 90 95 Gly Ala Ser Met Glu
Ile His Gln Ala Leu Thr Arg Ser Asn Thr Ile 100 105 110 Arg Asn Val
Leu Pro Arg His Glu Gln Gly Gly Ile Phe Ala Ala Glu 115 120 125 Gly
Tyr Ala Arg Ser Ser Gly Lys Pro Gly Ile Cys Ile Ala Thr Ser 130 135
140 Gly Pro Gly Ala Met Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Phe
145 150 155 160 Asp Ser Val Pro Leu Ile Ala Ile Thr Gly Gln Val Pro
Arg Arg Met 165 170 175 Ile Gly Thr Met Ala Phe Gln Glu Thr Pro Val
Val Glu Val Thr Arg 180 185 190 Thr Ile Thr Lys His Asn Tyr Leu Val
Met Glu Val Asp Asp Ile Pro 195 200 205 Arg Ile Val Arg Glu Ala Phe
Phe Leu Ala Thr Ser Val Arg Pro Gly 210 215 220 Pro Val Leu Ile Asp
Val Pro Lys Asp Val Gln Gln Gln Phe Ala Ile 225 230 235 240 Pro Asn
Trp Glu Gln Pro Met Arg Leu Pro Leu Tyr Met Ser Thr Met 245 250 255
Pro Lys Pro Pro Lys Val Ser His Leu Glu Gln Ile Leu Arg Leu Val 260
265 270 Ser Glu Ser Lys Arg Pro Val Leu Tyr Val Gly Gly Gly Cys Leu
Asn 275 280 285 Ser Ser Glu Glu Leu Arg Arg Phe Val Glu Leu Thr Gly
Ile Pro Val 290 295 300 Ala Ser Thr Phe Met Gly Leu Gly Ser Tyr Pro
Cys Asp Asp Glu Glu 305 310 315 320 Phe Ser Leu Gln Met Leu Gly Met
His Gly Thr Val Tyr Ala Asn Tyr 325 330 335 Ala Val Glu Tyr Ser Asp
Leu Leu Leu Ala Phe Gly Val Arg Phe Asp 340 345 350 Asp Arg Val Thr
Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala Lys Ile 355 360 365 Val His
Ile Asp Ile Asp Ser Thr Glu Ile Gly Lys Asn Lys Thr Pro 370 375 380
His Val Ser Val Cys Cys Asp Val Gln Leu Ala Leu Gln Gly Met Asn 385
390 395 400 Glu Val Leu Glu Asn Arg Arg Asp Val Leu Asp Phe Gly Glu
Trp Arg 405 410 415 Cys Glu Leu Asn Glu Gln Arg Leu Lys Phe Pro Leu
Arg Tyr Lys Thr 420 425 430 Phe Gly Glu Glu Ile Pro Pro Gln Tyr Ala
Ile Gln Leu Leu Asp Glu 435 440 445 Leu Thr Asp Gly Lys Ala Ile Ile
Thr Thr Gly Val Gly Gln His Gln 450 455 460 Met Trp Ala Ala Gln Phe
Tyr Arg Phe Lys Lys Pro Arg Gln Trp Leu 465 470 475 480 Ser Ser Gly
Gly Leu Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Met 485 490 495 Gly
Ala Ala Ile Ala Asn Pro Gly Ala Val Val Val Asp Ile Asp Gly 500 505
510 Asp Gly Ser Phe Ile Met Asn Ile Gln Glu Leu Ala Thr Ile Arg Val
515 520 525 Glu Asn Leu Pro Val Lys Val Leu Leu Ile Asn Asn Gln His
Leu Gly 530 535 540 Met Val Leu Gln Trp Glu Asp His Phe Tyr Ala Ala
Asn Arg Ala Asp 545 550 555 560 Ser Phe Leu Gly Asp Pro Ala Asn Pro
Glu Ala Val Phe Pro Asp Met 565 570 575 Leu Leu Phe Ala Ala Ser Cys
Gly Ile Pro Ala Ala Arg Val Thr Arg 580 585 590 Arg Glu Asp Leu Arg
Glu Ala Ile Gln Thr Met Leu Asp Thr Pro Gly 595 600 605 Pro Phe Leu
Leu Asp Val Val Cys Pro His Gln Asp His Val Leu Pro 610 615 620 Leu
Ile Pro Ser Gly Gly Thr Phe Lys Asp Ile Ile Val 625 630 635
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