U.S. patent application number 11/537495 was filed with the patent office on 2008-04-10 for rps gene family, primers, probes and detection methods.
Invention is credited to Frederick M. AUSUBEL, Barbara Baker, Andrew F. Bent, Douglas Dahlbeck, Jeffrey Ellis, Fumiaki Katagiri, Barbara N. Kunkel, Michael Nicholas, John Salmeron, Brian J. Staskawicz, Guo-Liang Yu.
Application Number | 20080085835 11/537495 |
Document ID | / |
Family ID | 22852788 |
Filed Date | 2008-04-10 |
United States Patent
Application |
20080085835 |
Kind Code |
A1 |
AUSUBEL; Frederick M. ; et
al. |
April 10, 2008 |
RPS GENE FAMILY, PRIMERS, PROBES AND DETECTION METHODS
Abstract
Disclosed is substantially pure DNA encoding an Arabidopsis
thaliana Rps2 polypeptide, substantially pure Rps2 polyneptide; and
methods of using such DNA to express the Rps2 polypeptide in plant
cells and whole plants to provide, in transgenic plants, disease
resistance to pathogens. Also disclosed are conserved regions
characteristic of the RPS family and primers and probes for the
identification and isolation of additional RPS disease-resistance
genes.
Inventors: |
AUSUBEL; Frederick M.;
(Newton, MA) ; Staskawicz; Brian J.; (Castro
Valley, CA) ; Bent; Andrew F.; (Madison, WI) ;
Dahlbeck; Douglas; (Castro Valley, CA) ; Katagiri;
Fumiaki; (San Diego, CA) ; Kunkel; Barbara N.;
(St. Louis, MO) ; Nicholas; Michael; (Menlo Park,
CA) ; Yu; Guo-Liang; (Berkeley, CA) ; Baker;
Barbara; (Berkeley, CA) ; Ellis; Jeffrey;
(Weetangera, AU) ; Salmeron; John; (Hillsborough,
NC) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
22852788 |
Appl. No.: |
11/537495 |
Filed: |
September 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10613765 |
Jul 2, 2003 |
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11537495 |
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09867852 |
May 29, 2001 |
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10613765 |
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09301085 |
Apr 28, 1999 |
6262248 |
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09867852 |
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08310912 |
Sep 22, 1994 |
5981730 |
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09301085 |
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08227360 |
Apr 13, 1994 |
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08310912 |
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Current U.S.
Class: |
506/2 ; 435/6.12;
435/6.13; 536/25.4 |
Current CPC
Class: |
C12N 15/8281 20130101;
C12Q 1/6895 20130101; C07K 14/415 20130101; C12Q 2600/13 20130101;
C12Q 2600/156 20130101 |
Class at
Publication: |
506/2 ; 435/6;
536/25.4 |
International
Class: |
C40B 20/00 20060101
C40B020/00; C07H 21/00 20060101 C07H021/00; C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0001] This invention was made in part with Government funding and
the Government therefore has certain rights in the invention.
Claims
1. A method of isolating a disease resistance gene or portion
thereof in plants having sequence identity to RPS2, said method
comprising: amplifying by PCR said disease resistance gene or
portion thereof using oligonucleotide primers wherein said primers
(a) are each greater than 13 nucleotides in length; (b) each have
regions of complementarily to opposite DNA strands in a region of
the nucleotide sequence of FIG. 2; and (c) optionally contain
sequences capable of producing restriction enzyme cut sites in the
amplified product; and isolating said disease resistance gene or
portion thereof.
2. A method of isolating a disease-resistance gene or fragment
thereof from a plant cell, comprising: (a) providing a sample of
plant cell DNA; (b) providing a pair of oligonucleotides having
sequence homology to a conserved region of an RPS
disease-resistance gene; (c) combining said pair of
oligonucleotides with said plant cell DNA sample under conditions
suitable for polymerase chain reaction-mediated DNA amplification;
and (d) isolating said amplified disease-resistance gene or
fragment thereof.
3. The method of claim 1, wherein said amplification is carried out
using a reverse-transcription polymerase chain reaction.
4. The method of claim 1, wherein said reverse-transcription
polymerase chain reaction is RACE.
5. A method of identifying a plant disease-resistance gene in a
plant cell, comprising: (a) providing a preparation of plant cell
DNA; (b) providing a detectably-labelled DNA sequence having
homology to a conserved region of an RPS gene; (c) contacting said
preparation of plant cell DNA with said detectablly-labelled DNA
sequence under hybridization conditions providing detection of
genes having 50% or greater sequence identity; and (d) identifying
a disease-resistance gene by its association with said detectable
label.
6. The method of claim 5, wherein said DNA sequence is produced
according to the method of claim 1.
7. The method of claim 5, wherein said preparation of plant cell
DNA is isolated from a plant genome.
8. A method of isolating a disease-resistance gene or portion
thereof from a plant cell, plant tissue sample, or recombinant
plant cell library, comprising the steps of: (a) providing DNA
sample from a plant cell, plant tissue, or recombinant plant cell
library; (b) providing a pair of oligonucleotides having sequence
homology to any one of SEQ ID NOs: 110-113, 145-156, 158-162,
209-211, or 213; (c) combining said pair of oligonucleotides with
said DNA sample under conditions suitable for polymerase chain
reaction-mediated DNA amplification; and (d) isolating said
amplified disease-resistance gene or portion thereof.
Description
BACKGROUND OF THE INVENTION
[0002] This application is a continuation-in-part of application
Ser. No. 08/227,360, filed Apr. 13, 1994.
[0003] The invention relates to recombinant plant nucleic acids and
polypeptides and uses thereof to confer disease resistance to
pathogens in transgenic plants.
[0004] Plants employ a variety of defensive strategies to combat
pathogens. One defense response, the so-called hypersensitive
response (HR), involves rapid localized necrosis of infected
tissue. In several host-pathogen interactions, genetic analysis has
revealed a gene-for-gene correspondence between a particular
avirulence (avr) gene in an avirulent pathogen that elicits an HR
in a host possessing a particular resistance gene.
SUMMARY OF THE INVENTION
[0005] In general, the invention features substantially pure DNA
(for example, genomic DNA, CDNA, or synthetic DNA) encoding an Rps
polypeptide as- defined below. In related aspects, the invention
also features a vector, a cell (e.g., a plant cell) , and a
transgenic plant or seed thereof which includes such a
substantially pure DNA encoding an Rps polypeptide.
[0006] In preferred embodiments, an RPS gene is the RPS2 gene of a
plant of the genus Arabidopsis. In various referred embodiments,
the cell is a transformed plant cell derived from a cell of a
transgenic plant. In related aspects, the invention, features a
transgenic plant containing a transgene which encodes an Rps
polypeptide that S is expressed in plant tissue susceptible to
infection by pathogens expressing the avrRpt2 avirulence gene or
pathogens expressing an avirulence signal similarly recognized by
an Rps polypeptide.
[0007] In a second aspect, the invention features a substantially
pure DNA which includes a promoter capable of expressing the RPS2
gene in plant tissue susceptible to infection by bacterial
pathogens expressing the avrRpt2 avirulence gene.
[0008] In preferred embodiments, the promoter is the promoter
native to an RPS gene. Additionally, transcriptional and
translational regulatory regions are preferably native to an RPS
gene.
[0009] The transgenic plants of the invention are preferably plants
which are susceptible to infection by a pathogen expressing an
avirulence gene, preferably the avrRpt2 avirulence gene. In
preferred embodiments the transgenic plant is from the group of
plants consisting of but not limited to Arabidopsis, tomato,
soybean, bean, maize, wheat and rice.
[0010] In another aspect, the invention features-a method of
providing resistance in a plant to a pathogen which involves. (a)
producing a transgenic plant cell having a transgene encoding an
Rps2 polypeptide wherein the transgene is integrated into the
genome of the transgenic plant and is positioned for expression in
the plant cell; and (b) growing a transgenic plant from the
transgenic plant cell wherein the RPS2 transgene is expressed in
the transgenic plant.
[0011] In another aspect, the invention features a method of
detecting a resistance gene in a plant cell involving: (a)
contacting the RPS2 gene or a portion thereof greater than 9
nucleic acids, pref erably greater than 18 nucleic acids in length
with a preparation of genomic DNA from the plant cell under
hybridization conditions providing detection of DNA sequences
having about 50% or greater sequence identity to the DNA sequence
of FIG. 2 encoding the RTps2 polypeptide.
[0012] In another aspect, the invention features a method of
producing an Rps2 polypeptide which involves: (a) providing a cell
transformed with DNA encoding an Rps2 polypeptide positioned for
expression in the cell; (b) culturing the transformed cell under
conditions for expressing the DNA, and (c) isolating the Rps2
polypeptide.
[0013] In another aspect, the invention features substantially pure
Rps2 polypeptide. Preferably, the polypeptide includes a greater
than 50 amino acid sequence substantially identical to a greater
than 50 amino acid sequence shown in FIG. 2, open reading frame
"a". Most preferably, the polypeptide is the Arabidopsis thaliana
Rps2 polypeptide.
