U.S. patent application number 14/606219 was filed with the patent office on 2015-07-30 for herbicide-resistant sunflower plants and methods of use.
The applicant listed for this patent is BASF SE. Invention is credited to Alberto Javier Leon, Monica Mariel Morata, Andres Daniel Zambelli.
Application Number | 20150211017 14/606219 |
Document ID | / |
Family ID | 39110694 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150211017 |
Kind Code |
A1 |
Leon; Alberto Javier ; et
al. |
July 30, 2015 |
HERBICIDE-RESISTANT SUNFLOWER PLANTS AND METHODS OF USE
Abstract
Novel herbicide-resistant sunflower plants designated as MUT31
and herbicide-resistant descendents thereof are provided. The MUT31
sunflower plants and the herbicide-resistant descendents thereof
comprise increased resistance to at least one imidazolinone
herbicide, when compared to wild-type sunflower plants. Methods for
controlling weeds in the vicinity of these herbicide-resistant
sunflower plants and methods for increasing the
herbicide-resistance of a sunflower plant are also provided.
Inventors: |
Leon; Alberto Javier; (Mar
Del Plata, AR) ; Morata; Monica Mariel; (Mar Del
Plata, AR) ; Zambelli; Andres Daniel; (Mar Del Plata,
AR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF SE |
Ludwigshafen Am Rhein |
|
DE |
|
|
Family ID: |
39110694 |
Appl. No.: |
14/606219 |
Filed: |
January 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12517919 |
Oct 11, 2011 |
9035133 |
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PCT/EP2007/063737 |
Dec 11, 2007 |
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14606219 |
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60874309 |
Dec 12, 2006 |
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Current U.S.
Class: |
800/278 ;
435/416; 504/253; 800/300 |
Current CPC
Class: |
A01H 1/06 20130101; A01H
5/10 20130101; A01H 1/00 20130101; A01N 43/50 20130101; C12N
15/8274 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 1/06 20060101 A01H001/06; A01H 5/10 20060101
A01H005/10; A01N 43/50 20060101 A01N043/50 |
Claims
1-48. (canceled)
49. A sunflower plant, or part thereof, comprising both: (a) the
MUT31 trait of sunflower line MUT31, a representative sample of
seed of said line having been deposited under ATCC Patent Deposit
No. PTA-7839, wherein said MUT31 trait was produced by mutation
breeding and comprises imidazolinone herbicide tolerance, even in
the absence of an herbicide tolerant acetohydroxyacid synthase
large subunit (AHASL) protein, and said herbicide tolerance is
reducible or inhibitable by malathion; and (b) at least one
herbicide tolerant AHASL gene wherein said sunflower plant exhibits
greater AHAS-inhibiting herbicide tolerance than a sunflower plant
containing, as the sole AHAS-inhibiting herbicide tolerance trait
thereof, either said MUT31 trait or said herbicide tolerant AHASL
gene.
50. The sunflower plant of claim 49, wherein the AHAS-inhibiting
herbicide tolerance comprises imidazolinone herbicide
tolerance.
51. The sunflower plant of claim 49, wherein said sunflower plant
is selected from the group consisting of: (a) sunflower plants that
are progeny of line MUT31; (b) sunflower plants that are mutants,
recombinants, or genetically engineered derivatives of line MUT31;
and (c) sunflower plants that are progeny of at least one of the
sunflower plants of (a)-(b).
52. The sunflower plant of claim 49, wherein said at least one
herbicide tolerant AHASL gene encodes an AHASL protein comprising
an alanine-to valine amino acid substitution at amino acid position
190 or equivalent position relative to the amino acid sequence of
SEQ ID NO:2.
53. The sunflower plant of claim 49, wherein said imidazolinone
herbicide comprises at least one of:
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)
quinoline-3-carboxylic acid,
5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic
acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)--
nicotinic acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic
acid, a mixture of methyl
6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and
methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate,
or a combination of any of the foregoing.
54. The sunflower plant of claim 49, wherein said imidazolinone
herbicide is imazamox, imazapyr, imazethapyr, or mixtures
thereof.
55. A seed of the sunflower plant of claim 49, wherein said seed is
an imidazolinone-tolerant descendent of a plant of line MUT31 and
comprises the imidazolinone herbicide tolerance trait of line
MUT31.
56. The seed of claim 55, wherein said seed is treated with an
imidazolinone herbicide.
57. A cell of the plant of claim 49, wherein said cell comprises
the imidazolinone herbicide tolerance trait of line MUT31
58. A pollen grain of the plant of claim 49, wherein said pollen
grain comprises the imidazolinone herbicide tolerance trait of line
MUT31.
59. A method for controlling weeds in a field, said method
comprising: growing in the field a sunflower plant comprising both
(a) the MUT31 trait of sunflower line MUT31, a representative
sample of seed of said line having been deposited under ATCC Patent
Deposit No. PTA-7839, wherein said MUT31 trait was produced by
mutation breeding and comprises imidazolinone herbicide tolerance,
even in the absence of an herbicide tolerant acetohydroxyacid
synthase large subunit (AHASL) protein, and said herbicide
tolerance is reducible or inhibitable by malathion; and (b) at
least one herbicide tolerant AHASL gene wherein said sunflower
plant exhibits greater AHAS-inhibiting herbicide tolerance than a
sunflower plant containing, as the sole AHAS-inhibiting herbicide
tolerance trait thereof, either said MUT31 trait or said herbicide
tolerant AHASL gene; and contacting said sunflower plant and weeds
in the field with an effective amount of an imidazolinone herbicide
to which the sunflower plant is tolerant and which would inhibit
the growth of the wild-type variety of the sunflower plant, thereby
controlling the weeds.
60. The method of claim 59, wherein the AHAS-inhibiting herbicide
tolerance comprises imidazolinone herbicide tolerance.
61. The method of claim 59, wherein said sunflower plant is
selected from the group consisting of: (a) sunflower plants that
are progeny of line MUT31; (b) sunflower plants that are mutants,
recombinants, or genetically engineered derivatives of line MUT31;
and (c) sunflower plants that are progeny of at least one of the
sunflower plants of (a)-(b).
62. The method of claim 59, wherein said imidazolinone herbicide
comprises at least one of:
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)
quinoline-3-carboxylic acid,
5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic
acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)--
nicotinic acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic
acid, a mixture of methyl
6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and
methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate,
or a combination of any of the foregoing.
63. A method for combating undesired vegetation comprising
contacting a sunflower seed of a sunflower plant of claim 49 before
sowing and/or after pregermination with an imidazolinone
herbicide.
64. The method of claim 63, further comprising planting said
sunflower seed in an environment favorable for growth of a
sunflower plant.
65. The methods of claim 64, wherein said environment comprises
undesired vegetation.
66. A sunflower plant, or part thereof, comprising the MUT31 trait
of sunflower line MUT31, a representative sample of seed of said
line having been deposited under ATCC Patent Deposit No. PTA-7839,
wherein said MUT31 trait was produced by mutation breeding and
comprises imidazolinone herbicide tolerance, even in the absence of
an herbicide tolerant acetohydroxyacid synthase large subunit
(AHASL) protein, and said tolerance is reducible or inhibitable by
malathion; and wherein said MUT31 trait being capable of enhancing
AHAS-inhibiting herbicide tolerance of a sunflower plant comprising
an herbicide tolerant AHASL gene, when the MUT31 trait is stacked
with said herbicide tolerant AHASL gene in the plant.
67. The sunflower plant of claim 66, wherein the AHAS-inhibiting
herbicide tolerance comprises imidazolinone herbicide
tolerance.
68. A method for enhancing the AHAS-inhibiting herbicide tolerance
of a sunflower plant, comprising: providing in said sunflower plant
both the MUT31 trait and an herbicide tolerant AHASL gene, wherein
said sunflower plant exhibits greater AHAS-inhibiting herbicide
tolerance than a sunflower plant containing, as the sole
AHAS-inhibiting herbicide tolerance trait thereof, either said
MUT31 trait or said herbicide tolerant AHASL gene.
69. The method of claim 68, wherein the AHAS-inhibiting herbicide
tolerance comprises imidazolinone herbicide tolerance.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of agricultural
biotechnology, particularly to herbicide-resistant sunflower plants
and novel polynucleotide sequences that encode wild-type and
herbicide-resistant sunflower acetohydroxyacid synthase large
subunit proteins.
BACKGROUND OF THE INVENTION
[0002] Acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as
acetolactate synthase or ALS), is the first enzyme that catalyzes
the biochemical synthesis of the branched chain amino acids valine,
leucine and isoleucine (Singh (1999) "Biosynthesis of valine,
leucine and isoleucine," in Plant Amino Acid, Singh, B. K., ed.,
Marcel Dekker Inc. New York, N.Y., pp. 227-247). AHAS is the site
of action of five structurally diverse herbicide families including
the sulfonylureas (LaRossa and Falco (1984) Trends Biotechnol.
2:158-161), the imidazolinones (Shaner et al. (1984) Plant Physiol.
76:545-546), the triazolopyrimidines (Subramanian and Gerwick
(1989) "Inhibition of acetolactate synthase by
triazolopyrimidines," in Biocatalysis in Agricultural
Biotechnology, Whitaker, J. R. and Sonnet, P. E. eds., ACS
Symposium Series, American Chemical Society, Washington, D.C., pp.
277-288), and the pyrimidyloxybenzoates (Subramanian et al. (1990)
Plant Physiol. 94: 239-244). Imidazolinone and sulfonylurea
herbicides are widely used in modern agriculture due to their
effectiveness at very low application rates and relative
non-toxicity in animals. By inhibiting AHAS activity, these
families of herbicides prevent further growth and development of
susceptible plants including many weed species. Several examples of
commercially available imidazolinone herbicides are PURSUIT.RTM.
(imazethapyr), SCEPTER.RTM. (imazaquin) and ARSENAL.RTM.
(imazapyr). Examples of sulfonylurea herbicides are chlorsulfuron,
metsulfuron methyl, sulfometuron methyl, chlorimuron ethyl,
thifensulfuron methyl, tribenuron methyl, bensulfuron methyl,
nicosulfuron, ethametsulfuron methyl, rimsulfuron, triflusulfuron
methyl, triasulfuron, primisulfuron methyl, cinosulfuron,
amidosulfuron, flazasulfuron, imazosulfuron, pyrazosulfuron ethyl
and halosulfuron.
[0003] Due to their high effectiveness and low-toxicity,
imidazolinone herbicides are favored for application by spraying
over the top of a wide area of vegetation. The ability to spray a
herbicide over the top of a wide range of vegetation decreases the
costs associated with plant establishment and maintenance, and
decreases the need for site preparation prior to use of such
chemicals. Spraying over the top of a desired tolerant species also
results in the ability to achieve maximum yield potential of the
desired species due to the absence of competitive species. However,
the ability to use such spray-over techniques is dependent upon the
presence of imidazolinone-resistant species of the desired
vegetation in the spray over area.
[0004] Among the major agricultural crops, some leguminous species
such as soybean are naturally resistant to imidazolinone herbicides
due to their ability to rapidly metabolize the herbicide compounds
(Shaner and Robinson (1985) Weed Sci. 33:469-471). Other crops such
as corn (Newhouse et al. (1992) Plant Physiol. 100:882-886) and
rice (Barrett et al. (1989) Crop Safeners for Herbicides, Academic
Press, New York, pp. 195-220) are somewhat susceptible to
imidazolinone herbicides. The differential sensitivity to the
imidazolinone herbicides is dependent on the chemical nature of the
particular herbicide and differential metabolism of the compound
from a toxic to a non-toxic form in each plant (Shaner et al.
(1984) Plant Physiol. 76:545-546; Brown et al., (1987) Pestic.
Biochem. Physiol. 27:24-29). Other Plant Physiological differences
such as absorption and translocation also play an important role in
sensitivity (Shaner and Robinson (1985) Weed Sci. 33:469-471).
[0005] Plants resistant to imidazolinones, sulfonylureas,
triazolopyrimidines, and pyrimidyloxybenzoates have been
successfully produced using seed, microspore, pollen, and callus
mutagenesis in Zea mays, Arabidopsis thaliana, Brassica napus
(i.e., canola) Glycine max, Nicotiana tabacum, sugarbeet (Beta
vulgaris) and Oryza sativa (Sebastian et al. (1989) Crop Sci.
29:1403-1408; Swanson et al., 1989 Theor. Appl. Genet. 78:525-530;
Newhouse et al. (1991) Theor. Appl. Genet. 83:65-70; Sathasivan et
al. (1991) Plant Physiol. 97:1044-1050; Mourand et al. (1993) J.
Heredity 84:91-96; Wright and Penner (1998) Theor. Appl. Genet.
96:612-620; U.S. Pat. No. 5,545,822). In all cases, a single,
partially dominant nuclear gene conferred resistance. Four
imidazolinone-resistant wheat plants were also previously isolated
following seed mutagenesis of Triticum aestivum L. cv. Fidel
(Newhouse et al. (1992) Plant Physiol. 100:882-886). Inheritance
studies confirmed that a single, partially dominant gene conferred
resistance. Based on allelic studies, the authors concluded that
the mutations in the four identified lines were located at the same
locus. One of the Fidel cultivar resistance genes was designated
FS-4 (Newhouse et al. (1992) Plant Physiol. 100:882-886).
[0006] Naturally occurring plant populations that were discovered
to be resistant to imidazolinone and/or sulfonylurea herbicides
have also been used to develop herbicide-resistant sunflower
breeding lines. Recently, two sunflower lines that are resistant to
a sulfonylurea herbicide were developed using germplasm originating
from a wild population of common sunflower (Helianthus annuus) as
the source of the herbicide-resistance trait (Miller and Al-Khatib
(2004) Crop Sci. 44:1037-1038). Previously, White et al. ((2002)
Weed Sci. 50:432-437) had reported that individuals from a wild
population of common sunflower from South Dakota, U.S.A. were
cross-resistant to an imidazolinone and a sulfonylurea herbicide.
Analysis of a portion of the coding region of the acetohydroxyacid
synthase large subunit (AHASL) genes of individuals from this
population revealed a point mutation that results in an Ala-to-Val
amino acid substitution in the sunflower AHASL protein that
corresponds to Ala205 in the wild-type Arabidopsis thaliana AHASL
protein (White et al. (2003) Weed Sci. 51:845-853).