[0014] In another aspect, the invention features a method of
providing resistance in a transgenic plant to infection by
pathogens which do not carry the avrRpt2 avirulence gene wherein
the method includes: (a) producing a transgenic plant cell having
transgenes encoding an Rps2 polypeptide as well as a transgene
encoding the avrRpt2 gene product wherein the transgenes are
integrated into the genome of the transgenic plant;- are positioned
for expression in the plant cell; and the avrRpt2 transgene and, if
desired, the RPS2 gene, are under the control of regulatory
sequences suitable for controlled expression of the gene(s); and
(b) growing a transgenic plant from the transgenic plant cell
wherein the RPS2 and avrRpt2 transgenes are expressed in the
transgenic plant.
[0015] In another aspect, the invention features a method of
providing resistance in a transgenic plant to infection by
pathogens in the absence of avirulence gene expression in the
pathogen wherein the method involves: (a) producing a transgenic
plant cell having integrated in the genome a transgene containing
the RPS2. gene under the control of a promoter providing
constitutive expression of the RPS2 gene; and (b) growing a
transgenic plant from the transgenic plant cell wherein the RPS2
transgene is expressed constitutively in the transgenic plant.
[0016] In another aspect, the invention features a method of
providing controllable resistance in a transgenic plant to
infection by pathogens in the absence of avirulence gene expression
in the pathogen wherein the method involves: (a) producing a
transgenic plant cell having integrated in the genome a transgene
containing the RPS2 gene under the control of a promoter providing
controllable expression of the RPS2 gene; and (b) growing a
transgenic plant from the transgenic plant cell wherein the RPS2
transgene is controllably expressed in the transgenic plant. In
preferred embodiments, the RPS2 gene is expressed using a
tissue-specific or cell type-specific promoter, or by a promoter
that is activated by the introduction of an external signal or
agent, such as a chemical signal or agent.
[0017] In other aspects, the invention features a substantially
pure oligonucleotide including one or a combination of the
sequences:
[0018] 5' GGNATGGGNGGNNTNGGNAARACNAC 3', wherein N is A, T, G, or
C; and R is A or G;
[0019] 5' NARNGGNARNCC 3', wherein N is A, T, G or C; and R is A or
G;
[0020] 5'NCGNGWNGTNAKDAWNCGNGA 3', wherein N is A, T, G or C; W is
A or T; D is A, G, or T; and K is G or T;
[0021] 5' GGWNTBGGWAARACHAC 3', wherein N is A, T, G or C; is G or
A; B is Cf G, or T; H is A, C, or T; and W is A or T;
[0022] 5' TYGAYGAYRTBKRBRA 3', wherein R is G( or A; B is C, G, or
T; D is A, G, or T; Y is T or C; and K is G or T;
[0023] 5' TYCCAVAYRTCRTCMA 3', wherein N is A, T, G or C; R is G or
A; V is G or C or A; and Y is T or C;
[0024] 5' GGWYTCCWYTGCHYT 3', wherein B is C, G, or T; E is A, C,
or T; W is A or T; and Y is T or C;
[0025] 5' ARDGCVARWGGVARNCC 3', wherein N is A, T, G or C; R is G
or A; W is A or T; D is A, G, or T; and V is G, C, or A; and
[0026] 5' ARRTTRCRTADSWRAWYTT 3', wherein R is G or A; W is A or T;
D is A, G, or T;. S is G or C: and Y is C or T.
[0027] In other aspects, the invention features a recombinant plant
gene including one or a combination of the DNA sequences:
[0028] 5' GGATGGGNGGNNTNGNAARACNAC 3', wherein N is A, T, G or C;
and R is A or G;
[0029] 5' NARNOCNARNCC 3', wherein N is A, T, G or C; and R is A or
G.;
[0030] 5' NCGNGWNGTNAKDAWNCGNOA 3', wherein N is A, T, G or C; W is
A or T; D is A, G or T; and K is G or T.
[0031] In another aspect, the invention features a substantially
pure plant polypeptide including one or a combination of the amino
acid sequences:
[0032] Gly Xaa.sub.1 Xaa.sub.2 Gly Xaa.sub.3 Gly Lys Thr Thr
Xaa.sub.4 Xaa.sub.5, wherein Xaa.sub.1 is Met or Pro; Xaa.sub.2 is
Gly or Pro; Xaa.sub.3 is Ile, Leu, or Val; Xaa.sub.4 is Ile, Leu,
or Thr; and Xaa.sub.5 is Ala or Met;
[0033] Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Leu Xaa.sub.4 Xaa.sub.5 Xaa,
Asp Asp Xaa.sub.7 Xaa.sub.8, wherein Xaa.sub.1 is Phe or Lys;
Xaa.sub.2 is Arg or Lys; Xaa.sub.3 is Ile, Val, or Phe; Xaa.sub.4
is Ile, Leu, or Val; Xaa.sub.5 is Ile or Leu; Xaa.sub.6 is Ile or
Val; Xaa.sub.7 is Ile, Leu, or Val; and Xaa.sub.8 is Asp or
Trp;
[0034] Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Thx
Xaa.sub.6 Arg, wherein Xaa.sub.1 is Ser or Cys; Xaa.sub.2 is Arg or
Lys; Xaa.sub.3 is Phe, Ile, or Val; Xaa.sub.4 is Ile, or Met;
Xaa.sub.5 is Ile, Leu, or Phe; Xaa.sub.6 is Ser, Cys, or Thr;
[0035] Gly Leu Pro Leu Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4,
wherein Xaa.sub.1 is Thr, Ala, or Ser; Xaa.sub.2 is Leu or Val;
Xaa.sub.3 is Ile, Val, or Lys; and Xaa.sub.4 is Val or Thr; and
[0036] Xaa.sub.1 Xaa.sub.2 Ser Tyr Xaa.sub.3 Xaa.sub.4 Leu, wherein
Xaa.sub.1 is Lys or Gly, Xaa.sub.2 is Ile or Phe; Xaa.sub.3 is Asp
or Lys; and Xaa.sub.4 is Ala, Gly, or Asn.
[0037] In another aspect, the invention features a method of
isolating a disease-resistance gene or fragment thereof from a
plant cell, involving: (a) providing a sample of plant cell DNA;
(b) providing a pair of oligonucleotides having sequence homology
to a conserved region of an RPS disease-resistance gene; (c)
combining the pair of oligonucleotides with the plant cell DNA
sample under conditions suitable for polymerase chain
reaction-mediated DNA amplification; and (d) isolating the
amplified disease-resistance gene or fragment thereof.
[0038] In preferred embodiments, the amplification is carried out
using a reverse-transcription polymerase chain reaction, for
example, the RACE method
[0039] In another aspect, the invention features a method of
identifying a plant disease-resistance gene in a plant cell,
involving: (a) providing a preparation of plant cell DNA (for
example, from the plant genome); (b) providing a
detectably-labelled DNA sequence (for example, prepared by the
methods of the invention) having homology to a conserved -region of
an RPS gene; (c) contacting the preparation of plant cell DNA with
the detectablly-labelled DNA sequence under hybridization
conditions providing detection of genes having 50% or greater
sequence identity; and (d) identifying a disease-resistance gene by
its association with the detectable label.
[0040] In another aspect, the invention features a method of
isolating a disease-resistance gene from a recombinant plant cell
library, involving: (a) providing a recombinant plant cell library;
(b) contacting the recombinant plant cell library with a
detectably-labelled gene fragment produced according to the PCR
method of the invention under hybridization conditions providing
detection of genes having 50% or greater sequence identity; and (c)
isolating a member of a disease-resistance gene by its association
with the detectable label.
[0041] In another aspect, the invention features a method of
isolating a disease-resistance gene from a recombinant plant cell
library, involving: (a) providing a recombinant plant cell library;
(b) contacting the recombinant plant cell library with a
detectably-labelled RPS oligonucleotide of the invention under
hybridization conditions providing detection of genes having 50% or
greater sequence identity; and (c) isolating a disease-resistance
gene by its association with the detectable label.
[0042] In another aspect, the invention features a recombinant
plant polypeptide capable of conferring disease-resistance wherein
the plant polypeptide includes a P-loop domain or nucleotide
binding site domain. Preferably, the polypeptide further includes a
leucine-rich repeating domain.
[0043] In another aspect, the invention features a recombinant
plant polypeptide capable of conferring disease-resistance wherein
the plant polypeptide contains a leucine-rich repeating domain.
[0044] In another aspect, the invention features a plant
disease-resistance gene isolated according to the method involving:
(a) providing a sample of plant cell DNA; (b) providing a pair of
oligonucleotides having sequence homology to a conserved region of
an RPS disease-resistance gene; (c) combining the pair of
oligonucleotides with the plant cell DNA sample-under-conditions
suitable for polymerase chain reaction-mediated DNA amplification;
and (d) isolating the amplified disease-resistance gene or fragment
thereof.