[0007] Computer-based modeling of the three dimensional
conformation of the AHAS-inhibitor complex predicts several amino
acids in the proposed inhibitor binding pocket as sites where
induced mutations would likely confer selective resistance to
imidazolinones (Ott et al. (1996) J. Mol. Biol. 263:359-368). Wheat
plants produced with some of these rationally designed mutations in
the proposed binding sites of the AHAS enzyme have in fact
exhibited specific resistance to a single class of herbicides (Ott
et al. (1996) J. Mol. Biol. 263:359-368).
[0008] Plant resistance to imidazolinone herbicides has also been
reported in a number of patents. U.S. Pat. Nos. 4,761,373,
5,331,107, 5,304,732, 6,211,438, 6,211,439 and 6,222,100 generally
describe the use of an altered AHAS gene to elicit herbicide
resistance in plants, and specifically discloses certain
imidazolinone-resistant corn lines. U.S. Pat. No. 5,013,659
discloses plants exhibiting herbicide resistance due to mutations
in at least one amino acid in one or more conserved regions. The
mutations described therein encode either cross-resistance for
imidazolinones and sulfonylureas or sulfonylurea-specific
resistance, but imidazolinone-specific resistance is not described.
U.S. Pat. No. 5,731,180 and U.S. Pat. No. 5,767,361 discuss an
isolated gene having a single amino acid substitution in a
wild-type monocot AHAS amino acid sequence that results in
imidazolinone-specific resistance. In addition, rice plants that
are resistant to herbicides that interfere with AHAS have been
developed by mutation breeding and also by the selection of
herbicide-resistant plants from a pool of rice plants produced by
anther culture. See, U.S. Pat. Nos. 5,545,822, 5,736,629,
5,773,703, 5,773,704, 5,952,553 and 6,274,796.
[0009] In plants, as in all other organisms examined, the AHAS
enzyme is comprised of two subunits: a large subunit (catalytic
role) and a small subunit (regulatory role) (Duggleby and Pang
(2000) J. Biochem. Mol. Biol. 33:1-36). The AHAS large subunit
(also referred to herein as AHASL) may be encoded by a single gene
as in the case of Arabidopsis, rice, and sugar beet or by multiple
gene family members as in maize, canola, and cotton. Specific,
single-nucleotide substitutions in the large subunit confer upon
the enzyme a degree of insensitivity to one or more classes of
herbicides (Chang and Duggleby (1998) Biochem J. 333:765-777).
[0010] For example, bread wheat, Triticum aestivum L., contains
three homologous acetohydroxyacid synthase large subunit genes.
Each of the genes exhibit significant expression based on herbicide
response and biochemical data from mutants in each of the three
genes (Ascenzi et al. (2003) International Society of Plant
Molecular Biologists Congress, Barcelona, Spain, Ref. No. 510-17).
The coding sequences of all three genes share extensive homology at
the nucleotide level (WO 03/014357). Through sequencing the AHASL
genes from several varieties of Triticum aestivum, the molecular
basis of herbicide tolerance in most IMI-tolerant
(imidazolinone-tolerant) lines was found to be the mutation
5653(At)N, indicating a serine to asparagine substitution at a
position equivalent to the serine at amino acid 653 in Arabidopsis
thaliana (WO 03/01436; WO 03/014357). This mutation is due to a
single nucleotide polymorphism (SNP) in the DNA sequence encoding
the AHASL protein.
[0011] Multiple AHASL genes are also know to occur in
dicotyledonous plants species. Recently, Kolkman et al. ((2004)
Theor. Appl. Genet. 109: 1147-1159) reported the identification,
cloning, and sequencing for three AHASL genes (AHASL1, AHASL2, and
AHASL3) from herbicide-resistant and wild type genotypes of
sunflower (Helianthus annuus L.). Kolkman et al. reported that the
herbicide-resistance was due either to the Pro197Leu (using the
Arabidopsis AHASL amino acid position nomenclature) substitution or
the Ala205Val substitution in the AHASL1 protein and that each of
these substitutions provided resistance to both imidazolinone and
sulfonylurea herbicides.
[0012] Given their high effectiveness and low-toxicity,
imidazolinone herbicides are favored for agricultural use. However,
the ability to use imidazolinone herbicides in a particular crop
production system depends upon the availability of
imidazolinone-resistant varieties of the crop plant of interest. To
produce such imidazolinone-resistant varieties, plant breeders need
to develop breeding lines with the imidazolinone-resistance trait.
Thus, additional imidazolinone-resistant breeding lines and
varieties of crop plants, as well as methods and compositions for
the production and use of imidazolinone-resistant breeding lines
and varieties, are needed.
SUMMARY OF THE INVENTION
[0013] The present invention provides sunflower plants having
increased resistance to at least one herbicide when compared to a
wild-type sunflower plant. In particular, the sunflower plants of
the invention have increased resistance to at least one
imidazolinone herbicide, particularly imazamox and/or imazapyr,
when compared to a wild-type sunflower plant. Unlike previously
described imidazolinone-resistant sunflower plants, the sunflower
plants of the present invention comprise a novel
herbicide-resistance mechanism that does not involve a mutation in
gene encoding an acetohydroxyacid synthase large subunit (AHASL)
protein. The sunflower plants of the invention also include seeds
and progeny plants that comprise at least one copy of a gene or
polynucleotide encoding a herbicide-resistant AHASL protein of the
invention.
[0014] In one embodiment, the present invention provides
herbicide-resistant sunflower plants that are from the sunflower
line that has been designated herein as MUT31. A sample of seeds of
the MUT31 line has been deposited with the American Type Culture
Collection (ATCC) as ATCC Patent Deposit No. PTA-7839. The present
invention further provides seeds, progeny plants and other
descendent plants that comprise the herbicide-resistance
characteristics of MUT31.
[0015] The present invention provides methods for controlling weeds
in the vicinity of the herbicide-resistant sunflower plants of the
invention. The methods comprises applying an effective amount of an
herbicide to the weeds and to the herbicide-resistant sunflower
plant, wherein the herbicide-resistant sunflower plant has
increased resistance to at least one herbicide, particularly an
imidazolinone herbicide, more particularly imazamox, when compared
to a wild-type sunflower plant. The herbicide can be, for example,
applied to the foliage of the plants, to the seeds of the plants
prior to planting, and/or to the soil before or after planting.
[0016] The present invention further provides methods for producing
a herbicide-resistant sunflower plant. The methods involve crossing
a first sunflower plant comprising resistance to a herbicide to a
second sunflower plant that is not resistant to the herbicide,
wherein the first sunflower plant comprises the
herbicide-resistance characteristics of MUT31, particularly a MUT31
sunflower plant or any herbicide-resistant descendent of MUT31.
Such a herbicide resistant thereof comprises the
herbicide-resistance characteristics of MUT31, representative seeds
of MUT 31 having been deposited with the ATCC as Patent Deposit
Number PTA-7839. The methods further involve selecting for a
progeny plant that is resistant to the herbicide.
[0017] Additionally, the present invention provides methods for
increasing the herbicide-resistance of a sunflower plant. The
methods involve crossing a first sunflower plant comprising
resistance to a herbicide to a second sunflower plant, wherein the
first sunflower plant comprises the herbicide-resistance
characteristics of MUT31, particularly a MUT31 sunflower plant or
any herbicide-resistant descendent of MUT31. The second sunflower
plant can, but is not required to, comprise resistance to at least
one herbicide. The methods further involve selecting for a progeny
plant that comprises increased herbicide resistance when compared
to the herbicide resistance of the second sunflower plant. In one
embodiment of the invention, the second sunflower plant comprises
at least one herbicide-resistant AHASL gene. Such a second
sunflower plant comprises enhanced resistance one or more
AHAS-inhibiting herbicides, particularly an imidazolinone
herbicide.
BRIEF DESCRIPTION THE DRAWINGS
[0018] FIG. 1 is a graphical representation of the results of a
2'3.times.2 factorial experiment to the test the effects of
sunflower genotype (RHA266 or MUT31), herbicide dose (0,
0.25.times., and, 0.50.times.; where X=50 g a.i./ha imazamox), and
malathion (0 or 1000 g a.i./ha). The sunflower plants were at the
3-4 leaf growth stage at time of malathion and herbicide spraying.
Malathion was sprayed on the plants 30 minutes prior to the
imazamox spraying. The sunflower plants were evaluated at 7 days
after herbicide spraying to determine damage scores. Additional
details are provided below in Example 3.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention relates to sunflower plants having
increased resistance to at least one herbicide when compared to
wild-type sunflower plants. Herbicide-resistant sunflower plants
were produced as described hereinbelow by exposing wild-type (with
respect to herbicide resistance) sunflower plants to a mutagen,
allowing the plants to mature and reproduce, and selecting progeny
plants that displayed enhanced resistance to the imidazolinone
herbicide, imazamox, when compared to the resistance of a wild-type
sunflower plant to the imidazolinone herbicide. The invention
provides the novel herbicide-resistant sunflower line that is
designated herein as MUT31. While the present invention is not
bound by any particular biological mechanism, the
herbicide-resistant sunflower plants of invention comprise a novel
herbicide-resistance mechanism that is independent of a mutation or
mutations in one or more AHASL genes. Thus, the present invention
provides a new source of imidazolinone resistance that finds use in
methods for controlling weeds and also methods for producing
herbicide-resistant sunflower plants or increasing the herbicide
resistance of sunflower plants that already comprise herbicide
resistance including, but not limited to,
imidazolinone-resistance.
[0020] For the present invention, the terms "herbicide-tolerant"
and "herbicide-resistant" are used interchangeable and are intended
to have an equivalent meaning and an equivalent scope. Similarly,
the terms "herbicide-tolerance" and "herbicide-resistance" are used
interchangeable and are intended to have an equivalent meaning and
an equivalent scope. Likewise, the terms "imidazolinone-resistant"
and "imidazolinone-resistance" are used interchangeable and are
intended to be of an equivalent meaning and an equivalent scope as
the terms "imidazolinone-tolerant" and "imidazolinone-tolerance",
respectively.
[0021] The present invention involves herbicide-tolerant or
herbicide-resistant plants and methods for making and using such
plants. A "herbicide-tolerant" or a "herbicide-resistant" plant is
a plant that is tolerant or resistant to at least one herbicide at
a level that would normally kill, or inhibit the growth of, a
normal or wild-type plant.
[0022] In one embodiment of the invention, the methods of the
present invention for increasing the herbicide resistance of a
plant involve the use of sunflower plants comprising
herbicide-resistant AHASL polynucleotides and herbicide-resistant
AHASL proteins. A "herbicide-resistant AHASL polynucleotide" is a
polynucleotide that encodes a herbicide-resistant AHASL protein,
wherein the protein comprises herbicide-resistant AHAS activity. A
"herbicide-resistant AHASL protein" is an AHASL protein that
displays higher AHAS activity, relative to the AHAS activity of a
wild-type AHASL protein, when in the presence of at least one
herbicide that is known to interfere with AHAS activity and at a
concentration or level of the herbicide that is to known to inhibit
the AHAS activity of the wild-type AHASL protein. Furthermore, the
AHAS activity of such a herbicide-tolerant or herbicide-resistant
AHASL protein may be referred to herein as "herbicide-tolerant" or
"herbicide-resistant" AHAS activity.
[0023] Furthermore, it is recognized that a herbicide-tolerant or
herbicide-resistant AHASL protein can be introduced into a plant by
transforming a plant or ancestor thereof with a nucleotide sequence
encoding a herbicide-tolerant or herbicide-resistant AHASL protein.
Such herbicide-tolerant or herbicide-resistant AHASL proteins are
encoded by the herbicide-tolerant or herbicide-resistant AHASL
polynucleotides. Alternatively, a herbicide-tolerant or
herbicide-resistant AHASL protein may occur in a plant as a result
of a naturally occurring or induced mutation in an endogenous AHASL
gene in the genome of a plant or progenitor thereof. Nucleotide
sequences encoding herbicide-resistant AHASL proteins and
herbicide-resistant plants comprising an endogenous gene that
encodes a herbicide-resistant AHASL protein are known in the art.
See, for example, U.S. Pat. Nos. 5,013,659, 5,731,180, 5,767,361,
5,545,822, 5,736,629, 5,773,703, 5,773,704, 5,952,553 and
6,274,796; all of which are herein incorporated by reference.
[0024] The present invention provides plants, plant tissues, and
plant cells with increased resistance or tolerance to at least one
herbicide, particularly an imidazolinone herbicide, more
particularly imazamox, imazapyr, or both imazamox and imazapyr. The
preferred amount or concentration of the herbicide is an "effective
amount" or "effective concentration." By "effective amount" and
"effective concentration" is intended an amount and concentration,
respectively, that is sufficient to kill or inhibit the growth of a
similar, wild-type, plant, plant tissue, or plant cell, but that
said amount does not kill or inhibit as severely the growth of the
herbicide-resistant plants, plant tissues, and plant cells of the
present invention. Typically, the effective amount of a herbicide
is an amount that is routinely used in agricultural production
systems to kill weeds of interest. Such an amount is known to those
of ordinary skill in the art, or can be easily determined using
methods known in the art.
[0025] The methods of the present invention find use in increasing
the herbicide resistance of a sunflower plant, including a
sunflower plant that comprises resistance to one or more
herbicides, such as, for example, those herbicides that inhibit or
otherwise interfere or with the activity of the wild-type AHAS
enzyme. Such herbicides may also be referred to herein as
"AHAS-inhibiting herbicides" or simply "AHAS inhibitors." As used
herein, an "AHAS-inhibiting herbicide" or an "AHAS inhibitor" is
not meant to be limited to single herbicide that interferes with
the activity of the AHAS enzyme. Thus, unless otherwise stated or
evident from the context, an "AHAS-inhibiting herbicide" or an
"AHAS inhibitor" can be one herbicide or a mixture of two, three,
four, or more herbicides, each of which interferes with the
activity of the AHAS enzyme.
[0026] By "wild-type, sunflower plant, plant tissue, or plant cell"
is intended a sunflower plant, plant tissue, or plant cell,
respectively, that lacks the herbicide-resistance characteristics
of the herbicide-resistant sunflower plants of the present
invention, particularly MUT31 and herbicide-resistant descendents
thereof. The use of the term "wild-type" is not, therefore,
intended to imply that a sunflower plant, plant tissue, or plant
cell lacks recombinant DNA in its genome, and/or lacks
herbicide-resistant characteristics that are different from those
disclosed herein.
[0027] As used herein unless clearly indicated otherwise, the term
"plant" intended to mean a plant at any developmental stage, as
well as any part or parts of a plant that may be attached to or
separate from a whole intact plant. Such parts of a plant include,
but are not limited to, organs, tissues, and cells of a plant.