[0045] In another aspect, the invention features a plant
disease-resistance gene isolated according to the method involving:
(a) providing a preparation of plant cell DNA; (b) providing a
detectably-labelled DNA sequence having homology to a conserved
region of an RPS gene; (c) contacting the preparation of plant cell
DNA with the detectably-labelled DNA sequence under hybridization
conditions providing detection of genes having 50% or greater
sequence identity; and (d) identifying a disease-resistance gene by
its association with the detectable label.
[0046] In another aspect, the invention features a plant
disease-resistance gene according to the method involving: (a)
providing a recombinant plant cell library; (b) contacting the
recombinant plant cell library with a detectably-labelled RPS gene
fragment produced according to the method of the invention under
hybridization conditions providing detection of genes having 50% or
greater sequence identity; and (c) isolating a disease-resistance
gene by its association with the detectable label.
[0047] In another aspect, the invention features a method of
identifying a plant disease-resistance gene involving (a) providing
a plant tissue sample; (b) introducing by biolistic transformation
into the plant tissue sample a candidate plant disease-resistance
gene; (c) expressing the candidate plant disease-resistance gene
within the plant tissue sample; and (d) determining whether the
plant tissue sample exhibits a disease-resistance response, whereby
a response identifies plant disease-resistance gene.
[0048] Preferably, the plant tissue sample is either leaf, root,
flower, fruit, or stem tissue; the candidate plant
disease-resistance gene is obtained from a cDNA expression library;
and the disease-resistance response is the hypersensitive
response.
[0049] In another aspect, the invention features a plant
disease-resistance gene isolated according to the method involving:
(a) providing a plant tissue sample; (b) introducing by biolistic
transformation into the plant tissue sample a candidate plant
disease-resistance gene; (c) expressing the candidate plant
disease-resistance gene within the plant tissue sample; and (d)
determining whether the plant tissue sample exhibits a
disease-resistance response, whereby a response identifies a plant
disease-resistance gene.
[0050] In another aspect, the invention features a purified
antibody which binds specifically to an rps family protein. Such an
antibody may be used in any standard itmmunodetection method for
the identification of an RPS polypeptide.
[0051] In another aspect, the invention features a DNA sequence
substantially identical to the DNA sequence shown in FIG. 12.
[0052] In another aspect, the invention features a substantially
pure polypeptide having a sequence substantially identical to a Prf
amino acid sequence shown in FIG. 5 (A or B).
[0053] By "disease resistance gene" is meant a gene encoding a
polypeptide capable of triggering the plant defense response in a
plant cell or plant tissue. An RPS gene is a disease resistance
gene having about 50% or greater sequence identity to the RPS2
sequence of FIG. 2 or a portion thereof. The gene, RPS2, is a
disease resistance gene encoding the Rps2 disease-resistance
polypeptide from Arabidopsis thaliana.
[0054] By "polypeptide" is meant any chain of amino acids,
regardless of length or post-translational modification (e.g.,
glycosylation or phosphorylation).
[0055] By "substantially identical" is meant a polypeptide or
nucleic acid exhibiting at least 50%, preferably 85%, more
preferably 90%, and most preferably 95% homology to a reference
amino acid or nucleic acid sequence. For polypeptides, the length
of comparison sequences will generally be at least 16 amino acids,
preferably at least 20 amino acids, more preferably at least 25
amino acids, and most preferably 35 amino acids. For nucleic acids,
the length of comparison sequences will generally be at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides.
[0056] Sequence identity is typically measured using sequence
analysis software (e.g., Sequence Analysis Software Package of the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wis. 53705). Such software
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, substitutions, and other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine alanine; valine,
isoleucine, leucine; aspartic acid, glutamic acid, asparagine,
glutamine; serine, threonine; lysine, arginine; and phenylalanine,
tyrosine.
[0057] By a "substantially pure polypeptide" is meant an Rps2
polypeptide which has been separated from components which
naturally accompany it. Typically, the polypeptide is substantially
pure when it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the preparation is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, Rps2 polypeptide. A substantially pure Rps2 polypeptide may
be obtained, for example, by extraction from a natural source
(e.g., a plant cell); by expression of a recombinant nucleic acid
encoding an Rps2 polypeptide; or by chemically synthesizing the
protein. Purity can be measured by any appropriate method, e.g.,
those described in column chromatography, polyacrylamide gel
electrophoresis, or by HPLC analysis.
[0058] A protein is substantially free of naturally associated
components when it is separated from those contaminants which
accompany it in its natural state. Thus, a protein which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those derived
from eukaryotic organisms but synthesized in E. coli or other
prokaryotes.
[0059] By "substantially pure DNA" is meant DNA that is free of the
genes which, in the naturally-occurring genome of the organism from
which the DNA of the invention is derived, flank the gene. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote; or which exists as a separate molecule (e.g., a cDNA or
a genomic or cDNA fragment produced by PCR or restriction
endonuclease digestion) independent of other sequences. It also
includes a recombinant DNA which is part of a hybrid encoding
additional polypeptide sequence.
[0060] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding (as used herein) an Rps2
polypeptide.
[0061] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, e.g., an Rps2 polypeptide, a recombinant protein
or a RNA molecule).
[0062] By "reporter gene" is meant a gene whose expression may be
assayed; such genes include, without limitation,
.beta.-glucuronidase (GUS), luciferase, chloramphenicol
transacetylase (CAT), and .beta.-galactosidase.
[0063] By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell-type specific, tissue-specific or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the native gene.
[0064] By "operably linked" is meant that a gene and a regulatory
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the regulatory sequence(s).
[0065] By "plant cell" is meant any self-propagating cell bounded
by a semi-permeable membrane and containing a plastid. Such a cell
also requires a cell wall if further propagation is desired. Plant
cell, as used herein includes, without limitation, algae,
cyanobacteria, seeds suspension cultures, embryos, meristematic
regions, callus tissue, leaves, roots, shoots, gametophytes,
sporophytes, pollen, and microspores.
[0066] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0067] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
plants and the DNA (transgene) is inserted by artifice into the
nuclear or plastidic genome.
[0068] By "pathogen" is meant an organism whose infection into the
cells of viable plant tissue elicits a disease response in the
plant tissue.
[0069] By an "RPS disease-resistance gene" is meant any member of
the family of plant genes characterized by their ability to trigger
a plant defense response and having at least 20%, preferably 30%,
and most preferably 50% amino acid sequence identity to one of the
conserved regions of one of the RPS members described herein (i.e.,
either the RPS2, L6, N, or Prf genes). Representative members of
the RPS gene family include, without limitation, the rps2 gene of
Arabidopsis, the L6 gene of flax, the Prf gene of tomato, and the N
gene of tobacco By "conserved region" is meant any stretch of six
or more contiguous amino acids exhibiting at least 30%, preferably
50%, and most preferably 70% amino acid sequence identity between
two or more of the RPS family members, RPS2, L6, N, or Prf.
Examples of preferred conserved regions are shown (as boxed or
designated sequences) in FIGS. 5 A and B, 6, 7, and 8 and include,
without limitation, nucleotide binding site domains, leucine-rich
repeats, leucine zipper domains, and P-loop domains.
[0070] By "detectably-labelled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide
probe or primer, a gene or fragment thereof, or a cDNA molecule.
Methods for detectably-labelling a molecule are well known in the
art and include, without limitation, radioactive labelling (e.g.,
with an isotope such as .sup.32P or .sup.35S) and nonradioactive
labelling (e.g., chemiluminescent labelling, e.g., fluorescein
labelling).
[0071] By "biolistic transformation" is meant any method for
introducing foreign molecules into a cell using velocity driven
microprojectiles such as tungsten or gold particles. Such
velocity-driven methods originate from pressure bursts which
include, but are not limited to, helium-driven, air-driven, and
gunpowder-driven techniques. Biolistic transformation may be
applied to the transformation or transfection of a wide variety of
cell types and intact tissues including, without limitation,
intracellular organelles (e.g., chloroplasts and mitochondria),
bacteria, yeast, fungi, algae, pollen, animal tissue, plant tissue
(e.g., leaf, seedling, embryo, epidermis, flower, meristem, and
root), pollen, and cultured cells.
[0072] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally-occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more preferably 90%, and most
preferably at least 99%, by weight, antibody, e.g., an
rps2-specific antibody. A purified rps antibody may be obtained,
for example, by affinity chromatography using
recombinantly-produced rps protein or conserved motif peptides and
standard techniques.
[0073] By "specifically binds" is meant an antibody which
recognizes and binds an rps protein but which does not
substantially recognize and bind other molecules in a sample, e.g.,
a biological sample, which naturally includes rps protein.
[0074] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DETAILED DESCRIPTION
[0075] The drawings will first be described.
DRAWINGS
[0076] FIGS. 1A-1F are a schematic summary of the physical and RFLP
analysis that led to the cloning of the RPS2 locus.