Examples of particular plant parts include a stem, a leaf, a root,
an inflorescence, a flower, a floret, a fruit, a pedicle, a
peduncle, a stamen, an anther, pollen, a stigma, a style, an ovary,
a petal, a sepal, a carpel, a root tip, a root cap, a root hair, a
leaf hair, a seed hair, a pollen grain, a microspore, a cotyledon,
a hypocotyl, an epicotyl, xylem, phloem, parenchyma, endosperm, a
companion cell, a guard cell, and any other known organs, tissues,
and cells of a plant. Furthermore, it is recognized that a seed is
a plant.
[0028] The sunflower plants of the present invention include both
non-transgenic plants and transgenic plants. By "non-transgenic
plant" is intended to mean a plant lacking recombinant DNA in its
genome. By "transgenic plant" is intended to mean a plant
comprising recombinant DNA in its genome. Such a transgenic plant
can be produced by introducing recombinant DNA into the genome of
the plant. When such recombinant DNA is incorporated into the
genome of the transgenic plant, progeny of the plant can also
comprise the recombinant DNA. A progeny plant that comprises at
least a portion of the recombinant DNA of at least one progenitor
transgenic plant is also a transgenic plant.
[0029] The present invention provides the herbicide-resistant
sunflower line that is referred to herein as MUT31. A deposit of at
least 2500 seeds from sunflower (Helianthus annuus L.) line MUT31
was made under the Budapest Treaty with the Patent Depository of
the American Type Culture Collection (ATCC), Manassas, Va. 20110
USA on Aug. 22, 2006 and assigned ATCC Patent Deposit Number
PTA-7839. The deposit of sunflower line MUT31 was made for a term
of at least 30 years and at least 5 years after the most recent
request for the furnishing of a sample of the deposit is received
by the ATCC. The deposit will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure. The deposit of
sunflower line MUT31 was made for a term of at least 30 years and
at least 5 years after the most recent request for the furnishing
of a sample of the deposit is received by the ATCC. Additionally,
Applicants have satisfied all the requirements of 37 C.F.R.
.sctn..sctn.1.801-1.809, including providing an indication of the
viability of the sample. Applicants impose no restrictions on the
availability of the deposited material from the ATCC after the
issuance of a patent from this application. However, Applicants
have no authority to wave any restrictions imposed by law on the
transfer of biological material or its transportation in commerce.
Applicants do not waive any infringement of Applicants' rights
granted under such patent.
[0030] The present invention provides herbicide-resistant sunflower
plants of the MUT31 line that were produced by a mutation breeding.
Wild-type sunflower plants were mutagenized by exposing the plants
to a mutagen, particularly a chemical mutagen, more particularly
ethyl methanesulfonate (EMS). However, the present invention is not
limited to herbicide-resistant sunflower plants that are produced
by a mutagenesis method involving the chemical mutagen EMS. Any
mutagenesis method known in the art may be used to produce the
herbicide-resistant sunflower plants of the present invention. Such
mutagenesis methods can involve, for example, the use of any one or
more of the following mutagens: radiation, such as X-rays, Gamma
rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g., product of
nuclear fission by uranium 235 in an atomic reactor), Beta
radiation (e.g., emitted from radioisotopes such as phosphorus 32
or carbon 14), and ultraviolet radiation (preferably from 2500 to
2900 nm), and chemical mutagens such as base analogues (e.g.,
5-bromo-uracil), related compounds (e.g., 8-ethoxy caffeine),
antibiotics (e.g., streptonigrin), alkylating agents (e.g., sulfur
mustards, nitrogen mustards, epoxides, ethylenamines, sulfates,
sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous
acid, or acridines. Herbicide-resistant plants can also be produced
by using tissue culture methods to select for plant cells
comprising herbicide-resistance mutations and then regenerating
herbicide-resistant plants therefrom. See, for example, U.S. Pat.
Nos. 5,773,702 and 5,859,348, both of which are herein incorporated
in their entirety by reference.
[0031] Further details of mutation breeding can be found in
"Principals of Cultivar Development" Fehr, 1993 Macmillan
Publishing Company the disclosure of which is incorporated herein
by reference.
[0032] Sunflower plants and seeds of the present invention comprise
the herbicide resistance characteristics of MUT31. Such plants and
seeds may be referred to herein as comprising the MUT31 trait. The
MUT31 trait confers resistance to imidazolinone herbicides to a
plant or seed possessing this trait whether the trait is in the
heterozygous or homozygous state in the plant. In particular, the
MUT31 trait confers to a sunflower plant or seed resistance to at
least one imidazolinone herbicide, particularly imazamox and/or
imazapyr, wherein the imidazolinone resistance is reduced or
inhibited by the organophosphate insecticide malathion. As
described hereinbelow, the resistance or tolerance of MUT31
sunflower plants to the imidazolinone herbicide imazamox was found
to be reduced or inhibited following the application of malathion
to the MUT31 sunflower plants prior to the application of imazamox.
Thus, sunflower plants and seeds comprising the MUT31 trait
comprise malathion-inhibitable, imidazolinone resistance. While the
present invention is not bound to a particular biological
mechanism, the herbicide resistance of MUT31 sunflower plants or
the MUT31 trait is believed to be due to an induced mutation in a
single gene within the sunflower nuclear genome.
[0033] The sunflower plants of the present invention include, for
example, descendents of the MUT31 line that are heterozygous or
homozygous for the MUT31 trait. It is recognized that such
descendents can be produced via sexual reproduction or by any
asexual reproduction methods known in the art such as for example,
tissue culture. Descendents of the MUT31 line that comprise the
MUT31 trait can be identified by determining if a descendent
sunflower plant comprises malathion-inhibitable, imidazolinone
resistance. The present invention does not depend on a particular
method for determining if a descendent sunflower plant comprises
malathion-inhibitable, imidazolinone resistance. Any method known
in the art can be used including the method disclosed in Example 3
below to determine if a descendent of MUT31 comprises
malathion-inhibitable, imidazolinone resistance. The method
involves applying malathion to a descendent of MUT31 prior to the
application of the imidazolinone herbicide and determining whether
malathion reduces or inhibits the resistance of the descendent to
the imidazolinone herbicide. Descendents that comprise the MUT31
trait comprise malathion-inhibitable, imidazolinone resistance.
Thus, the sunflower plants of the present invention include, for
example, those sunflower plants that are descendents of MUT31 and
comprise malathion-inhibitable, imidazolinone resistance.
[0034] The present invention provides methods for enhancing the
tolerance or resistance of a sunflower plant, plant tissue, plant
cell, or other host cell to at least one herbicide, particularly an
imidazolinone herbicide or mixture two or more imidazolinone
herbicides. For the present invention, the imidazolinone herbicides
include, but are not limited to, PURSUIT.RTM. (imazethapyr),
CADRE.RTM. (imazapic), RAPTOR.RTM. (imazamox), SCEPTER.RTM.
(imazaquin), ASSERT.RTM. (imazethabenz), ARSENAL.RTM. (imazapyr), a
derivative of any of the aforementioned herbicides, and a mixture
of two or more of the aforementioned herbicides, for example,
imazapyr/imazamox (ODYSSEY.RTM.). More specifically, the
imidazolinone herbicide can be selected from, but is not limited
to, 2-(4-isopropyl-4-methyl-5-oxo-2-imidiazolin-2-yl) -nicotinic
acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylic
acid,
5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic
acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)--
nicotinic acid,
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinic
acid, and a mixture of methyl
6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and
methyl 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate.
The use of 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-
2-imidazolin-2-yl) -nicotinic acid and
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-ni-
cotinic acid is preferred. The use of
2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicoti-
nic acid is particularly preferred.
[0035] The herbicide-resistant plants of the invention find use in
methods for controlling weeds. Thus, the present invention further
provides a method for controlling weeds in the vicinity of a
herbicide-resistant sunflower plant of the invention. The method
comprises applying an effective amount of a herbicide to the weeds
and to the herbicide-resistant sunflower plant, wherein the plant
has increased resistance to at least one herbicide, particularly an
imidazolinone or sulfonylurea herbicide, when compared to a
wild-type plant. In such a method for controlling weeds, the
herbicide-resistant plants of the invention are preferably crop
plants, including, but not limited to, sunflower, alfalfa, Brassica
sp., soybean, cotton, safflower, peanut, tobacco, tomato, potato,
wheat, rice, maize, sorghum, barley, rye, millet, and sorghum.
[0036] By providing plants having increased resistance to
herbicides, particularly imidazolinone and sulfonylurea herbicides,
a wide variety of formulations can be employed for protecting
plants from weeds, so as to enhance plant growth and reduce
competition for nutrients. A herbicide can be used by itself for
pre-emergence, post-emergence, pre-planting and at planting control
of weeds in areas surrounding the plants described herein or an
imidazolinone herbicide formulation can be used that contains other
additives. The herbicide can also be used as a seed treatment. That
is an effective concentration or an effective amount of the
herbicide, or a composition comprising an effective concentration
or an effective amount of the herbicide can be applied directly to
the seeds prior to or during the sowing of the seeds. Additives
found in an imidazolinone or sulfonylurea herbicide formulation or
composition include other herbicides, detergents, adjuvants,
spreading agents, sticking agents, stabilizing agents, or the like.
The herbicide formulation can be a wet or dry preparation and can
include, but is not limited to, flowable powders, emulsifiable
concentrates and liquid concentrates. The herbicide and herbicide
formulations can be applied in accordance with conventional
methods, for example, by spraying, irrigation, dusting, coating,
and the like.
[0037] The present invention provides seeds with increased
resistance to at least one herbicide, particularly an imidazolinone
herbicide. Such seeds include, for example, sunflower seeds that
are herbicide-resistant descendents of the MUT31 sunflower
line.
[0038] A "descendent" of the MUT31 sunflower line comprises any
plant, plant cell, or plant part that is derived by sexual and/or
asexual propagation from MUT31, representative seeds of MUT31
having been deposited with the ATCC and assigned ATCC Patent
Deposit No. PTA-7839. For example, such a descendent includes a
plant produced by crossing a first plant with a second plant to
produce a seed of the third plant (i.e., a descendent), wherein the
first plant is a MUT31 sunflower plant and the second is another
sunflower plant that is not a MUT31 sunflower plant. Such a
descendent also includes any plants that are descended from the
third plant whether produced by sexual and/or asexual propagation.
For example, cells, tissue or an organ from the third plant could
be used to produce a fourth plant by an asexual propagation method
including, but not limited to, in vitro plant cell, tissue, and
organ culture methods and methods involving the rooting of stem
cuttings.
[0039] For the present invention, a herbicide-resistant descendent
of MUT31 is a descendent of MUT31 that comprises the
herbicide-resistance characteristics of MUT31 by way of being
descended from one or more MUT31 sunflower plants. In other words,
such a herbicide-resistant descendent has inherited the
herbicide-resistance characteristics of a MUT31 sunflower plant by
sexual reproduction, asexual reproduction, or combination
thereof.
[0040] Unless otherwise clearly indicated or apparent from the
context, the "progeny" of a plant includes a plant of any
subsequent generation whose ancestry can be traced to that plant.
Similar, unless otherwise clearly indicated by context, the
"herbicide resistant progeny" of MUT31 includes a plant of any
subsequent generation whose ancestry can be traced to MUT31 and
that comprises the herbicide-resistance characteristics of MUT31 by
way of that ancestry.
[0041] The present invention provides methods for producing a
herbicide-resistant sunflower plant, particularly a
herbicide-resistant sunflower plant, through conventional plant
breeding involving sexual reproduction. The methods comprise
crossing a first plant comprises resistance to a herbicide to a
second plant that is not resistant to the herbicide. The first
plant can be any of the herbicide-resistant plants of the present
invention including, for example, sunflower plants that comprise
the herbicide-resistance characteristics of the MUT31 sunflower
plant, particularly MUT31 sunflower plants and herbicide-resistant
descendents of MUT31. The second plant can be any plant that is
capable of producing viable progeny plants (i.e., seeds) when
crossed with the first plant. Typically, but not necessarily, the
first and second plants are of the same species. The methods of the
invention can further involve one or more generations of
backcrossing the progeny plants of the first cross to a plant of
the same line or genotype as either the first or second plant.
Alternatively, the progeny of the first cross or any subsequent
cross can be crossed to a third plant that is of a different line
or genotype than either the first or second plant. The methods of
the invention can additionally involve selecting plants that
comprise the herbicide-resistance characteristics of the first
plant.
[0042] The present invention further provides methods for
increasing the herbicide-resistance of a plant, particularly a
herbicide-resistant sunflower plant, through conventional plant
breeding involving sexual reproduction. The methods comprise
crossing a first plant comprises resistance to a herbicide to a
second plant that may or may not be resistant to the herbicide or
may be resistant to a different herbicide or herbicides than the
first plant. The first plant can be any of the herbicide-resistant
sunflower plants of the present invention including, for example, a
MUT31 sunflower plants and a herbicide-resistant descendent
thereof. The second plant can be any plant that is capable of
producing viable progeny plants (i.e., seeds) when crossed with the
first plant. Typically, but not necessarily, the first and second
plants are of the same species. The progeny plants produced by this
method of the present invention have increased or enhanced
resistance to a herbicide when compared to either the first or
second plant or both. When the first and second plants are
resistant to different herbicides, the progeny plants will have the
combined herbicide-resistance characteristics of the first and
second plants. The methods of the invention can further involve one
or more generations of backcrossing the progeny plants of the first
cross to a plant of the same line or genotype as either the first
or second plant. Alternatively, the progeny of the first cross or
any subsequent cross can be crossed to a third plant that is of a
different line or genotype than either the first or second plant.
The methods of the invention can additionally involve selecting
plants that comprise the herbicide-resistance characteristics of
the first plant, the second plant, or both the first and the second
plant.
[0043] The present invention provides methods for enhancing or
increasing the resistance of a sunflower plant to at least one
imidazolinone herbicide. Imidazolinone herbicides are known as
AHAS-inhibiting herbicides because of their recognized ability to
inhibit AHAS activity in vivo and in vitro. In addition to
imidazolinone herbicides, AHAS-inhibiting herbicides include, for
example, sulfonylurea herbicides, triazolopyrimidine herbicides,
pyrimidinyloxybenzoate herbicides, and
sulfonylamino-carbonyltriazolinone herbicides.