[0077] FIG. 1A is a diagram showing the alignment of the genetic
and the RFLP maps of the relevant portion of Arabidopsis thaliana
chromosome IV adapted from the map published by Lister and Dean
(1993) Plant J. 4:745-750. The RFLP marker L11F11 represents the
left anr of the YUP11F11 YAC clone.
[0078] FIG. 1B is a diagram showing the alignment of relevant YACs
around the RPS2 locus. YAC constructs designated YUP16G5, YUP18G9
and YUP11F11 were provided by J. Ecker, University of Pennsylvania.
YAC constructs designated EW3H7. EW11D4, EW1E4; and EW9C3 were
provided by E. Ward, Ciba-Geigy, Inc.
[0079] FIG. 1C is a diagram showing the alignment of cosmid clones
around the APS2 locus. Cosmid clones with the designation H are
detivatives or the EW3H7 YAC-aclone whereas those with the
designation E are -derimtives of the EW11E4 YAC clone. Vertical
arrows indicate the relative positions of RFLP markers between the
ecotypes La-er and the rps2-101N plant. The RFLP markers were
identified by screening a Southern blot containing more than 50
different restriction enzyme digests using either the entire part
or pieces of the corresponding cosmid clones as probes. The cosmid
clones described in FIG. 1C were provided by J. Giraudat, C.N.R.S.,
Gif-sur-Yvette, France.
[0080] FIGS. 1D and 1E are maps of EcoRI restriction endonuclease
sites in the cosmids E4-4 and E4-6, respectively. The recombination
break points surrounding the RPS2 locus are located within the 4.5
and 7.5 kb EcoRI restriction endonuclease fragments.
[0081] FIG. 1F is a diagram showing the approximate location of
genes which encode the RNA transcripts which have been identified
by polyA+RNA blot analysis. The sizes of the transcripts are given
in kilobase pairs below each transcript.
[0082] FIG. 2 is the complete nucleotide sequence of cDNA-4
comprising the RPS2 gene locus. The three reading frames are shown
below the nucleotide sequence. The deduced amino acid sequence of
reading frame "a" is provided and contains 909 amino acids. The
methionine encoded by the ATG start codon is circled in open
reading frame "a" of FIG. 2. The A of the ATG start codon is
nucleotide 31 of FIG. 2.
[0083] FIG. 3 is the nucleotide sequence of the avrRpt2 gene and
its deduced amino acid sequence. A potential ribosome binding site
is underlined. An inverted repeat is indicated by horizontal arrows
at the 3' end of the open reading frame. The deduced amino acid
sequence is provided below the a nucleotide sequence of the open
reading frame.
[0084] FIG. 4 is a schematic summary of the complementation
analysis that allowed functional confirmation that the DNA carried
on p4104 and p4115 (encoding cDNA-4) confers RPS2 disease
resistance activity to Arabidopsis thaliana plants previously
lacking RPS2 disease resistance activity. Small vertical marks
along the "genome" line represent restriction enzyme EcoRI
recognition sites, and the numbers above this line represent the
size, in kilobase pairs (kb), of the resulting DNA fragments (see
also FIG. 1E). Opposite "cDNAs" are the approximate locations of
the coding sequences for RNA transcripts (See also FIG. 1F);
arrowheads indicate the direction of transcription for cDNAs 4, 5,
and 6. For functional complementation experiments,
rps2-201C/rps2-201C plants were genetically transformed with the
Arabidopsis thaliana genomic DNA sequences indicated; these
sequences were carried on the named plasmids (derivatives of the
binary cosmid vector pSLJ4541) and delivered to the plant via
Agrobacterium-mediated transformation methods. The disease
resistance phenotype of the resulting transformants following
inoculation with P. syingae expressing avrRpt2 is given as "Sus."
(susceptible, no resistance response) or "Res.f" (disease
resistant).
[0085] FIG. 5A shows regions of sequence similarity between the L-6
protein of flax, N protein of tobacco, Prf protein of tomato, and
rps2 protein of Arabidopsis.
[0086] FIG. 5B shows sequence similarity between the N and L-6
proteins.
[0087] FIG. 6 shows a sequence analysis of RPS2 polypeptide showing
polypeptide regions corresponding to an N-terminal hydrophobic
region, a leucine zipper, NBSs (kinase-1a, kinase-2, and kinase-3
motifs), and a predicted membrane integrated region
[0088] FIG. 7 shows the amino acid sequence of the RPS2 LRR (amino
acids 505-867). The top line indicates the consensus sequences for
the RPS2 LRR. An "X" stands for an arbitrary amino acid sequence
and an "a" stands for an aliphatic amino acid residue. The
consensus sequence for the RPS2 LRR is closely related to the
consensus for the yeast adenylate cyclase CYRI LRR (PX Xa XXL XXL
XXLXL XXNXaXXa). The amino acid residues that match the consensus
sequence are shown in bold. Although this figure shows 14 LRRs, the
C-terminal boundary of the LRR is not very clear because the LRR
closer to the C-terminus does not fit the consensus sequence very
well.
[0089] FIG. 8 shows a sequence analysis of RPS2, indicating regions
with similarity to leucine zipper, P-loop, membrane-spanning, and
leucine-rich repeat motifs. Regions with similarity to defined
functional domains are indicated with a line over the relevant
amino acids. Potential N-glycosylation sequences are marked with a
dot, and the location of the rps2-201 Thr to Pro mutation at animo
acid 668 is marked with an asterisk.
[0090] FIG. 9 is a schematic representation of the transient assay
method. The top panel shows the essential principles of the assay.
The bottom panel shows a schematic representation of the actual
transient assay procedure. Psp NP53121 is used because it is a weak
Arabidopsis pathogen, but potent in causing the HR when carrying an
avirulence gene. In the absence of an HR, the damage to plant cells
infected with NP53121 is minimal, enhancing the difference of GUS
accumulation in cells that undergo the HR in comparison to those
that do not. Prior to bombardment, one half of an Arabidopsis leaf
is infiltrated with P. syringae (stippled side of leaf); the other
half of the leaf serves as a noninfected control, an "internal"
reference for the infected side, and as a measure of transformation
efficiency.
[0091] FIG. 10, panels A-B, are photographs showing the
complementation of the rps2 mutant phenotype using the biolistic
transient expression assay. The left sides of rps2-101C mutant
leaves were infiltrated with Psp 3121/avrRpt2. Infiltrated leaves
were cobombarded with either 35S-uidA plus .DELTA.GUS (Panel A) or
35S-uidA plus 35S-RPS2 (cDNA-2 clone 4) (Panel B). Note that in
Panel B the infected side of the leaf shows less GUS activity than
the uninfected side, indicating that the transformed cells on the
infected side underwent an HR and that 35S-RPS2 complemented the
mutant phenotype (see FIG. 9).
[0092] FIG. 11 is a schematic representation of pKEx4tr showing the
structure of this cDNA expression vector. For convenience, the
multiple cloning site contains the Bbp recognition sequences for
PmeI and NotI and is flanked by T7 and T3 promoters. The region
spanning the modified 35S promoter to the nopaline synthase 3'
sequences (nos 3') was cloned into the Hind III-EcoRI site of
pUC18, resulting in the loss of the EcoRI site.
[0093] FIG. 12 shows a nucleic acid sequence of the tomato Prf
gene.
THE GENETIC BASIS FOR RESISTANCE TO PATHOGENS
[0094] An overview of the interaction between a plant host and a
microbial pathogen is presented. The invasion of a plant by a
potential pathogen can have a range of outcomes delineated by the
following outcomes: either the pathogen successfully proliferates
in the host, causing associated disease symptoms, or its growth is
halted by the host defenses. In some plant-pathogen interactions,
the visible hallmark of an active defense response is the so-called
hypersensitive response or "HR". The HR involves rapid necrosis of
cells near the site of the infection and may include the formation
of a visible dry brown lesion.
[0095] Pathogens which elicit an lR on a given host are said to be
avirulent on that host, the host is said to be resistant, and the
plant-pathogen interaction is said to be incompatible. Strains
which proliferate and cause disease on a particular host are said
to be virulent; in this case the host is said to be susceptible,
and the plant-pathogen interaction is said to be compatible
[0096] "Classical" genetic analysis has been used successfully to
help elucidate the genetic basis of plant-pathogen recognition for
those cases in which a series of strains (races) of a particular
fungal or bacterial pathogen are either virulent or avirulent on a
series of cultivars (or different wild accessions) of a particular
host species. In many such cases, genetic analysis of both the host
and the pathogen revealed that many avirulent fungal and bacterial
strains differ from virulent ones by the possession of one or more
avirulence (avr) genes that have corresponding "resistance" genes
in the host. This avirulence gene-resistance gene correspondence is
termed the "gene-for-gene" model (Crute, et al., (1985) pp 197-309
in: Mechanisms of Resistance to Plant Disease. R.S.S. Fraser, ed.;
Ellingboe, (1981) Annu. Rev. Phytopathol. 19:125-143; Flor, (1971)
Annu. Rev. Phytopathol. 9:275-296; Keen and Staskawicz, (1988)
supra; and Keen et al. in: Application of Biotechnology to Plant
Patbhoen Control. I. Chet, ed., John Wiley & Sons, 1993, pp.