[0044] In an embodiment of the invention, the methods involve
enhancing or increasing the resistance of a herbicide-resistant
sunflower plant that comprises resistance to an AHAS-inhibiting
herbicide, wherein the resistance to the AHAS-inhibiting herbicide
is due to one or more herbicide-resistant AHASL proteins. Such a
herbicide-resistant sunflower plant can be resistant to one or more
AHAS-inhibiting herbicides such as, for example, an imidazolinone
herbicide, a sulfonylurea herbicide, a triazolopyrimidine
herbicide, a pyrimidinyloxybenzoate herbicide, a
sulfonylamino-carbonyltriazolinone herbicide, or mixture thereof.
Examples of some suitable imidazolinone herbicides are described
above. Sulfonylurea herbicides include, but are not limited to,
chlorsulfuron, metsulfuron methyl, sulfometuron methyl, chlorimuron
ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron
methyl, nicosulfuron, ethametsulfuron methyl, rimsulfuron,
triflusulfuron methyl, triasulfuron, primisulfuron methyl,
cinosulfuron, amidosulfuron, flazasulfuron, imazosulfuron,
pyrazosulfuron ethyl, halosulfuron, azimsulfuron, cyclosulfuron,
ethoxysulfuron, flazasulfuron, flupyrsulfuron methyl,
foramsulfuron, iodosulfuron, oxasulfuron, mesosulfuron,
prosulfuron, sulfosulfuron, trifloxysulfuron, tritosulfuron, a
derivative of any of the aforementioned herbicides, and a mixture
of two or more of the aforementioned herbicides. The
triazolopyrimidine herbicides include, but are not limited to,
cloransulam, diclosulam, florasulam, flumetsulam, metosulam, and
penoxsulam. The pyrimidinyloxybenzoate herbicides of the invention
include, but are not limited to, bispyribac, pyrithiobac,
pyriminobac, pyribenzoxim and pyriftalid. The
sulfonylamino-carbonyltriazolinone herbicides include, but are not
limited to, flucarbazone and propoxycarbazone.
[0045] It is recognized that pyrimidinyloxybenzoate herbicides are
closely related to the pyrimidinylthiobenzoate herbicides and are
generalized under the heading of the latter name by the Weed
Science Society of America. Accordingly, the herbicides of the
present invention further include pyrimidinylthiobenzoate
herbicides, including, but not limited to, the
pyrimidinyloxybenzoate herbicides described above.
[0046] The sunflower plants of the present invention can be
non-transgenic or transgenic. Examples of non-transgenic sunflower
plants having increased resistance to at least one imidazolinone
herbicide include the MUT31 sunflower plant, representative seeds
of MUT31 having been deposited with the ATCC as Patent Deposit No.
PTA-7839; or mutant, recombinant, or a genetically engineered
derivative of MUT31; or of any progeny of MUT31; or a plant that is
a progeny of any of these plants; or a plant that comprises the
herbicide-resistance characteristics of MUT31, particularly a
herbicide-resistant descendent of MUT31. An example of a transgenic
sunflower plant having increased resistance to at least one
imidazolinone herbicide is a sunflower plant that is a genetically
engineered derivative of MUT31 that comprises the
herbicide-resistance characteristics of MUT31. Such a genetically
engineered derivative can comprises in its genome, for example, a
transgene of interest including, but not limited to, a
herbicide-resistant AHASL gene, a gene conferring disease
resistance, and a gene conferring insect resistance.
[0047] The present invention provides methods that involve the use
of an imidazolinone herbicide. In these methods, the imidazolinone
herbicide can be applied by any method known in the art including,
but not limited to, seed treatment, soil treatment, and foliar
treatment.
[0048] Prior to application, the imidazolinone herbicide can be
converted into the customary formulations, for example solutions,
emulsions, suspensions, dusts, powders, pastes and granules. The
use form depends on the particular intended purpose; in each case,
it should ensure a fine and even distribution of the compound
according to the invention.
[0049] The formulations are prepared in a known manner (see e.g.,
for review U.S. Pat. No. 3,060,084, EP-A 707 445 (for liquid
concentrates), Browning, "Agglomeration", Chemical Engineering,
Dec. 4, 1967, 147-48, Perry's Chemical Engineer's Handbook, 4th
Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO
91/13546, U.S. Pat. Nos. 4,172,714, 4,144,050, 3,920,442,
5,180,587, 5,232,701, 5,208,030, GB 2,095,558, U.S. Pat. no.
3,299,566, Klingman, Weed Control as a Science, John Wiley and
Sons, Inc., New York, 1961, Hance et al., Weed Control Handbook,
8th Ed., Blackwell Scientific Publications, Oxford, 1989 and
Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag
GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and
Technology of Agrochemical Formulations, Kluwer Academic
Publishers, Dordrecht, 1998 (ISBN 0-7514-0443-8), for example by
extending the active compound with auxiliaries suitable for the
formulation of agrochemicals, such as solvents and/or carriers, if
desired emulsifiers, surfactants and dispersants, preservatives,
antifoaming agents, anti-freezing agents, for seed treatment
formulation also optionally colorants and/or binders and/or gelling
agents.
[0050] Examples of suitable solvents are water, aromatic solvents
(for example Solvesso products, xylene), paraffins (for example
mineral oil fractions), alcohols (for example methanol, butanol,
pentanol, benzyl alcohol), ketones (for example cyclohexanone,
gamma-butyrolactone), pyrrolidones (NMP, NOP), acetates (glycol
diacetate), glycols, fatty acid dimethylamides, fatty acids and
fatty acid esters. In principle, solvent mixtures may also be
used.
[0051] Examples of suitable carriers are ground natural minerals
(for example kaolins, clays, talc, chalk) and ground synthetic
minerals (for example highly disperse silica, silicates).
[0052] Suitable emulsifiers are nonionic and anionic emulsifiers
(for example polyoxyethylene fatty alcohol ethers, alkylsulfonates
and arylsulfonates).
[0053] Examples of dispersants are lignin-sulfite waste liquors and
methylcellulose.
[0054] Suitable surfactants used are alkali metal, alkaline earth
metal and ammonium salts of lignosulfonic acid, naphthalenesulfonic
acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid,
alkylarylsulfonates, alkyl sulfates, alkylsulfonates, fatty alcohol
sulfates, fatty acids and sulfated fatty alcohol glycol ethers,
furthermore condensates of sulfonated naphthalene and naphthalene
derivatives with formaldehyde, condensates of naphthalene or of
naphthalenesulfonic acid with phenol and formaldehyde,
polyoxyethylene octylphenol ether, ethoxylated isooctylphenol,
octylphenol, nonylphenol, alkylphenol polyglycol ethers,
tributylphenyl polyglycol ether, tristearylphenyl polyglycol ether,
alkylaryl polyether alcohols, alcohol and fatty alcohol ethylene
oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl
ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycol
ether acetal, sorbitol esters, lignosulfite waste liquors and
methylcellulose.
[0055] Substances which are suitable for the preparation of
directly sprayable solutions, emulsions, pastes or oil dispersions
are mineral oil fractions of medium to high boiling point, such as
kerosene or diesel oil, furthermore coal tar oils and oils of
vegetable or animal origin, aliphatic, cyclic and aromatic
hydrocarbons, for example toluene, xylene, paraffin,
tetrahydronaphthalene, alkylated naphthalenes or their derivatives,
methanol, ethanol, propanol, butanol, cyclohexanol, cyclohexanone,
isophorone, highly polar solvents, for example dimethyl sulfoxide,
N-methylpyrrolidone or water.
[0056] Also anti-freezing agents such as glycerin, ethylene glycol,
propylene glycol and bactericides such as can be added to the
formulation.
[0057] Suitable antifoaming agents are for example antifoaming
agents based on silicon or magnesium stearate.
[0058] Suitable preservatives are for example Dichlorophen and
enzylalkoholhemiformal.
[0059] Seed Treatment formulations may additionally comprise
binders and optionally colorants.
[0060] Binders can be added to improve the adhesion of the active
materials on the seeds after treatment. Suitable binders are block
copolymers EO/PO surfactants but also polyvinylalcohols,
polyvinylpyrrolidones, polyacrylates, polymethacrylates,
polybutenes, polyisobutylenes, polystyrene, polyethyleneamines,
polyethyleneamides, polyethyleneimines (Lupasol.RTM.,
Polymin.RTM.), polyethers, polyurethanes, polyvinylacetate, tylose
and copolymers derived from these polymers.
[0061] Optionally, also colorants can be included in the
formulation. Suitable colorants or dyes for seed treatment
formulations are Rhodamin B, C.I. Pigment Red 112, C.I. Solvent Red
1, pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment
blue 15:1, pigment blue 80, pigment yellow 1, pigment yellow 13,
pigment red 112, pigment red 48:2, pigment red 48:1, pigment red
57:1, pigment red 53:1, pigment orange 43, pigment orange 34,
pigment orange 5, pigment green 36, pigment green 7, pigment white
6, pigment brown 25, basic violet 10, basic violet 49, acid red 51,
acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red
10, basic red 108.
[0062] An examples of a suitable gelling agent is carrageen
(Satiagel.RTM.).
[0063] Powders, materials for spreading, and dustable products can
be prepared by mixing or concomitantly grinding the active
substances with a solid carrier.
[0064] Granules, for example coated granules, impregnated granules
and homogeneous granules, can be prepared by binding the active
compounds to solid carriers. Examples of solid carriers are mineral
earths such as silica gels, silicates, talc, kaolin, attaclay,
limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous
earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground
synthetic materials, fertilizers, such as, for example, ammonium
sulfate, ammonium phosphate, ammonium nitrate, ureas, and products
of vegetable origin, such as cereal meal, tree bark meal, wood meal
and nutshell meal, cellulose powders and other solid carriers.
[0065] In general, the formulations comprise from 0.01 to 95% by
weight, preferably from 0.1 to 90% by weight, of the imidazolinone
herbicide. In this case, the imidazolinone herbicides are employed
in a purity of from 90% to 100% by weight, preferably 95% to 100%
by weight (according to NMR spectrum). For seed treatment purposes,
respective formulations can be diluted 2-10 fold leading to
concentrations in the ready to use preparations of 0.01 to 60% by
weight active compound by weight, preferably 0.1 to 40% by
weight.
[0066] The imidazolinone herbicide can be used as such, in the form
of their formulations or the use forms prepared therefrom, for
example in the form of directly sprayable solutions, powders,
suspensions or dispersions, emulsions, oil dispersions, pastes,
dustable products, materials for spreading, or granules, by means
of spraying, atomizing, dusting, spreading or pouring. The use
forms depend entirely on the intended purposes; they are intended
to ensure in each case the finest possible distribution of the
imidazolinone herbicide according to the invention.
[0067] Aqueous use forms can be prepared from emulsion
concentrates, pastes or wettable powders (sprayable powders, oil
dispersions) by adding water. To prepare emulsions, pastes or oil
dispersions, the substances, as such or dissolved in an oil or
solvent, can be homogenized in water by means of a wetter,
tackifier, dispersant or emulsifier. However, it is also possible
to prepare concentrates composed of active substance, wetter,
tackifier, dispersant or emulsifier and, if appropriate, solvent or
oil, and such concentrates are suitable for dilution with
water.
[0068] The active compound concentrations in the ready-to-use
preparations can be varied within relatively wide ranges. In
general, they are from 0.0001 to 10%, preferably from 0.01 to 1%
per weight.
[0069] The imidazolinone herbicide may also be used successfully in
the ultra-low-volume process (ULV), it being possible to apply
formulations comprising over 95% by weight of active compound, or
even to apply the active compound without additives.
[0070] The following are examples of formulations:
[0071] 1. Products for dilution with water for foliar applications.
For seed treatment purposes, such products may be applied to the
seed diluted or undiluted. [0072] A) Water-soluble concentrates
(SL, LS) Ten parts by weight of the imidazolinone herbicide are
dissolved in 90 parts by weight of water or a water-soluble
solvent. As an alternative, wetters or other auxiliaries are added.
The imidazolinone herbicide dissolves upon dilution with water,
whereby a formulation with 10% (w/w) of imidazolinone herbicide is
obtained. [0073] B) Dispersible concentrates (DC) Twenty parts by
weight of the imidazolinone herbicide are dissolved in 70 parts by
weight of cyclohexanone with addition of 10 parts by weight of a
dispersant, for example polyvinylpyrrolidone. Dilution with water
gives a dispersion, whereby a formulation with 20% (w/w) of
imidazolinone herbicide is obtained. [0074] C) Emulsifiable
concentrates (EC) Fifteen parts by weight of the imidazolinone
herbicide are dissolved in 7 parts by weight of xylene with
addition of calcium dodecylbenzenesulfonate and castor oil
ethoxylate (in each case 5 parts by weight). Dilution with water
gives an emulsion, whereby a formulation with 15% (w/w) of
imidazolinone herbicide is obtained. [0075] D) Emulsions (EW, EO,
ES) Twenty-five parts by weight of the imidazolinone herbicide are
dissolved in 35 parts by weight of xylene with addition of calcium
dodecylbenzenesulfonate and castor oil ethoxylate (in each case 5
parts by weight). This mixture is introduced into 30 parts by
weight of water by means of an emulsifier machine (e.g.,
Ultraturrax) and made into a homogeneous emulsion. Dilution with
water gives an emulsion, whereby a formulation with 25% (w/w) of
imidazolinone herbicide is obtained. [0076] E) Suspensions (SC, OD,
FS) In an agitated ball mill, 20 parts by weight of the
imidazolinone herbicide are comminuted with addition of 10 parts by
weight of dispersants, wetters and 70 parts by weight of water or
of an organic solvent to give a fine imidazolinone herbicide
suspension. Dilution with water gives a stable suspension of the
imidazolinone herbicide, whereby a formulation with 20% (w/w) of
imidazolinone herbicide is obtained. [0077] F) Water-dispersible
granules and water-soluble granules (WG, SG) Fifty parts by weight
of the imidazolinone herbicide are ground finely with addition of
50 parts by weight of dispersants and wetters and made as
water-dispersible or water-soluble granules by means of technical
appliances (for example extrusion, spray tower, fluidized bed).