65-88). According to a simple formulation of this model, plant
resistance genes encode specific receptors for molecular signals
generated by avr genes. Signal transduction pathway(s) then carry
the signal to a set of target genes that initiate the HR and other
host defenses (Gabriel and Rolfe, (1990) Annu. Rev. Phytopathol.
28:365-391). Despite this simple predictive model, the molecular
basis of the avr-resistance gene interaction is still unknown.
[0097] One basic prediction of the gene-for-gene hypothesis has
been convincingly confirmed at the molecular level by the cloning
of a variety of bacterial avr genes (Innes, et al., (1993) J.
Bacteriol. 175:4859-4869; Dong, et al., (1991) Plant Cell 3:61-72;
Whelan et al., (1991) Plant Cell 3:49-59; Staskawicz et al., (1987)
J. Bacteriol. 169:5789-5794; Gabriel et al., (1986) P.N.A.S.,.sub.1
USA 83:6415-6419; Keen and Staskawicz, (1988) Annu. Rev. Microbiol.
42:421-440; Kobayashi et al., (1990) Mol. Plant-Micrbobe Interact.
3:94-102 and (1990) Mol. Plant-Microbe Interact. 3:103-111). Many
of these cloned avirulence genes have been shown to correspond to
individual resistance genes in the cognate host plants and have
been shown to conf er an avirulent phenotype when transferred to an
otherwise virulent strain. The avrRpt2 locus was isolated from
Pseudomonas syringae pv. tomato and sequenced by Innes et al.
(Innes, R. et al. (1993) J. Bacteriol. 175:4859-4869). FIG. 3,is
the nucleotide sequence and deduced amino acid sequence of the
avrlRpt2 gene.
[0098] Exaples of known signals to which plants respond when
infected by pathogens include harpins from Erwinia (Wei et al.
(1992) Science 257:85-88) and Pseudomonas (He et al. (1993) Cell
73:1255-1266); avr4 (Joosten et al. (1994) Nature 367:384-386) and
avr9 peptides (van den Ackerveken et al (1992) Plant J. 2:359-366)
from Cladosporium; PopAl from Pseudomonas (Arlat et al. (1994) EMBO
J. 13:5 43-553); avrd-generated lipopolysaccharide (Midland et al.
(1993) J. Org. Chem. 58:2940-2945); and NIPI from Rhynchosporium
(Hahn et al. (1993) Mol. Plant Microbe Interacet. 6:745-754).
[0099] Compared to a genes, considerably less is known about plant
resistance genes that correspond to specific avr-generated signals.
The plant resistance gene, RPS2 (rps for resistance to Pseudomonas
syringae), the first gene of a new, previously unidentified class
of plant disease resistance genes corresponds to a specific avr
gene (avrRpt2). Some of the work leading up to the cloning of RPS2
is described in Yu, et al., (1993), Molecular Plant-Microbe
Interactions 6:434-443 and in Kunkel, et al., (1993) Plant Cell
5:865-875.
[0100] An apparently unrelated avirulence gene which corresponds
specifically to plant disease resistance gene, Pto, has been
isolated from tomato (Lycopersicon esculentum) (Martin et al.,
(1993) Science 262:1432-1436).
[0101] Tomato plants expressing the Pto gene are resistant to
infection by strains of Pseudomonas syringae pv. tomato that
express the avrPto avirulence gene. The amino acid sequence
inferred from the Pto gene DNA sequence displays strong similarity
to serine-threonine protein kinases, implicating Pto in signal
transduction. No similarity to the tomato Pto locus or any known
protein kinases was observed for RPS2, suggesting that RPS2 is
representative of a new class of plant disease resistance
genes.
[0102] The isolation of a race-specific resistance gene from Zea
mays (corn) known as Hm1 has been reported (Johal and Briggs (1992)
Science 258:985-987). Hm1 confers resistance against specific races
of the fungal pathogen Cochliobolus carbonum by controlling
degradation of a fungal toxin, a strategy that is mechanistically
distinct from the avirulence-gene specific resistance of the
RPS2-avrRpt2 resistance mechanism.
[0103] The cloned RPS2 gene of the invention can be used to
facilitate the construction of plants that are resistant to
specific pathogens and to overcome the inability to transfer
disease resistance genes between species using classical breeding
techniques (Keen et al., (1993), supra). There now follows a
description of the cloning and characterization of an Arabidopsis
thaliana RPS2 genetic locus, the RPS2 genomic DNA, and the RPS2
cDNA. The avrRpt2 gene and the RPS2 gene, as well as mutants
rps2-101C, rps2-102C, and rps2-201C (also designated rps2-201), are
described in Dong, et al., (1991) Plant Cell 3:61-72; Yu, et al.,
(1993) supra; Kunkel et al., (1993) supra; Whalen et al., (1991),
supra; and Innes et al., (1993) supra). A mutant designated
rps2-101N has also been isolated. The identification and cloning of
the RPS2 gene is described below.
RPS2 Overcomes Sensitivity to Pathogens Carrying the avrfpt2
Gene
[0104] To demonstrate the genetic relationship between an
avirulence gene in the pathogen and a resistance gene in the host,
it was necessary first to isolate an avirulence gene. By screening
Pseudomonas strains that are known pathogens of crop plants related
to Arabidopsis, highly virulent strains, P. syringae pv. maculicola
(Psm) ES4326, P. syringae pv. tomato (Pst) DC3000, and an avirulent
strain, Pst MM1065 were identified and analyzed as to their
respective abilities to grow in wild type Arabidopsis thaliana
plants (Dong et al., (1991) Plant Cell, 3:61-72; Whalen et al.,
(1991) Plant Cell 3:49-59; MM1065 is designated JL1065 in Whalen et
al.). PSn ES4326 or Pst DC3000 can multiply 10.sup.4 fold in
Arabidopsis thaliana leaves and cause water-soaked lesions that
appear over the course of two days. Pst MM1065 multiplies a maximum
of 10 fold in Arabidopsis thaliana leaves and causes the appearance
of a mildly chlorotic dry lesion after 48 hours. Thus, disease
resistance is associated with severely inhibited growth of the
pathogen.
[0105] An avirulence gene (avr) of the Pst MM1065 strain was cloned
using standard techniques as described in Dong et al. (1991), Plant
Cell 3:61-72; Whalen et al., (1991) supra; and Innes et al.,
(1993), supra. The isolated avirulence gene from this strain was
designated avrRpt2. Normally, the virulent strain Psm ES4326 or Pst
DC3000 causes the appearance of disease symptoms after 48 hours as
described above. In contrast, Psm ES4326/avrRpt2 or Pst
DC30001avrRpt2 elicits the appearance of a visible necrotic
hypersensitivity response (HR) within 16 hours and multiplies 50
fold less than Psm ES4326 or Pst DC3000 in wild type Arabidopsis
thaliana leaves (Dong et al., (1991), supra; and Whalen et al.,
(1991), supra). Thus, disease resistance in a wild type Arabidopsis
plant requires, in part, an avirulence gene in the pathogen or a
signal generated by the avirulence gene.
[0106] The isolation of four Arabidopsis thaliana disease
resistance mutants has been described using the cloned avrRpt2 gene
to search for the host gene required for disease resistance to
pathogens carrying the avrfpt2 gene (Yu et al., (1993), supra;
Kunkel et al., (1993), supra). The four Arabidopsis thaliana
mutants failed to develop an HR when infiltrated with Psm
ES4326/avrRpt2 or Pst DC3000/avrRpt2 as expected for plants having
lost their disease resistance capacity. In the case of one of these
mutants, approximately 3000 five to six week old M.sub.2 ecotype
Columbia (Col-0 plants) plants generated by ethyl methanesulfonic
acid (EMS) mutagenesis were hand-inoculated with Psm ES4326/avrRpt2
and a single mutant, rps2-101C, was identified (resistance to
Pseudomonas syringae) (Yu et al., (1993), supra.
[0107] The second mutant was isolated using a procedure that
specifically enriches for mutants unable to mount an HR (Yu et al.,
(1993), supra). When 10-day old Arabidopsis thaliana seedlings
growing on petri plates are infiltrated with Pseudomonas syringae
pv. phaseolicola (Psp) NPS3121 versus Psp NPS3121/avrRpt2, about
90% of the plants infiltrated with Psp NPS3121 survive, whereas
about 90%-95% of the plants infiltrated with Psp NPS3121/avrRpt2
die.
[0108] Apparently, vacuum infiltration of an entire small
Arabidopsis thaliana seedling with Psp NPS3121/avrfRpt2 elicits a
systemic HR. which usually kills the seedling. In contrast,
seedlings infiltrated with Psp NPS3121 survive because Psp NPS3121
is a weak pathogen on Arabidopsis thaliana. The second disease
resistance mutant was isolated by infiltrating 4000 EMS-mutagenized
Columbia M.sub.2 seedlings with Psp NPS3121/avrRpt2. Two hundred
survivors were obtained. These were transplanted to soil and
re-screened by hand inoculation when the plants reached maturity of
these 200 survivors, one plant failed to give an HR when
hand-infiltrated with Psm ES4326/avrRpt2. This mutant was
designated rps2-102C (Yu et al., (1993), supra).