Dilution with water gives a stable dispersion or solution of the
imidazolinone herbicide, whereby a formulation with 50% (w/w) of
imidazolinone herbicide is obtained. [0078] G) Water-dispersible
powders and water-soluble powders (WP, SP, SS, WS) Seventy-five
parts by weight of the imidazolinone herbicide are ground in a
rotor-stator mill with addition of 25 parts by weight of
dispersants, wetters and silica gel. Dilution with water gives a
stable dispersion or solution of the imidazolinone herbicide,
whereby a formulation with 75% (w/w) of imidazolinone herbicide is
obtained. [0079] I) Gel-Formulation (GF) In an agitated ball mill,
20 parts by weight of the imidazolinone herbicide are comminuted
with addition of 10 parts by weight of dispersants, 1 part by
weight of a gelling agent wetter and 70 parts by weight of water or
of an organic solvent to give a fine imidazolinone herbicide
suspension. Dilution with water gives a stable suspension of the
imidazolinone herbicide, whereby a formulation with 20% (w/w) of
imidazolinone herbicide is obtained. This gel formulation is
suitable for us as a seed treatment.
[0080] 2. Products to be applied undiluted for foliar applications.
For seed treatment purposes, such products may be applied to the
seed diluted. [0081] A) Dustable powders (DP, DS) Five parts by
weight of the imidazolinone herbicide are ground finely and mixed
intimately with 95 parts by weight of finely divided kaolin. This
gives a dustable product having 5% (w/w) of imidazolinone
herbicide. [0082] B) Granules (GR, FG, GG, MG) One-half part by
weight of the imidazolinone herbicide is ground finely and
associated with 95.5 parts by weight of carriers, whereby a
formulation with 0.5% (w/w) of imidazolinone herbicide is obtained.
Current methods are extrusion, spray-drying or the fluidized bed.
This gives granules to be applied undiluted for foliar use.
[0083] Conventional seed treatment formulations include for example
flowable concentrates FS, solutions LS, powders for dry treatment
DS, water dispersible powders for slurry treatment WS,
water-soluble powders SS and emulsion ES and EC and gel formulation
GF. These formulations can be applied to the seed diluted or
undiluted. Application to the seeds is carried out before sowing,
either directly on the seeds.
[0084] In a preferred embodiment a FS formulation is used for seed
treatment. Typically, a FS formulation may comprise 1-800 g/l of
active ingredient, 1-200 g/l Surfactant, 0 to 200 g/l antifreezing
agent, 0 to 400 g/l of binder, 0 to 200 g/l of a pigment and up to
1 liter of a solvent, preferably water.
[0085] The present invention provides seeds of the
herbicide-resistant plants of the present invention, particularly
seeds that are herbicide-resistant descendents of MUT31. For seed
treatment, seeds of the present invention are treated with
herbicides, preferably herbicides selected from the group
consisting of AHAS-inhibiting herbicides such as amidosulfuron,
azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron,
cinosulfuron, cyclosulfamuron, ethametsulfuron, ethoxysulfuron,
flazasulfuron, flupyrsulfuron, foramsulfuron, halosulfuron,
imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron,
nicosulfuron, oxasulfuron, primisulfuron, prosulfuron,
pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron,
thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron,
triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic,
imazapyr, imazaquin, imazethapyr, cloransulam, diclosulam,
florasulam, flumetsulam, metosulam, penoxsulam, bispyribac,
pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxim,
pyriftalid, pyrithiobac, and mixtures thereof, or with a
formulation comprising a AHAS-inhibiting herbicide. Preferably, the
AHAS-inhibiting herbicides of the present invention are
imidazolinone herbicides.
[0086] The term seed treatment comprises all suitable seed
treatment techniques known in the art, such as seed dressing, seed
coating, seed dusting, seed soaking, and seed pelleting.
[0087] In accordance with one variant of the present invention, a
further subject of the invention is a method of treating soil by
the application, in particular into the seed drill: either of a
granular formulation containing the imidazolinone herbicide as a
composition/formulation (e.g., a granular formulation, with
optionally one or more solid or liquid, agriculturally acceptable
carriers and/or optionally with one or more agriculturally
acceptable surfactants. This method is advantageously employed, for
example, in seedbeds of cereals, maize, cotton, and sunflower.
[0088] The present invention also comprises seeds coated with or
containing with a seed treatment formulation comprising at least
one ALS inhibitor selected from the group consisting of
amidosulfuron, azimsulfuron, bensulfuron, chlorimuron,
chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron,
ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron,
halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron,
metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron,
pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron,
thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron,
triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic,
imazapyr, imazaquin, imazethapyr, cloransulam, diclosulam,
florasulam, flumetsulam, metosulam, penoxsulam, bispyribac,
pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxim,
pyriftalid and pyrithiobac.
[0089] The term seed embraces seeds and plant propagules of all
kinds including but not limited to true seeds, seed pieces,
suckers, corms, bulbs, fruit, tubers, grains, cuttings, cut shoots
and the like and means in a preferred embodiment true seeds.
[0090] The term "coated with and/or containing" generally signifies
that the active ingredient is for the most part on the surface of
the propagation product at the time of application, although a
greater or lesser part of the ingredient may penetrate into the
propagation product, depending on the method of application. When
the said propagation product is (re)planted, it may absorb the
active ingredient.
[0091] The seed treatment application with the imidazolinone
herbicide or with a formulation comprising the imidazolinone
herbicide is carried out by spraying or dusting the seeds before
sowing of the plants and before emergence of the plants.
[0092] In the treatment of seeds, the corresponding formulations
are applied by treating the seeds with an effective amount of the
imidazolinone herbicide or a formulation comprising the
imidazolinone herbicide. Herein, the application rates are
generally from 0.1 g to 10 kg of the a.i. (or of the mixture of
a.i. or of the formulation) per 100 kg of seed, preferably from 1 g
to 5 kg per 100 kg of seed, in particular from 1 g to 2.5 kg per
100 kg of seed. For specific crops such as lettuce the rate can be
higher.
[0093] The present invention provides a method for combating
undesired vegetation or controlling weeds comprising contacting the
seeds of the resistant plants according to the present invention
before sowing and/or after pregermination with an imidazolinone
herbicide. The method can further comprise sowing the seeds, for
example, in soil in a field or in a potting medium in greenhouse.
The method finds particular use in combating undesired vegetation
or controlling weeds in the immediate vicinity of the seed.
[0094] The control of undesired vegetation is understood as meaning
the killing of weeds and/or otherwise retarding or inhibiting the
normal growth of the weeds. Weeds, in the broadest sense, are
understood as meaning all those plants which grow in locations
where they are undesired.
[0095] The weeds of the present invention include, for example,
dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds
include, but are not limited to, weeds of the genera: Sinapis,
Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga,
Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium,
Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium,
Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium,
Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver,
Centaurea, Trifolium, Ranunculus, and Taraxacum. Monocotyledonous
weeds include, but are not limited to, weeds of the genera:
Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca,
Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum,
Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria,
Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea,
Dactyloctenium, Agrostis, Alopecurus, and Apera.
[0096] In addition, the weeds of the present invention can include,
for example, crop plants that are growing in an undesired location.
For example, a volunteer maize plant that is in a field that
predominantly comprises soybean plants can be considered a weed, if
the maize plant is undesired in the field of soybean plants.
[0097] The articles "a" and "an" are used herein to refer to one or
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one or more
elements.
[0098] As used herein, the word "comprising," or variations such as
"comprises" or "comprising," will be understood to imply the
inclusion of a stated element, integer or step, or group of
elements, integers or steps, but not the exclusion of any other
element, integer or step, or group of elements, integers or steps.
The following examples are offered by way of illustration and not
by way of limitation.
EXAMPLE 1
Mutagenesis of Helianthus annuus Line RHA266 and Selection of
Imidazolinone-Resistant Plants
[0099] In the spring of the first growing season, forty rows of
sunflower (Helianthus annuus) line RHA266 were sown outdoors at the
Advanta Semillas Biotech Research Station in Balcarce, BsAs,
Argentina and then a portion of the plants were treated with ethyl
methanesulfonate (EMS, also referred to as methanesulfonic acid
ethyl ester). EMS is a known mutagen that typically induces
GC-to-AT transitions in DNA (Jander et al. (2003) Plant Physiol.
131:139-146). Plants were treated with a solution comprising 0.5%,
5%, or 10%, (w/v) EMS. For each EMS treatment, 13 rows of sunflower
plants were treated. Before flowering, all M.sub.0 plants were
bagged in order to ensure that the resulting M.sub.1 seeds were the
product of self-pollination. The seed heads from each EMS treatment
were harvested and threshed in bulk. In the following growing
season, the M.sub.1 seeds were sown outdoors with each treatment in
a separate plot. Twenty days later, when the plants were at the 2-4
leaf pair growth stage, all of the EMS-treated plants were sprayed
with 2.times. of SWEEPER 70DG (100 g a.i./ha). The active
ingredient in SWEEPER is imazamox. After the herbicide spraying, a
total of 54 plants survived and were selected as putative
resistant. Forty-four resistant plants reached flowering, produced
pollen, and M.sub.2 seed. The distribution of the forty-four
fertile resistant plants per EMS treatment is indicated in Table
1.
TABLE-US-00001 TABLE 1 Number of M1 Imidazolinone-Resistant
Sunflower Plants Recovered from Each EMS Treatment EMS
Concentration (%) No. of Resistant Plants Recovered 0.5 19 5.0 9 10
16
[0100] Tissue samples were taken from each individual surviving M1
plant and DNA from each sample was extracted for PCR amplification
and sequencing studies described below in Example 2.
[0101] M2 seeds that were produced by each of the forty-four
fertile M1 plants were sown in individual plots in Fargo, N.D. then
sprayed with 0.5.times. of SWEEPER 70DG (25 g ai/ha imazamox) at
the 2-4 leaf pair growth stage. One of the plots was selected as
homozygous tolerant and designated as MUT31. Nineteen M2 plants of
MUT31 were harvested, their M2:3 progenies sown in Balcarce in the
summer of 2003-2004, and the resulting plants allowed to mature and
then selfed. M4 seed from one plot was harvested and declared
breeder seed on the basis of phenotypic observations. (Breeder seed
is seed produced by the direct control of the plant breeder and is
the basis of the first and recurring increase of foundation
seeds.)
EXAMPLE 2
PCR Amplification and Sequencing of Sunflower Polynucleotides
Encoding Imidazolinone-Resistant and Wild-Type AHASL1 Proteins
[0102] To attempt to determine the origin of the imidazolinone
tolerance in the sunflower plants of Example 1, polymerase chain
reaction (PCR) amplification of genomic DNA was employed to amplify
the entire coding regions of each to the sunflower AHASL 1, AHASL2,
and AHASL3 genes. For the PCR amplifications, genomic DNA was
isolated from tissue of the M.sub.1 MUT31 sunflower plant. Control,
wild-type genomic DNA was also isolated from tissue of an RHA266
sunflower plant for PCR amplifications of each of the wild-type
AHASL genes. The resulting PCR products were sequenced to determine
the DNA sequences of the AHASL1, AHASL2, and AHASL3 genes from the
MUT31 and RHA266 plants.
[0103] Surprisingly, when the DNA sequences of the AHASL1, AHASL2,
and AHASL3 genes from MUT31 were aligned and compared to their
corresponding DNA sequences from RHA266, no differences were
detected (data not shown). While the present invention is not bound
by any particular biological mechanism, these results indicate that
the sunflower plants of the MUT31 comprise a novel
herbicide-resistance mechanism that is independent of a mutation or
mutations in one or more AHASL genes.
EXAMPLE 3
Analysis of Herbicide Detoxification by MUT31
[0104] To evaluate the detoxification ability of MUT31 sunflower
plants, an experiment was conducted in the greenhouse. The
objective of the experiment was to determine if the imazamox
tolerance of MUT31 plants is associated with detoxification
mechanism that is mediated by a P450 monooxygenase enzyme (referred
to herein as "P450 enzyme"). It was previously reported that the
organophosphate insecticide malathion
(diethyl-dimethoxythiophosphorylthio-succinate) specifically
inhibits P450 enzymes by blocking the herbicide detoxification
activity (Yu et al. (2004) Pest. Biochem. Physiol. 78:21-30). Thus,
plants comprising enhanced herbicide tolerance that is due to an
altered P450 enzyme are expected to become less tolerant or
susceptible to the herbicide when malthion is applied to the plants
before they are treated with the herbicide.
[0105] A factorial experiment with three factors: genotypes (MUT31
and RHA266), herbicide dose (Control, 0.25.times., and 0.50.times.;
where X=50 g ai/ha imazamox) and malathion (with or without
malathion) was arranged in a randomized split-split plot block
design. Imazamox (SWEEPER) was sprayed at the 3-4 leaf growth
stage. The P450 inhibitor malathion was sprayed at a rate of 1000 g
ai/ha 30 minutes prior to herbicide spraying. The evaluation of the
plants was carried out seven days after herbicide spraying, using
the criteria set forth in Table 2.
TABLE-US-00002 TABLE 2 Criteria for Herbicide Damage Scores for
Plant Evaluations Symptom Damage score Chlorosis, yellow flash
5-10% Growth rate reduction, internodes shortening 10-20% Leaf
deformations 20-30% Necrosis 30-45% Dead plant +50%
[0106] The application of malathion in the absence of herbicide had
no effect on the response of the MUT31 and RHA266 sunflower lines;
both the mean and variance were zero (Table 3). Both lines were
more tolerant (lower damage % score) when malathion was not sprayed
before imazamox. When the plants were treated with 0.5.times.
imazamox alone, MUT31 showed a significant increase in herbicide
tolerance with respect to the control RHA266. The herbicide
tolerance of MUT31 significantly decreased (higher score) after
malathion treatment (Table 3, FIG. 1). The results of the
statistical analysis are presented in Table 4.
[0107] The results of this factorial experiment indicate that
malathion inhibited the herbicide tolerance exhibited by MUT31 and
suggest that the herbicide tolerant phenotype of MUT31 may be due
to detoxification mechanism mediated by one or more altered P450
enzymes. Although the present invention does not depend on any
particular biological mechanism for enhanced herbicide resistance,
these results further suggest that the MUT31 sunflower plant
comprises in its genome one or more mutations in one or more genes
encoding P450 enzymes.
TABLE-US-00003 TABLE 3 Mean Herbicide Damage Score Values Herbicide
dose 0X (control) 0.25X 0.50X Malathion No Yes No Yes No Yes MUT31
0.00 0.00 9.50 30.35 20.13 40.31 RHA266 0.00 0.00 27.95 45.30 42.35
45.50
TABLE-US-00004 TABLE 4 Statistical Analysis of the Factorial
Experiment MUT-31 and RHA266 Source Df MeanSq F Pr (>F)
Significance Replicates 3 38.2 Malathion 1 1893.43 122.01 0.001589
** Error (a) 3 15.52 Herbicide dose 1 619.08 34.966 0.001041 **
Malathion .times. dose 1 110.45 6.2381 0.046678 * Error (b) 6 17.71
Line 1 1849.08 101.6794 3.27E-07 *** Malathion .times. Line 1
210.89 11.5969 0.005217 ** Dose .times. Line 1 17.93 0.9857
0.340397 Malathion .times. Dose .times. 1 91.63 5.0388 0.044418 *
Line Residuals 12 18.19
EXAMPLE 4
Herbicide Tolerance of Sunflower Lines with MUT31 and a
Herbicide-Tolerant AHASL Gene
[0108] A field trial was conducted to compare the herbicide
tolerance of sunflower hybrids carrying the MUT31 trait and the
A205V mutation in a sunflower AHASL gene (A205V/A205V). A sunflower
AHASL gene with the A205V mutation encodes a AHASL protein which
has a valine at amino acid that corresponds to amino acid position
205 in the Arabidopsis thaliana AHASL protein. The amino acid at
the same position in a wild-type sunflower AHASL protein is
alanine. In the amino acid sequence of the sunflower AHASL protein,
this alanine-to-valine amino acid substitution is at amino acid
position 190. By convention, the sites of amino acid substitutions
that are known to give rise to herbicide resistance in plant AHASL
proteins are typically referred to by the position of the
substitution in the amino acid sequence of the Arabidopsis AHASL
protein.
TABLE-US-00005 TABLE 5 Description of Sunflower Lines Tested Entry
Entry Type of Material Mut event Zygosity Description 1 IMI
Restorer A205V homo hybrid 2 IMI cms .times. IMI A205V homo hybrid
restorer 3 WT .times. IMI restorer A205V hetero hybrid 4 WT .times.
IMI restorer A205V hetero hybrid 5 A837 cms .times. IMI A205V
hetero hybrid restorer 6 IMI cms .times. MUT31 A205V/MUT31 hetero
hybrid restorer 7 WT -- -- line 8 MUT31 Restorer MUT31 homo
line
[0109] Seed from each entry was produced under optimum seed
production conditions in South America in 2005/2006. The field
trial was conducted at one location in North Dakota, USA in 2006.
The entries were organized in a randomized complete block using a
split plot design consisting of 3 replications for each treatment
combination. Factor A was the herbicide treatment, and factor B was
the sunflower entry. The plot size was 4 rows.times.12 ft and the
seeding rate was consistent with local agronomic practices. The
herbicide rates for each treatment for Entries 1-6 are shown in
Table 6. The herbicide rates for each treatment for Entries 8 are
shown in Table 7. The spray volume was 10 gallons per acre (GPA)
(or 100 liters/ha) for a backpack sprayer or 20 GPA (or 200
liters/ha) for a tractor mounted boom. The herbicide treatments
were applied at the 2-4 leaf growth stage.
TABLE-US-00006 TABLE 6 Factor A, Herbicide Treatment List for
Entries 1-6: Treatment No. Treatment 1 Untreated 2 50 g ai/ha
imazamox + 0.25% (v/v) NIS 3 100 g ai/ha imazamox + 0.25% (v/v) NIS
4 200 g ai/ha imazamox + 0.25% (v/v) NIS 5 160 g ai/ha imazapyr +
0.25% (v/v) NIS NIS = non-ionic surfactant
TABLE-US-00007 TABLE 7 Factor A, Herbicide Treatment List for Entry
8: Treatment No. Treatment 1 Untreated 2 12.5 g ai/ha imazamox +
0.25% (v/v) NIS 3 25 g ai/ha imazamox + 0.25% (v/v) NIS 4 37.5 g
ai/ha imazamox + 0.25% (v/v) NIS 5 80 g ai/ha imazapyr + 0.25%
(v/v) NIS NIS = non-ionic surfactant
[0110] Entry 7 (WT Maintainer line) was left unsprayed in all
treatment blocks. Each herbicide treatment was tested on a WT
border plot to ensure efficacy of the product (100% crop injury at
21 days post spray).
[0111] Phytotoxicity ratings were assessed at 7 days and 21 days
following herbicide application. Phytotoxicity was recorded as the
amount of plant damage (in percent), where a rating of `0`
indicated no damage to the plants in the plot relative to the
untreated plot. A rating of `100` indicated complete necrosis
(death) of the plants in the plot relative to the untreated
plot.
[0112] The data was subjected to an ANOVA analysis and the means
from the 3 repetitions are presented in Table 8 (phytotoxicity at
21 days post-treatment).
TABLE-US-00008 TABLE 8 Phytotoxicity Ratings (% Crop Injury)
recorded 21 Days after Treatment (DAT) 21 DAT 21 DAT 21 DAT 21 DAT
Type of Mut 50 G 100 G 200 G 160 G 21 DAT Entry Material event
Zygosity IMAZAMOX IMAZAMOX IMAZAMOX IMAZAPYR UNTREATED 1 IMI
Restorer A205 homo 5.00 6.67 15.00 6.67 0.00 2 IMI cms x A205 homo
0.00 3.33 21.67 3.33 0.00 IMI restorer 3 WT x IMI A205 hetero 6.67
25.00 73.33 20.00 0.00 restorer 4 WT x IMI A205 hetero 11.67 46.67
76.67 43.33 0.00 restorer 5 A837 cms x A205 hetero 3.33 40.00 78.33
36.67 0.00 IMI restorer 6 IMI cms x A205/ hetero 1.67 10.00 43.33
10.00 0.00 MUT31 MUT31 restorer 7 WT 0.00 0.00 0.00 0.00 0.00 21
DAT 21 DAT 21 DAT 21 DAT Type of Mut 12.5 G 25 G 37.5 G 80 G 21 DAT
Entry Material event Zygosity IMAZAMOX IMAZAMOX IMAZAMOX IMAZAPYR
UNTREATED 8 MUT31 MUT31 homo 15.00 25.00 46.67 88.33 0.00 Restorer
LSD = 8.89 CV = 56.78 St Dev = 5.55
[0113] The phytotoxicity in the heterozygous A205V entries (entries
3-5) was significantly higher than the double heterozygous
A205V/MUT31 entry (entry 6) at 21 days after treatment with
imazamox and imazapyr. The homozygous A205V entries (entries 1-2)
demonstrated the lowest levels of phytotoxicity or crop injury
(Table 1). At 100 g ai/ha of imazamox the range in phytotoxicity of
the A205V heterozygous entries was between 25% and 47% compared to
an injury rating of 10% for the A205V/MUT31 heterozygous entry. At
200 g ai/ha of imazamox the range in phytotoxicity of the A205V
heterozygous entries was between 73% and 78% injury compared to an
injury rating of 43% for the A205V/MUT31 heterozygous entry. With
160 g ai/ha of imazapyr the range in phytotoxicity of the A205V
heterozygous entries was between 20% and 43% injury compared to an
injury rating of 10% for the A205V/MUT31 heterozygous entry.
[0114] When MUT31 alone (entry 8) was challenged with 37.5 g ai/ha
of imazamox, it demonstrated an injury rating of 47% at 21 days
after treatment. From previous studies (data not shown), MUT31 has
demonstrated 100% crop injury at rates of 75 g ai of imazamox per
hectare and 100 g ai of imazapyr per hectare.
[0115] The double heterozygous A205V/MUT31 entries demonstrated
significantly higher herbicide tolerance to both imazamox and
imazapyr treatments versus the heterozygous A205V/-entries and
versus the MUT31 entry on its own.
[0116] Based on this data, MUT31 when stacked with the A205V
mutation in the heterozygous state provides stronger (enhanced)
herbicide tolerance than the A205V mutation in the heterozygous
state. Having a product that works in the heterozygous state at
2.times. x the commercial product rate (100 g ai imazamox/ha and
160 g ai imazapyr/ha) is a great advantage to sunflower hybrid
plant breeders over the current homozygous A205V/A205V product,
saving both time and resources in the breeding of imidazolinone
tolerant sunflowers.
[0117] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0118] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
611968DNAHelianthus annuusCDS(1)..(1968) 1atg gcg gct cct ccc aac
cct tcc atc tcc ttc aaa cca ccg tca ccc 48Met Ala Ala Pro Pro Asn
Pro Ser Ile Ser Phe Lys Pro Pro Ser Pro 1 5 10 15 gcc gcc gca ctg
cca cca cgc tcc gcc ttc ctc ccc cgt ttc gca tta 96Ala Ala Ala Leu
Pro Pro Arg Ser Ala Phe Leu Pro Arg Phe Ala Leu 20 25 30 ccc atc
act tcc act acc caa aaa cga cac cgt ctt cac atc tcc aat 144Pro Ile
Thr Ser Thr Thr Gln Lys Arg His Arg Leu His Ile Ser Asn 35 40 45
gtt ctc tcc gac tcc aaa tcc acc acc acc acc acc acc acc act caa
192Val Leu Ser Asp Ser Lys Ser Thr Thr Thr Thr Thr Thr Thr Thr Gln
50 55 60 cga ccg tta ccg gtg cag cct ttt gtc tcc cgt tac gcg cca
gat caa 240Arg Pro Leu Pro Val Gln Pro Phe Val Ser Arg Tyr Ala Pro
Asp Gln 65 70 75 80 ccg aga aaa ggc gca gac gtg ttg gtg gaa gct ctg
gaa cgg gaa ggt 288Pro Arg Lys Gly Ala Asp Val Leu Val Glu Ala Leu
Glu Arg Glu Gly 85 90 95 gtc acc gac gtc ttc gcc tac ccc ggc ggc
gcg tca atg gag atc cac 336Val Thr Asp Val Phe Ala Tyr Pro Gly Gly
Ala Ser Met Glu Ile His 100 105 110 caa gct ctc acg cgc tca agc act
atc cgc aat gtg ctc ccc cgt cac 384Gln Ala Leu Thr Arg Ser Ser Thr
Ile Arg Asn Val Leu Pro Arg His 115 120 125 gaa cag ggc ggc gtg ttc
gcc gcc gaa ggc tac gcg cgc gcc tcc ggt 432Glu Gln Gly Gly Val Phe
Ala Ala Glu Gly Tyr Ala Arg Ala Ser Gly 130 135 140 ctt ccc ggc gtg
tgt atc gcc act tcc ggt ccc gga gct acg aac cta 480Leu Pro Gly Val
Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu 145 150 155 160 gtt
agt ggt ctt gct gac gcg ctg tta gac agt gtc ccc atg gtg gca 528Val
Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Met Val Ala 165 170
175 atc acc ggt caa gtt ccc cgg aga atg atc gga acc gat gcg ttt caa
576Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln
180 185 190 gaa acc cca att gtt gag gta aca cgt tcg atc act aaa cat
aat tat 624Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His
Asn Tyr 195 200 205 ctt gtg ttg gat gtt gag gat att ccc aga att gtt
cgt gag gct ttt 672Leu Val Leu Asp Val Glu Asp Ile Pro Arg Ile Val
Arg Glu Ala Phe 210 215 220 tat ctt gcg agt tcg ggt cga ccc ggc ccg
gtt ttg ata gat gta ccg 720Tyr Leu Ala Ser Ser Gly Arg Pro Gly Pro
Val Leu Ile Asp Val Pro 225 230 235 240 aaa gat ata cag caa cag tta
gtg gtg ccg aaa tgg gat gaa ccg atg 768Lys Asp Ile Gln Gln Gln Leu
Val Val Pro Lys Trp Asp Glu Pro Met 245 250 255 agg tta ccg ggt tat
ttg tct aga atg ccg aag cct caa tat gat ggg 816Arg Leu Pro Gly Tyr
Leu Ser Arg Met Pro Lys Pro Gln Tyr Asp Gly 260 265 270 cat ttg gaa
cag att gtt agg ttg gtg ggg gaa gcg aag agg ccg gtt 864His Leu Glu
Gln Ile Val Arg Leu Val Gly Glu Ala Lys Arg Pro Val 275 280 285 ttg
tat gtg ggt ggt ggg tgt ttg aat tcg gat gat gag ttg agg cgg 912Leu
Tyr Val Gly Gly Gly Cys Leu Asn Ser Asp Asp Glu Leu Arg Arg 290 295
300 ttt gtg gag ctt acg ggg att ccg gtt gcg agt act ttg atg ggg ctc
960Phe Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu
305 310 315 320 gga gcg tac cct gct tcg agt gat ttg tcg ctt cat atg
ctt ggg atg 1008Gly Ala Tyr Pro Ala Ser Ser Asp Leu Ser Leu His Met
Leu Gly Met 325 330 335 cat ggt acg gtt tat gcg aat tat gcg gtt gat
aag agt gat ttg ttg 1056His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp
Lys Ser Asp Leu Leu 340 345 350 ctt gcg ttt ggg gtg cgg ttt gat gat
cgt gtg acg ggg aag ctt gag 1104Leu Ala Phe Gly Val Arg Phe Asp Asp
Arg Val Thr Gly Lys Leu Glu 355 360 365 gcg ttt gct agt agg gcg aag
att gtt cat att gat att gat cct gct 1152Ala Phe Ala Ser Arg Ala Lys