[0109] A third mutant, rps2-201C, was isolated in a screen of
approximately 7500 M.sub.2 plants derived from seed of Arabidopsis
thaliana ecotype Col-0 that had been mutagenized with diepoxybutane
(Kunkel et al., (1993), supra). Plants were inoculated by dipping
entire leaf rosettes into a solution containing Pst DC3000/avrRpt2
bacteria and the surfactant Silwet L-77 (Whalen et al., (1991),
supra), incubating plants in a controlled environment growth
chamber for three to four days, and then visually observing disease
symptom development. This screen revealed four mutant lines
(carrying the rpb2-201C, rps2-202C, rps2-203C, and rps2-204C
alleles), and plants homozygous for rps2-201C were a primary
subject for further study (Kunkel et al., (1993), supra and the
instant application).
[0110] Isolation of the fourth rps2 mutant, rps2-101N, has not yet
been published. This fourth isolate is either a mutant or a
susceptible Arabidopsis ecotype. Seeds of the Arabidopsis Nossen
ecotype were gamma-irradiated and then sown densely in flats and
allowed to germinate and grow through a nylon mesh. When the plants
were five to six weeks old, the flats were inverted, the plants
were partially submerged in a tray containing a culture of Psm
ES4326/avrfpt2, and the plants were vacuum infiltrated in a vacuum
desiccator. Plants inoculated this way develop an HR within 24
hours. Using this procedure, approximately 40,000 plants were
screened and one susceptible plant was identified. Subsequent RFLP
analysis of this plant suggested that it may not be a Nossen mutant
but rather a different Arabidopsis ecotype that is susceptible to
Psm ES4326/avrRpt2. This plant is referred to as rps2-21N. The
isolated mutants rps2-101C, rps2-102C, rps2-201C, and rps2-101N are
referred to collectively as the "rps2 mutants".
The rps2 Mutants Fail to Specifically Respond to the Cloned
Avirulence Gene, avrRpt2
[0111] The RPS2 gene product is specifically required for
resistance to pathogens carrying the avirulence gene, avrRpt2. A
mutation in Rps2 polypeptide that eliminates or reduces its
function would be observable as the absence of a hypersensitive
response upon infiltration of the pathogen. The rps2 mutants
displayed disease symptoms or a null response when infiltrated with
Psm ES4326/avrRpt2, Pst DC3000/avrRpt2 or Psp NPS3121/avrRpt2,
respectively. Specifically, no HR response was elicited, indicating
that the plants were susceptible and had lost resistance to the
pathogen despite the presence of the avrRpt2 gene in the
pathogen.
[0112] Pathogen growth in rps2 mutant plant leaves was similar in
the presence and absence of the avrRpt2 gene. Psm ES4326 and Psm
ES4326/avrfpt2 growth in rps2 mutants was compared and found to
multiply equally well in the rps2 mutants, at the same rate that
Psm Es4326 multiplied in wild-type Arabidopsis leaves. Similar
results were observed for Pst DC3000 and Pst DC3000/avrRpt2 growth
in rps2 mutants.
[0113] The rps2 mutants displayed a HR when infiltrated with
Pseudomonas pathogens carrying other avr genes, Psm ES4326/avrB,
Pst DC3000/avrB, Psm ES4326/avrRfpm1, Pst DC3000/avrRpm1. The
ability to mount an HR to an avr gene other than avrRpt2 indicates
that the zps2 mutants isolated by selection with avrRpt2 are
specific to avrRpt2.
Mapping and Cloning of the RPS2 Gene
[0114] Genetic analysis of rps2 mutants rps2-101C, rps2-102C,
rps-201C and rps-101N showed that they all corresponded to genes
that segregated as expected for a single Mendelian locus and that
all four were most likely allelic. The four rps2 mutants were
mapped to the bottom of chromosome IV using standard RFLP mapping
procedures including polymerase chain reaction (PCR)-based markers
(Yu et al., (1993), supra; Kunkel et al., (1993), supra; and
Mindrinos, M., unpublished). Segregation analysis showed that
rps2-101C and rps2-102C are tightly linked to the PCR marker, PG11,
while the RFLP marker M600 was used to define the chromosome
location of the rps2-201C mutation (FIG. 1A) (Yu et al., (1993),
supra; Kunkel et al., (1993), supra). RPS2 has subsequently been
mapped to the centromeric side of PG11.
[0115] Heterozygous RPS2/rps2 plants display a defense response
that is intermediate between those displayed by the wild-type and
homozygous rps2/rps2 mutant plants (Yu, et al., (1993), supra; and
Kunkel et al., (1993), supra). The heterozygous plants mounted an
HR in response to Psm ES4326/avrRpt2 or Pst DC3000/avrRpt2
infiltration; however, the HR appeared later than in wild type
plants and required a higher minimum inoculum (Yu, et al., (1993),
supra; and Kunkel et al., (1993), supra).
High Resolution Mapping of the RPS2 Gene and RPS2 cDNA
Isolation
[0116] To carry out map-based cloning of the RPS2 gene,
rps2-101N/rps2-101N was crossed with Landsberg erecta RPS2/RPS2.
Plants of the F.sub.1 generation were allowed to self pollinate (to
"self") and 165 F.sub.2 plants were selfed to generate F3 families.
Standard RFLP mapping procedures showed that rps2-101N maps close
to and on the centromeric side of the RFLP marker, PG11. To obtain
a more detailed map position, rps2-101N/rps-101N was crossed with a
doubly marked Landsberg erecta strain containing the recessive
mutations, cer2 and ap2. The genetic distance between cer2 and ap2
is approximately 15 cM, and the rps2 locus is located within this
interval. F.sub.2 plants that displayed either a CER2 ap2 or a cer2
AP2 genotype were collected, selfed, and scored for RPS2 by
inoculating at least 20 F.sub.3 plants for each F.sub.2with Psm
ES4326/avrRpt2. DNA was also prepared from a pool of approximately
20 F.sub.3 plants for each F.sub.2 line. The CER2 ap2 and cer2 AP2
recombinants were used to carry out a chromosome walk that is
illustrated in FIG. 1.
[0117] As shown in FIG. 1, RPS2 was mapped to a 28-35 kb region
spanned by cosmid clones E4-4 and E4-6. This region contains at
least six genes that produce detectable transcripts. There were no
significant differences in the sizes of the transcripts or their
level of expression in the rps2 mutants as determined by RNA blot
analysis. cDNA clones of each of these transcripts were isolated
and five of these were sequenced. As is described below, one of
these transcripts, cDNA-4, was shown to correspond to the RPS2
locus. From this study, three independent cDNA clones (cDNA-4-4,
cDNA-4-5, and cDNA-4-11) were obtained corresponding to RPS2 from
Columbia ecotype wild type plants. The apparent sizes of RPS2
transcripts were 3.8 and 3.1 kb as determined by RNA blot
analysis.
[0118] A fourth independent cDNA-4 clone (cDNA-4-2453) was obtained
using map-based isolation of RPS2 in a separate study. Yeast
artificial chromosome (YAC) clones were identified that carry
contiguous, overlapping inserts of Arabidopsis thaliana ecotype
Col-O genomic DNA from the M600 region spanning approximately 900
kb in the RPS2 region. Arabidopsis YAC libraries were obtained from
J. Ecker and E. Ward, supra and from E. Grill (Grill and Somerville
(1991) Mol. Gen. Genet. 226:484-490). Cosmids designated "H", and
"E" were derived from the YAC inserts and were used in the
isolation of RPS2 (FIG. 1).
[0119] The genetic and physical location of RPS2 was more precisely
defined using physically mapped RFLP, RAPD (random amplified
polymorphic DNA) and CAPS (cleaved amplified polymorphic sequence)
markers. Segregating populations from crosses between plants of
genotype RPS2/RPS2 (No-O wild type) and rps2-201/rps2-201 (Col-O
background) were used for genetic mapping. The RPS2 locus was
mapped using markers 17B7LE, PG11, M600 and other markers. For
high-resolution genetic mapping, a set of tightly linked RFLP
markers was generated using insert end fragments from YAC and
cosmid clones (FIG. 1) (Kunkel et al. (1993), spra; Konieczny and
Ausubel (1993) Plant J. 4:403-410; and Chang et al. (1988) PNAS USA
85:6856-6860). Cosmid clones E4-4 and E4-6 were then used to
identify expressed transcripts (designated cDNA-4, -5, -6, -7, -8
of FIG. 1F) from this region, including the cDNA-4-2453 clone.