Ile Val His Ile Asp Ile Asp Pro Ala 370 375 380 gaa att ggg aag aat
aag cag cct cat gtg tcg att tgt ggt gat att 1200Glu Ile Gly Lys Asn
Lys Gln Pro His Val Ser Ile Cys Gly Asp Ile 385 390 395 400 aag gtc
gcg tta cag ggt ttg aac aag att ttg gag gaa aag aat tcg 1248Lys Val
Ala Leu Gln Gly Leu Asn Lys Ile Leu Glu Glu Lys Asn Ser 405 410 415
gtg act aat ctt gat ttt tcg acc tgg aga aag gaa ttg gat gaa caa
1296Val Thr Asn Leu Asp Phe Ser Thr Trp Arg Lys Glu Leu Asp Glu Gln
420 425 430 aaa atg aag ttc ccg ttg agc ttt aaa acg ttt ggc gaa gcg
att cct 1344Lys Met Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala
Ile Pro 435 440 445 cca cag tat gct att caa gtt ctt gat gag tta acg
ggc ggg aat gca 1392Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr
Gly Gly Asn Ala 450 455 460 att att agc acc ggt gtc ggg caa cat cag
atg tgg gct gct cag ttt 1440Ile Ile Ser Thr Gly Val Gly Gln His Gln
Met Trp Ala Ala Gln Phe 465 470 475 480 tac aaa tac aac aaa cct aga
caa tgg ctg acg tcg ggc ggg cta ggg 1488Tyr Lys Tyr Asn Lys Pro Arg
Gln Trp Leu Thr Ser Gly Gly Leu Gly 485 490 495 gca atg ggt ttc ggc
ctg ccc gct gct atc ggg gcg gcc gtt gca aga 1536Ala Met Gly Phe Gly
Leu Pro Ala Ala Ile Gly Ala Ala Val Ala Arg 500 505 510 cct gat gcg
gta gta gtt gac atc gac ggt gac gga agc ttt atg atg 1584Pro Asp Ala
Val Val Val Asp Ile Asp Gly Asp Gly Ser Phe Met Met 515 520 525 aat
gtt caa gag tta gcc aca atc cgt gtt gaa aat ctg ccg gtt aag 1632Asn
Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys 530 535
540 att tta tta ctt aac aac cag cat ttg ggt atg gtg gtt cag tgg gag
1680Ile Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp Glu
545 550 555 560 gat cgg ttt tac aag gcg aat cgg gct cat acc tac tta
gga aac ccg 1728Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu
Gly Asn Pro 565 570 575 tca aaa gag tcg gaa ata ttc cct aac atg gtg
aag ttt gct gaa gcc 1776Ser Lys Glu Ser Glu Ile Phe Pro Asn Met Val
Lys Phe Ala Glu Ala 580 585 590 tgt gat atc ccg gct gct cga gtg acc
caa aag gcg gat cta cga gca 1824Cys Asp Ile Pro Ala Ala Arg Val Thr
Gln Lys Ala Asp Leu Arg Ala 595 600 605 gct att cag aag atg ttg gat
aca ccc ggg cct tac ttg ttg gat gtg 1872Ala Ile Gln Lys Met Leu Asp
Thr Pro Gly Pro Tyr Leu Leu Asp Val 610 615 620 att gtg ccg cat caa
gaa cac gtg ttg ccc atg atc ccg gct ggc gga 1920Ile Val Pro His Gln
Glu His Val Leu Pro Met Ile Pro Ala Gly Gly 625 630 635 640 ggt ttc
tcg gat gtg atc acc gag ggt gat ggc aga acg aaa tat tga 1968Gly Phe
Ser Asp Val Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr 645 650 655
2655PRTHelianthus annuus 2Met Ala Ala Pro Pro Asn Pro Ser Ile Ser
Phe Lys Pro Pro Ser Pro 1 5 10 15 Ala Ala Ala Leu Pro Pro Arg Ser
Ala Phe Leu Pro Arg Phe Ala Leu 20 25 30 Pro Ile Thr Ser Thr Thr
Gln Lys Arg His Arg Leu His Ile Ser Asn 35 40 45 Val Leu Ser Asp
Ser Lys Ser Thr Thr Thr Thr Thr Thr Thr Thr Gln 50 55 60 Arg Pro
Leu Pro Val Gln Pro Phe Val Ser Arg Tyr Ala Pro Asp Gln 65 70 75 80
Pro Arg Lys Gly Ala Asp Val Leu Val Glu Ala Leu Glu Arg Glu Gly 85
90 95 Val Thr Asp Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile
His 100 105 110 Gln Ala Leu Thr Arg Ser Ser Thr Ile Arg Asn Val Leu
Pro Arg His 115 120 125 Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr
Ala Arg Ala Ser Gly 130 135 140 Leu Pro Gly Val Cys Ile Ala Thr Ser
Gly Pro Gly Ala Thr Asn Leu 145 150 155 160 Val Ser Gly Leu Ala Asp
Ala Leu Leu Asp Ser Val Pro Met Val Ala 165 170 175 Ile Thr Gly Gln
Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln 180 185 190 Glu Thr
Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr 195 200 205
Leu Val Leu Asp Val Glu Asp Ile Pro Arg Ile Val Arg Glu Ala Phe 210
215 220 Tyr Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu Ile Asp Val
Pro 225 230 235 240 Lys Asp Ile Gln Gln Gln Leu Val Val Pro Lys Trp
Asp Glu Pro Met 245 250 255 Arg Leu Pro Gly Tyr Leu Ser Arg Met Pro
Lys Pro Gln Tyr Asp Gly 260 265 270 His Leu Glu Gln Ile Val Arg Leu
Val Gly Glu Ala Lys Arg Pro Val 275 280 285 Leu Tyr Val Gly Gly Gly
Cys Leu Asn Ser Asp Asp Glu Leu Arg Arg 290 295 300 Phe Val Glu Leu
Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu 305 310 315 320 Gly
Ala Tyr Pro Ala Ser Ser Asp Leu Ser Leu His Met Leu Gly Met 325 330
335 His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ser Asp Leu Leu
340 345 350 Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys
Leu Glu 355 360 365 Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp
Ile Asp Pro Ala 370 375 380 Glu Ile Gly Lys Asn Lys Gln Pro His Val
Ser Ile Cys Gly Asp Ile 385 390 395 400 Lys Val Ala Leu Gln Gly Leu
Asn Lys Ile Leu Glu Glu Lys Asn Ser 405 410 415 Val Thr Asn Leu Asp
Phe Ser Thr Trp Arg Lys Glu Leu Asp Glu Gln 420 425 430 Lys Met Lys
Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro 435 440 445 Pro
Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr Gly Gly Asn Ala 450 455
460 Ile Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe
465 470 475 480 Tyr Lys Tyr Asn Lys Pro Arg Gln Trp Leu Thr Ser Gly
Gly Leu Gly 485 490 495 Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly
Ala Ala Val Ala Arg 500 505 510 Pro Asp Ala Val Val Val Asp Ile Asp
Gly Asp Gly Ser Phe Met Met 515 520 525 Asn Val Gln Glu Leu Ala Thr
Ile Arg Val Glu Asn Leu Pro Val Lys 530 535 540 Ile Leu Leu Leu Asn
Asn Gln His Leu Gly Met Val Val Gln Trp Glu 545 550 555 560 Asp Arg
Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro 565 570 575
Ser Lys Glu Ser Glu Ile Phe Pro Asn Met Val Lys Phe Ala Glu Ala 580
585 590 Cys Asp Ile Pro Ala Ala Arg Val Thr Gln Lys Ala Asp Leu Arg
Ala 595 600 605 Ala Ile Gln Lys Met Leu Asp Thr Pro Gly Pro Tyr Leu
Leu Asp Val 610 615 620 Ile Val Pro His Gln Glu His Val Leu Pro Met
Ile Pro Ala Gly Gly 625 630 635 640 Gly Phe Ser Asp Val Ile Thr Glu
Gly Asp Gly Arg Thr Lys Tyr 645 650 655 3655PRTHelianthus annuus
3Met Ala Ala Pro Pro Asn Pro Ser Ile Ser Phe Lys Pro Pro Ser Pro 1
5 10 15 Ala Ala Ala Leu Pro Pro Arg Ser Ala Phe Leu Pro Arg Phe Ala
Leu 20 25 30 Pro Ile Thr Ser Thr Thr Gln Lys Arg His Arg Leu His
Ile Ser Asn 35 40 45 Val Leu Ser Asp Ser Lys Ser Thr Thr Thr Thr
Thr Thr Thr Thr Gln 50 55 60 Arg Pro Leu Pro Val Gln Pro Phe Val
Ser Arg Tyr Ala Pro Asp Gln 65 70 75 80 Pro Arg Lys Gly Ala Asp Val
Leu Val Glu Ala Leu Glu Arg Glu Gly 85 90 95 Val Thr Asp Val Phe
Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile His 100 105 110 Gln Ala Leu
Thr Arg Ser Ser Thr Ile Arg Asn Val Leu Pro Arg His 115 120 125 Glu
Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ala Ser Gly 130 135
140 Leu Pro Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu
145 150 155 160 Val Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val Pro
Met Val Ala 165 170 175 Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly
Thr Asp Ala Phe Gln 180 185 190 Glu Thr Pro Ile Val Glu Val Thr Arg
Ser Ile Thr Lys His Asn Tyr 195 200 205 Leu Val Leu Asp Val Glu Asp
Ile Pro Arg Ile Val Arg Glu Ala Phe 210 215 220 Tyr Leu Ala Ser Ser
Gly Arg Pro Gly Pro Val Leu Ile Asp Val Pro 225 230 235 240 Lys Asp
Ile Gln Gln Gln Leu Val Val Pro Lys Trp Asp Glu Pro Met 245 250 255
Arg Leu Pro Gly Tyr Leu Ser Arg Met Pro Lys Pro Gln Tyr Asp Gly 260
265 270 His Leu Glu Gln Ile Val Arg Leu Val Gly Glu Ala Lys Arg Pro
Val 275 280 285 Leu Tyr Val Gly Gly Gly Cys Leu Asn Ser Asp Asp Glu
Leu Arg Arg 290 295 300 Phe Val Glu Leu Thr Gly Ile Pro Val Ala Ser
Thr Leu Met Gly Leu 305 310 315 320 Gly Ala Tyr Pro Ala Ser Ser Asp
Leu Ser Leu His Met Leu Gly Met 325 330 335 His Gly Thr Val Tyr Ala
Asn Tyr Ala Val Asp Lys Ser Asp Leu Leu 340 345 350 Leu Ala Phe Gly
Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu 355 360 365 Ala Phe
Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Pro Ala 370 375 380
Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser Ile Cys Gly Asp Ile 385
390 395 400 Lys Val Ala Leu Gln Gly Leu Asn Lys Ile Leu Glu Glu Lys
Asn Ser 405 410 415 Val Thr Asn Leu Asp Phe Ser Thr Trp Arg Lys Glu
Leu Asp Glu Gln 420 425 430 Lys Met Lys Phe Pro Leu Ser Phe Lys Thr
Phe Gly Glu Ala Ile Pro 435 440 445 Pro Gln Tyr Ala Ile Gln Val Leu
Asp Glu Leu Thr Gly Gly Asn Ala 450 455 460 Ile Ile Ser Thr Gly Val
Gly Gln His Gln Met Trp Ala Ala Gln Phe 465 470 475 480 Tyr Lys Tyr
Asn Lys Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly 485 490 495 Ala
Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ala Val Ala Arg 500
505
510 Pro Asp Ala Val Val Val Asp Ile Asp Gly Asp Gly Ser Phe Met Met
515 520 525 Asn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro
Val Lys 530 535 540 Ile Leu Leu Leu Asn Asn Gln His Leu Gly Met Val
Val Gln Trp Glu 545 550 555 560 Asp Arg Phe Tyr Lys Ala Asn Arg Ala
His Thr Tyr Leu Gly Asn Pro 565 570 575 Ser Lys Glu Ser Glu Ile Phe
Pro Asn Met Val Lys Phe Ala Glu Ala 580 585 590 Cys Asp Ile Pro Ala
Ala Arg Val Thr Gln Lys Ala Asp Leu Arg Ala 595 600 605 Ala Ile Gln
Lys Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val 610 615 620 Ile
Val Pro His Gln Glu His Val Leu Pro Met Ile Pro Ala Gly Gly 625 630
635 640 Gly Phe Ser Asp Val Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr
645 650 655 41968DNAHelianthus annuusCDS(1)..(1968) 4atg gcg gct
cct ccc aac cct tcc atc tcc ttc aaa cca ccg tca ccc 48Met Ala Ala
Pro Pro Asn Pro Ser Ile Ser Phe Lys Pro Pro Ser Pro 1 5 10 15 gcc
gcc gca ctg cca cca cgc tcc gcc ttc ctc ccc cgt ttc gca tta 96Ala
Ala Ala Leu Pro Pro Arg Ser Ala Phe Leu Pro Arg Phe Ala Leu 20 25
30 ccc atc act tcc act acc caa aaa cga cac cgt ctt cac atc tcc aat
144Pro Ile Thr Ser Thr Thr Gln Lys Arg His Arg Leu His Ile Ser Asn
35 40 45 gtt ctc tcc gac tcc aaa tcc acc acc acc acc acc acc acc
act caa 192Val Leu Ser Asp Ser Lys Ser Thr Thr Thr Thr Thr Thr Thr
Thr Gln 50 55 60 cga ccg tta ccg gtg cag cct ttt gtc tcc cgt tac
gcg cca gat caa 240Arg Pro Leu Pro Val Gln Pro Phe Val Ser Arg Tyr
Ala Pro Asp Gln 65 70 75 80 ccg aga aaa ggc gca gac gtg ttg gtg gaa
gct ctg gaa cgg gaa ggt 288Pro Arg Lys Gly Ala Asp Val Leu Val Glu
Ala Leu Glu Arg Glu Gly 85 90 95 gtc acc gac gtc ttc gcc tac ccc
ggc ggc gcg tca atg gag atc cac 336Val Thr Asp Val Phe Ala Tyr Pro
Gly Gly Ala Ser Met Glu Ile His 100 105 110 caa gct ctc acg cgc tca
agc act atc cgc aat gtg ctc ccc cgt cac 384Gln Ala Leu Thr Arg Ser
Ser Thr Ile Arg Asn Val Leu Pro Arg His 115 120 125 gaa cag ggc ggc
gtg ttc gcc gcc gaa ggc tac gcg cgc gcc tcc ggt 432Glu Gln Gly Gly
Val Phe Ala Ala Glu Gly Tyr Ala Arg Ala Ser Gly 130 135 140 ctt ccc
ggc gtg tgt atc gcc act tcc ggt ccc gga gct acg aac cta 480Leu Pro
Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu 145 150 155
160 gtt agt ggt ctt gct gac gcg ctg tta gac agt gtc ccc atg gtg gca
528Val Ser Gly Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Met Val Ala
165 170 175 atc acc ggt caa gtt ccc cgg aga atg atc gga acc gat gtg
ttt caa 576Ile Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Val
Phe Gln 180 185 190 gaa acc cca att gtt gag gta aca cgt tcg atc act
aaa cat aat tat 624Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr
Lys His Asn Tyr 195 200 205 ctt gtg ttg gat gtt gag gat att ccc aga
att gtt cgt gag gct ttt 672Leu Val Leu Asp Val Glu Asp Ile Pro Arg
Ile Val Arg Glu Ala Phe 210 215 220 tat ctt gcg agt tcg ggt cga ccc
ggc ccg gtt ttg ata gat gta ccg 720Tyr Leu Ala Ser Ser Gly Arg Pro
Gly Pro Val Leu Ile Asp Val Pro 225 230 235 240 aaa gat ata cag caa
cag tta gtg gtg ccg aaa tgg gat gaa ccg atg 768Lys Asp Ile Gln Gln
Gln Leu Val Val Pro Lys Trp Asp Glu Pro Met 245 250 255 agg tta ccg
ggt tat ttg tct aga atg ccg aag cct caa tat gat ggg 816Arg Leu Pro
Gly Tyr Leu Ser Arg Met Pro Lys Pro Gln Tyr Asp Gly 260 265 270 cat
ttg gaa cag att gtt agg ttg gtg ggg gaa gcg aag agg ccg gtt 864His
Leu Glu Gln Ile Val Arg Leu Val Gly Glu Ala Lys Arg Pro Val 275 280
285 ttg tat gtg ggt ggt ggg tgt ttg aat tcg gat gat gag ttg agg cgg
912Leu Tyr Val Gly Gly Gly Cys Leu Asn Ser Asp Asp Glu Leu Arg Arg
290 295 300 ttt gtg gag ctt acg ggg att ccg gtt gcg agt act ttg atg
ggg ctc 960Phe Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met
Gly Leu 305 310 315 320 gga gcg tac cct gct tcg agt gat ttg tcg ctt
cat atg ctt ggg atg 1008Gly Ala Tyr Pro Ala Ser Ser Asp Leu Ser Leu
His Met Leu Gly Met 325 330 335 cat ggt acg gtt tat gcg aat tat gcg
gtt gat aag agt gat ttg ttg 1056His Gly Thr Val Tyr Ala Asn Tyr Ala
Val Asp Lys Ser Asp Leu Leu 340 345 350 ctt gcg ttt ggg gtg cgg ttt
gat gat cgt gtg acg ggg aag ctt gag 1104Leu Ala Phe Gly Val Arg Phe
Asp Asp Arg Val Thr Gly Lys Leu Glu 355 360 365 gcg ttt gct agt agg
gcg aag att gtt cat att gat att gat cct gct 1152Ala Phe Ala Ser Arg
Ala Lys Ile Val His Ile Asp Ile Asp Pro Ala 370 375 380 gaa att ggg
aag aat aag cag cct cat gtg tcg att tgt ggt gat att 1200Glu Ile Gly
Lys Asn Lys Gln Pro His Val Ser Ile Cys Gly Asp Ile 385 390 395 400
aag gtc gcg tta cag ggt ttg aac aag att ttg gag gaa aag aat tcg
1248Lys Val Ala Leu Gln Gly Leu Asn Lys Ile Leu Glu Glu Lys Asn Ser
405 410 415 gtg act aat ctt gat ttt tcg acc tgg aga aag gaa ttg gat
gaa caa 1296Val Thr Asn Leu Asp Phe Ser Thr Trp Arg Lys Glu Leu Asp
Glu Gln 420 425 430 aaa atg aag ttc ccg ttg agc ttt aaa acg ttt ggc
gaa gcg att cct 1344Lys Met Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly
Glu Ala Ile Pro 435 440 445 cca cag tat gct att caa gtt ctt gat gag
tta acg ggc ggg aat gca 1392Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu
Leu Thr Gly Gly Asn Ala 450 455 460 att att agc acc ggt gtc ggg caa
cat cag atg tgg gct gct cag ttt 1440Ile Ile Ser Thr Gly Val Gly Gln
His Gln Met Trp Ala Ala Gln Phe 465 470 475 480 tac aaa tac aac aaa
cct aga caa tgg ctg acg tcg ggc ggg cta ggg 1488Tyr Lys Tyr Asn Lys
Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly 485 490 495 gca atg ggt
ttc ggc ctg ccc gct gct atc ggg gcg gcc gtt gca aga 1536Ala Met Gly
Phe Gly Leu Pro Ala Ala Ile Gly Ala Ala Val Ala Arg 500 505 510 cct
gat gcg gta gta gtt gac atc gac ggt gac gga agc ttt atg atg 1584Pro
Asp Ala Val Val Val Asp Ile Asp Gly Asp Gly Ser Phe Met Met 515 520
525 aat gtt caa gag tta gcc aca atc cgt gtt gaa aat ctg ccg gtt aag
1632Asn Val Gln Glu Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys
530 535 540 att tta tta ctt aac aac cag cat ttg ggt atg gtg gtt cag
tgg gag 1680Ile Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Val Gln
Trp Glu 545 550 555 560 gat cgg ttt tac aag gcg aat cgg gct cat acc
tac tta gga aac ccg 1728Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr
Tyr Leu Gly Asn Pro 565 570 575 tca aaa gag tcg gaa ata ttc cct aac
atg gtg aag ttt gct gaa gcc 1776Ser Lys Glu Ser Glu Ile Phe Pro Asn
Met Val Lys Phe Ala Glu Ala 580 585 590 tgt gat atc ccg gct gct cga
gtg acc caa aag gcg gat cta cga gca 1824Cys Asp Ile Pro Ala Ala Arg
Val Thr Gln Lys Ala Asp Leu Arg Ala 595 600 605 gct att cag aag atg
ttg gat aca ccc ggg cct tac ttg ttg gat gtg 1872Ala Ile Gln Lys Met
Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val 610 615 620 att gtg ccg
cat caa gaa cac gtg ttg ccc atg atc ccg gct ggc gga 1920Ile Val Pro
His Gln Glu His Val Leu Pro Met Ile Pro Ala Gly Gly 625 630 635 640
ggt ttc tcg gat gtg atc acc gag ggt gat ggc aga acg aaa tat tga
1968Gly Phe Ser Asp Val Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr 645
650 655 5655PRTHelianthus annuus 5Met Ala Ala Pro Pro Asn Pro Ser
Ile Ser Phe Lys Pro Pro Ser Pro 1 5 10 15 Ala Ala Ala Leu Pro Pro
Arg Ser Ala Phe Leu Pro Arg Phe Ala Leu 20 25 30 Pro Ile Thr Ser
Thr Thr Gln Lys Arg His Arg Leu His Ile Ser Asn 35 40 45 Val Leu
Ser Asp Ser Lys Ser Thr Thr Thr Thr Thr Thr Thr Thr Gln 50 55 60
Arg Pro Leu Pro Val Gln Pro Phe Val Ser Arg Tyr Ala Pro Asp Gln 65
70 75 80 Pro Arg Lys Gly Ala Asp Val Leu Val Glu Ala Leu Glu Arg
Glu Gly 85 90 95 Val Thr Asp Val Phe Ala Tyr Pro Gly Gly Ala Ser
Met Glu Ile His 100 105 110 Gln Ala Leu Thr Arg Ser Ser Thr Ile Arg
Asn Val Leu Pro Arg His 115 120 125 Glu Gln Gly Gly Val Phe Ala Ala
Glu Gly Tyr Ala Arg Ala Ser Gly 130 135 140 Leu Pro Gly Val Cys Ile
Ala Thr Ser Gly Pro Gly Ala Thr Asn Leu 145 150 155 160 Val Ser Gly
Leu Ala Asp Ala Leu Leu Asp Ser Val Pro Met Val Ala 165 170 175 Ile
Thr Gly Gln Val Pro Arg Arg Met Ile Gly Thr Asp Val Phe Gln 180 185
190 Glu Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr
195 200 205 Leu Val Leu Asp Val Glu Asp Ile Pro Arg Ile Val Arg Glu
Ala Phe 210 215 220 Tyr Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu
Ile Asp Val Pro 225 230 235 240 Lys Asp Ile Gln Gln Gln Leu Val Val
Pro Lys Trp Asp Glu Pro Met 245 250 255 Arg Leu Pro Gly Tyr Leu Ser
Arg Met Pro Lys Pro Gln Tyr Asp Gly 260 265 270 His Leu Glu Gln Ile
Val Arg Leu Val Gly Glu Ala Lys Arg Pro Val 275 280 285 Leu Tyr Val
Gly Gly Gly Cys Leu Asn Ser Asp Asp Glu Leu Arg Arg 290 295 300 Phe
Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu 305 310
315 320 Gly Ala Tyr Pro Ala Ser Ser Asp Leu Ser Leu His Met Leu Gly
Met 325 330 335 His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ser
Asp Leu Leu 340 345 350 Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val
Thr Gly Lys Leu Glu 355 360 365 Ala Phe Ala Ser Arg Ala Lys Ile Val
His Ile Asp Ile Asp Pro Ala 370 375 380 Glu Ile Gly Lys Asn Lys Gln
Pro His Val Ser Ile Cys Gly Asp Ile 385 390 395 400 Lys Val Ala Leu
Gln Gly Leu Asn Lys Ile Leu Glu Glu Lys Asn Ser 405 410 415 Val Thr
Asn Leu Asp Phe Ser Thr Trp Arg Lys Glu Leu Asp Glu Gln 420 425 430
Lys Met Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro 435
440 445 Pro Gln Tyr Ala Ile Gln Val Leu Asp Glu Leu Thr Gly Gly Asn
Ala 450 455 460 Ile Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala
Ala Gln Phe 465 470 475 480 Tyr Lys Tyr Asn Lys Pro Arg Gln Trp Leu
Thr Ser Gly Gly Leu Gly 485 490 495 Ala Met Gly Phe Gly Leu Pro Ala
Ala Ile Gly Ala Ala Val Ala Arg 500 505 510 Pro Asp Ala Val Val Val
Asp Ile Asp Gly Asp Gly Ser Phe Met Met 515 520 525 Asn Val Gln Glu
Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys 530 535 540 Ile Leu
Leu Leu Asn Asn Gln His Leu Gly Met Val Val Gln Trp Glu 545 550 555
560 Asp Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro
565 570 575 Ser Lys Glu Ser Glu Ile Phe Pro Asn Met Val Lys Phe Ala
Glu Ala 580 585 590 Cys Asp Ile Pro Ala Ala Arg Val Thr Gln Lys Ala
Asp Leu Arg Ala 595 600 605 Ala Ile Gln Lys Met Leu Asp Thr Pro Gly
Pro Tyr Leu Leu Asp Val 610 615 620 Ile Val Pro His Gln Glu His Val
Leu Pro Met Ile Pro Ala Gly Gly 625 630 635 640 Gly Phe Ser Asp Val
Ile Thr Glu Gly Asp Gly Arg Thr Lys Tyr 645 650 655
6655PRTHelianthus annuus 6Met Ala Ala Pro Pro Asn Pro Ser Ile Ser
Phe Lys Pro Pro Ser Pro 1 5 10 15 Ala Ala Ala Leu Pro Pro Arg Ser
Ala Phe Leu Pro Arg Phe Ala Leu 20 25 30 Pro Ile Thr Ser Thr Thr
Gln Lys Arg His Arg Leu His Ile Ser Asn 35 40 45 Val Leu Ser Asp
Ser Lys Ser Thr Thr Thr Thr Thr Thr Thr Thr Gln 50 55 60 Arg Pro
Leu Pro Val Gln Pro Phe Val Ser Arg Tyr Ala Pro Asp Gln 65 70 75 80
Pro Arg Lys Gly Ala Asp Val Leu Val Glu Ala Leu Glu Arg Glu Gly 85
90 95 Val Thr Asp Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile
His 100 105 110 Gln Ala Leu Thr Arg Ser Ser Thr Ile Arg Asn Val Leu
Pro Arg His 115 120 125 Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr
Ala Arg Ala Ser Gly 130 135 140 Leu Pro Gly Val Cys Ile Ala Thr Ser
Gly Pro Gly Ala Thr Asn Leu 145 150 155 160 Val Ser Gly Leu Ala Asp
Ala Leu Leu Asp Ser Val Pro Met Val Ala 165 170 175 Ile Thr Gly Gln
Val Pro Arg Arg Met Ile Gly Thr Asp Val Phe Gln 180 185 190 Glu Thr
Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr 195 200 205
Leu Val Leu Asp Val Glu Asp Ile Pro Arg Ile Val Arg Glu Ala Phe 210
215 220 Tyr Leu Ala Ser Ser Gly Arg Pro Gly Pro Val Leu Ile Asp Val
Pro 225 230 235 240 Lys Asp Ile Gln Gln Gln Leu Val Val Pro Lys Trp
Asp Glu Pro Met 245 250 255 Arg Leu Pro Gly Tyr Leu Ser Arg Met Pro
Lys Pro Gln Tyr Asp Gly 260 265 270 His Leu Glu Gln Ile Val Arg Leu
Val Gly Glu Ala Lys Arg Pro Val 275 280 285 Leu Tyr Val Gly Gly Gly
Cys Leu Asn Ser Asp Asp Glu Leu Arg Arg 290 295 300 Phe Val Glu Leu
Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu 305 310 315 320 Gly
Ala Tyr Pro Ala Ser Ser Asp Leu Ser Leu His Met Leu Gly Met 325 330
335 His Gly Thr Val Tyr Ala Asn Tyr Ala Val Asp Lys Ser Asp Leu Leu
340 345 350 Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys
Leu Glu 355
360 365 Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Pro
Ala 370 375 380 Glu Ile Gly Lys Asn Lys Gln Pro His Val Ser Ile Cys
Gly Asp Ile 385 390 395 400 Lys Val Ala Leu Gln Gly Leu Asn Lys Ile
Leu Glu Glu Lys Asn Ser 405 410 415 Val Thr Asn Leu Asp Phe Ser Thr
Trp Arg Lys Glu Leu Asp Glu Gln 420 425 430 Lys Met Lys Phe Pro Leu
Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro 435 440 445 Pro Gln Tyr Ala
Ile Gln Val Leu Asp Glu Leu Thr Gly Gly Asn Ala 450 455 460 Ile Ile
Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe 465 470 475
480 Tyr Lys Tyr Asn Lys Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly
485 490 495 Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ala Val
Ala Arg 500 505 510 Pro Asp Ala Val Val Val Asp Ile Asp Gly Asp Gly
Ser Phe Met Met 515 520 525 Asn Val Gln Glu Leu Ala Thr Ile Arg Val
Glu Asn Leu Pro Val Lys 530 535 540 Ile Leu Leu Leu Asn Asn Gln His
Leu Gly Met Val Val Gln Trp Glu 545 550 555 560 Asp Arg Phe Tyr Lys
Ala Asn Arg Ala His Thr Tyr Leu Gly Asn Pro 565 570 575 Ser Lys Glu
Ser Glu Ile Phe Pro Asn Met Val Lys Phe Ala Glu Ala 580 585 590 Cys
Asp Ile Pro Ala Ala Arg Val Thr Gln Lys Ala Asp Leu Arg Ala 595 600
605 Ala Ile Gln Lys Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val
610 615 620 Ile Val Pro His Gln Glu His Val Leu Pro Met Ile Pro Ala
Gly Gly 625 630 635 640 Gly Phe Ser Asp Val Ile Thr Glu Gly Asp Gly
Arg Thr Lys Tyr 645 650 655
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