[0120] RPS2 DNA Sequence Analysis
[0121] DNA sequence analysis of cDNA-4 from wild-type Col-O plants
and from mutants rps2-101C, rps2-102C, rps2-201C and rps2-101N
showed that cDNA-4 corresponds to RPS2. DNA sequence analysis of
rps2-101C, rps2-102C and rps2-201C revealed changes from the
wild-type sequence as shown in Table 1. The numbering system in
Table 1 starts at the ATG start codon encoding the first methionine
where A is nucleotide 1. DNA sequence analysis of cDNA-4
corresponding to mutant rps2-102C showed that it differed from the
wild type sequence at amino acid residue 476. Moreover, DNA
sequence analysis of the cDNA corresponding to cDNA-4 from
rps2-101N showed that it contained a 10 bp insertion at amino acid
residue 581, a site within the leucine-rich repeat region which
causes a shift in the RPS2 reading frame. Mutant rps2-101C contains
a mutation that leads to the formation of a chain termination
codon. The DNA sequence of mutant allele rps2-201C revealed a
mutation altering a single amino acid within a segment of the LRR
region that also has similarity to the helix-loop-helix motif,
further supporting the designation of this locus as the RPS2 gene.
The DNA and amino acid sequences are shown in FIG. 2.
TABLE-US-00001 TABLE 1 position of Mutant Wild type mutation Change
rps2-101C 703 TGA 705 704 TAA Stop Codon rps2-101N 1741 GTG 1743
1741 GTGGAGTTGTATG Insertion rps2-102C 1426 AGA 1428 1427 AAA Amino
acid 476 arg lys rps2-201C 2002 ACC 2004 2002 CCC Amino acid thr
pro
[0122] DNA sequence analysis of cDNA-4 corresponding to RPS2 from
wild-type Col-O plants revealed an open reading frame (between two
stop codons) spanning 2,751 bp. There are 2,727 bp between the
first methionine codon of this reading frame and the 3'-stop codon,
which corresponds to a deduced 909 amino acid polypeptide (See open
reading frame "a" of FIG. 2). The amino acid sequence has a
relative molecular weight of 104,460 and a pI of 6.51.
[0123] As discussed below, RPS2 belongs to a new class of disease
resistance genes; the structure of the Rps2 polypeptide does not
resemble the protein structure of the product of the previously
cloned and publicized avirulence gene-specific plant disease
resistance gene, Pto, which has a putative protein kinase domain.
Fran the above analysis of the deduced amino acid sequence, RPS2
contains several distinct protein domains conserved in other
proteins from both eukaryotes and prokaryotes. These domains
include, but are not limited, to Leucine Rich Repeats (LRR) (Kobe
and Deisenhofer, (1994) Nature 366:751-756); nucleotide binding
site, e.g. the kinase la motif (P-loop) (Saraste et al. (1990)
Trends in Biological Sciences TIBS 15:430-434; Helix-Loop-Helix
(Murre et al. (1989) Cell 56:777-783; and Leucine Zipper (Rodrigues
and Park (1993) Mol. Cell Biol. 13:6711-6722). The amino acid
sequence of Rps2 contains a LRR motif (LRR motif from amino acid
residue 505 to amino acid residue 867), which is present in many
known proteins and which is thought to be involved in
protein-protein interactions and may thus allow interaction with
other proteins that are involved in plant disease resistance. The
N-terminal portion of the Rps2 polypeptide LRR is, for example,
related to the LRR of yeast (Saccharomyces cerevisiae) adenylate
cyclase, CYRI. A region predicted to be a transmembrane spanning
domain (Klein et al. (1985) Biochim., Diophys. Acta 815:468-476) is
located from amino acid residue 350 to amino acid residue 365,
N-terminal to the LRR. An ATP/GTP binding site motif (P-loop) is
predicted to be located between amino acid residue 177 and amino
acid residue 194, inclusive. The motifs are discussed in more
detail below.
[0124] From the above analysis of the deduced amino acid sequence,
the Rps2 polypeptide may have a membrane-receptor structure which
consists of an N-terminal extracellular region and a C-terminal
cytoplasmic region. Alternatively, the topology of the Rps2 may be
the opposite: an N-terminal cytoplasmic region and a C-terminal
extracellular region. LRR motifs are extracellular in many cases
and the Rps2 LRR contains five potential N-glycosylation sites.
Identification of RPS2 by Functional Complementation
[0125] Complementation of rps2-201 Homozygotes with genomic DNA
corresponding to Arabidopsis thaliana functionally confirmed that
the genomic region encoding cDNA-4 carries RPS2 activity. Cosmids
were constructed that contained overlapping contiguous sequences of
wild type Arabidopsis thaliana DNA from the RPS2 region contained
in YACs EW11D4, EW9C3, and YUP11F1 of FIG. 1 and FIG. 4. The cosmid
vectors were constructed from pSLJ4541 (obtained from J. Jones,
Sainsbury Institute, Norwich, England) which contains sequences
that allow the inserted sequence to be integrated into the plant
genome via Agrobacterium-mediated transformation (designated
"binary cosmid"). "H" and "E" cosmids (FIG. 1) were used to
identify clones carrying DNA from the Arabidopsis thaliana genomic
RPS2 region.
[0126] More than forty binary cosmids containing inserted RPS2
region DNA were used to transform rps2-201 homozygous mutants
utilizing Agrobacterium-mediated transformation (Chang et al.
((1990) p. 28, Abstracts of the Fourth International Conference on
Arabidopsis Research, Vienna, Austria). Transformants which
remained susceptible (determined by methods including the observed
absence of an HR following infection to P. syringae pv.
phaseolicola strain 3121 carrying avrRpt2 and Psp 3121 without
avrRpt2) indicated that the inserted DNA did not contain functional
RPS2. These cosrids conferred the "Sus." or susceptible phenotype
indicated in FIG. 4. Transformants which had acquired
avrRpt2-specific disease resistance (determined by methods
including the display of a strong hypersensitive response (HR) when
inoculated with Psp 3121 with avrRpt2, but not following
inoculation with Psp 3121 without avrRpt2) suggested that the
inserted DNA contained a functional RPS2 gene capable of conferring
the "Res." or resistant phenotype indicated in FIG. 4.
Transformants obtained using the pD4 binary cosmid displayed a
strong resistance phenotype as described above. The presence of the
insert DNA in the transformants was confirmed by classical genetic
analysis (the tight genetic linkage of the disease resistance
phenotype and the kanamycin resistance phenotype conferred by the
cotransformed selectable marker) and Southern analysis. These
results indicated that RPS2 is encoded by a segment of the 18 kb
Arabidopsis thaliana genomic region carried on cosmid pD4 (FIG.
4).
[0127] To further localize the RPS2 locus and confirm its ability
to confer a resistance phenotype on the rps2-201 homozygous
mutants, a set of six binary costnids containing partially
overlapping genomic DNA inserts were tested. The overlapping
inserts pD2, pD4, pD14, pD15, pD27, and pD47 were chosen based on
the location of the transcription corresponding to the five cDNA
clones in the RPS2 region (FIG. 4). These transformation
experiments utilized a vacuum infiltration procedure (Bechtold et
al. (1993) C.R. Acad. Sci. Paris 316:1194-1199) for
Agrobacteriun-mediated transformation. Agrobacterium-mediated
transformations with cosmids pD2, pD14 pD15, pD39, and pD46 were
performed using a root transformation/regeneration protocol
(Valveekens et al. (1988), PNAS 85:5536-5540). The results of
pathogen inoculation experiments assaying for RPS2 activity in
these transformants is indicated in FIG. 4.
[0128] These experiments were further confirmed using a
modification of the vacuum filtration procedure. In particular, the
procedure of Bechtold et al. (sra) was modified such that plants
were grown in peat-based potting soil covered with a screen,
primary inflorescences were removed, and plants with secondary
inflorescences (approximately 3 to 15 cm in length) were inverted
directly into infiltration medium, infiltrated, and then grown to
seed-harvest without removal from soil (detailed protocol available
on the AAtDB computer database (43). The presence of introduced
sequences in the initial pD4 transformant was verified by DNA blot
analysis with a pD4 vector and insert sequences (separately) as
probes. The presence of the expected sequences in transformants
obtained with the vacuum infiltration protocol was also confirmed
by DNA blot analysis. Root transformation experiments (19) were
performed with an easily regenerable rps2-201/rps2-201.times.No-O
mapping population. Transformants were obtained for pD4 with in
plant transformation, for pD2, 14, 16, 39, and 49 with root
transformation, and for pD2, 4, 14, 15, 27, and 47 with vacuum
infiltration as modified.
[0129] Additional transformation experiments utilized binary
cosmids carrying the complete coding region and more than 1 kb of
upstream genomic sequence for only cDNA-4 or cDNA-6. Using the
vacuum infiltration transformation method, three independent
transformants were obtained that carried the wild-type cDNA-6
genomic region in a rps2-201c homozygous background (pAD431 of FIG.
4). None of these plants displayed avrRpt2-dependent disease
resistance. Homozygous rps2-201c mutants were transformed with
wild-type genomic cDNA-4 (p4104 and p4115, each carrying Col-O
genomic sequences corresponding to all of the cDNA-4 open reading
frame, plus approximately 1.7 kb of 5' upstream sequence and
approximately 0.3 kb of 3' sequence downstream of the stop codon).
These p4104 and p4115 transformants displayed a disease resistance
phenotype similar to the wild-type RPS2 homozygotes from which the
rps2 were derived. Additional mutants (rps2-101N and rps2-100C
homozygotes) also displayed avrRpt2-dependent -resistance when
transformed with the cDNA-4 genomic region.
RPS2 Sequences Allow Detection of Other Resistance Genes
[0130] DNA blot analysis of Arabidopsis thaliana genomic DNA using
RPS2 cDNA as the probe showed that Arabidopsis contains several DNA
sequences that hybridize to RPS2 or a portion thereof, suggesting
that there are several related genes in the Arabidopsis genome.
[0131] From the aforementioned description and the nucleic acid
sequence shown in FIG. 2, it is possible to isolate other plant
disease resistance genes having about 50% or greater sequence
identity to the RPS2 gene. Detection and isolation can be carried
out with an oligonucleotide probe containing the RPS2 gene Or a
portion thereof greater than 9 nucleic acids in length, and
preferably greater than about 18 nucleic acids in length. Probes to
sequences encoding specific structural features of the Rps2
polypeptide are preferred as they provide a means of isolating
disease resistance genes having similar structural domains.
Hybridization can be done using standard techniques such as are
described in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, (1989).
[0132] For example, high stringency conditions for detecting the
RPS2 gene include hybridization at about 42.degree. C., and about
50% formamide; a first wash at about 65.degree. C., about 2.times.
SSC, and 1% SDS; followed by a second wash at about 65.degree. C.
and about 0.1%.times.SSC. Lower stringency conditions for detecting
RPS genes having about 50% sequence identity to the RPS2 gene are
detected by, for example, hybridization at about 42.degree. C. in
the absence of formamide; a first wash at about 42.degree. C.,
about 6.times. SSC, and about 1% SDS; and a second wash at about
50.degree. C., about 6.times. SSC, and about 1% SDS. An
approximately 350 nucleotide-DNA probe encoding the middle portion
of the LRR region of Rps2 was used as a probe in the above example.
Under lower stringency conditions, a minimum of 5 DNA bands were
detected in BamHI digested Arabidopsis thaliana genomic DNA as
sequences having sufficient sequence identity to hybridize to DNA
encoding the middle portion of the LtR motif of Rps2. Similar
results were obtained using a probe containing a 300 nucleotide
portion of the RPS2 gene encoding the extreme N-terminus of Rps2
outside of the LRE motif.
[0133] Isolation of other disease resistance genes is performed by
PCR amplification techniques well known to those skilled in the art
of molecular biology using oligonucleotide primers designed to
amplify only sequences flanked by the oligonucleotides in genes
having sequence identity to RPS2. The primers are optionally
designed to allow cloning of the amplified product into a suitable
vector.
The RPS Disease-Resistance Gene Family
[0134] As discussed above, we have discovered that the Arabidopsis
RPS2 gene described herein is representative of a new class of
plant resistance genes. Analysis of the derived amino acid sequence
for RPS2 revealed several regions of similarity with known
polypeptide motifs (see, e.g., Schneider et al., Genes Dev. 6:797
(1991)). Most prominent among these is a region of multiple,
leucine-rich repeats (LRRs). The LRR motif has been implicated in
protein-protein interactions and ligand binding in a diverse array
of proteins (see, e.g., Kornfield et al., Annu. Rev. Biochem.
64:631 (1985); Alber, Curr. Opin. Gen. Dev. 2:205 (1992); Lupas et
al., Science 252:116 2 (1991); Saraste et al., Trend Biochem. Sci.
15:430 (1990)). In one example, LRRs form the hormone binding sites
of mammalian gonadotropin hormone receptors (see, e.g, Lupas et
al., Science 252:1162 (1991)) and, in another example, a domain of
yeast adenylate cyclase that interacts with the RAS2 protein
(Kornfield et al., Annu. Rev. Biochem. 64:631 (1985)). In RPS2, the
LRR domain spans amino acids 503-867 and contains fourteen repeat
units of length 22-26 amino acids. A portion of each repeat
resembles the LRR consensus sequence (I/L/V)XXLXXLXX(I/L)XL. In
FIG. 7, the LRRs from RPS2 are shown, as well as an RPS2 consensus
sequence. Within the RPS2 LRR region, five (of six) sequences
matching the N-glycosylation consensus sequence [NX(S/T)] were
observed (FIG. 8, marked with a dot). In particular,
N-glycosylation is predicted to occur at amino acids 158, 543, 666,
757, 778, 787. Interestingly, the single nucleotide difference
between functional RPS2 and mutant allele rps2-201 is within the
LRR coding region, and this mutation disrupts one of the potential
glycosylation sites.
[0135] Also observed in the deduced amino acid sequence for RPS2 is
a second potential protein-protein interaction domain, a leucine
zipper (see, e.g., von Heijne, J. Mol. Biol. 225:487 (1992)), at
amino acids 30-57. This region contains four contiguous heptad
repeats that match the. leucine zipper consensus sequence
(I/R)XDLXXX. Leucine zippers facilitate the dimerization of
transcription factors by formation of coiled-coil structures, but
no sequences suggestive of an adjacent DNA binding domain (such as
a strongly basic region or a potential zinc-finger) were detected
in RPS2. Coiled-coil regions also promote specific interactions
between proteins that are not transcription factors (see, e.g.,
Ward et al., Plant Mol. Biol. 14:561 (1990); Ecker, Methods 1:186
(1990); Grill et al., Mol. Gen. Genet. 226:484 (1991)), and
computer-database similarity searches with the region spanning
amino acids 30-57 of RPS2 revealed highest similarity to the
coiled-coil regions of numerous myosin and paramyosin proteins.
[0136] A third RPS2 motif was found at the sequence GPGGVGKT at
deduced amino acids 182-189. This portion of RPS2 precisely matches
the generalized consensus for the phosphate-binding loop (P-loop)
of numerous ATP- and GTP-binding proteins (see, e.g., Saraste et
al., supra)). The postulated RPS2 P-loop is similar to those found
in RAS proteins and ATP synthase .beta.-subunits (Saraste et al.,
supra), but surprisingly is most similar to the published P-loop
sequences for the nifh and chvd genes, respectively. The presence
of this P-loop sequence strongly suggests nucleotide triphosphate
binding as one aspect of RPS2 function. This domain is also
referred to as a kinase-1a motif (or a nucleotide binding site, or
NES). Other conserved NBSs are present in the RPS2 sequence; these
NMSs include a kinase-2 motif at amino acids 258-262 and a
kinase-3a motif at amino acids 330-335.
[0137] Finally, inspection of the RPS2 sequence reveals a fourth
RPS2 motif, a potential membrane-spanning domain located at amino
acids 340-360. Within this region, a conserved GLPLAL motif is
found at amino acids 347-352. The presence of the membrane-spanning
domain raises the possibility that the RPS2 protein is membrane
localized, with the N-terminal leucine zipper and P-loop domains
residing together on the opposite side of the membrane from the LRR
region. An orientation in which the C-terminal LRR domain is
extracellular is suggested by the fact that five of the six
potential N-linked glycosylation sites occur C-terminal to the
proposed membrane-spanning domain, as well as by the overall more
positive charge of the N-terminal amino acid residues (see,
etg-&, Kornfield et al., supra; von Heijne, supra). A number of
proteins- that-contain LRRs are postulated or known to be
membrane-spanning receptors in which the LRRs are displayed
extracellularly as a ligand-binding domain (see, e.g., Lopez et
al., Proc. Natl. Acad. Sci. 84:5615 (1987); Braun et al., EMBO J.
10:1885 (1991); Schneider et al., supra).
[0138] The plant kingdom contains hundreds of resistance genes that
are necessarily divergent since they control different resistance
specificities. However, plant defense responses such as production
of activated oxygen species, PR-protein gene expression, and the
hypersensitive response are common to diverse plant-pathogen
interactions. This implies that there are points of convergence in
the defense signal transduction pathways downstream of initial
pathogen recognition, and also suggests that similar functional
motifs may exist among diverse resistance gene products. Indeed,
RPS2 is dissimilar from previously described disease resistance
genes such as Inn or Pto (see, e.g., Johal et al., supra; Martin et
al., supra), and thus represents a new class of genes having
disease resistance capabilities.
Isolation of Other Members of the RPS Disease-Resistance Gene
FamilY Usinc Conserved Motif Probes and Primers
[0139] We have discovered that the RPS2 motifs described above are
conserved in other disease-resistance genes, including, without
limitation, the N protein, the L6 protein, and the Prf protein. As
shown in FIG. 5 (A and B), we have determined that the L6
polypeptide of flax, the N polypeptide of tobacco, and the Prf
polypeptide of tomato each share unique regions of similarity
(including, but not limited to, the leucine-rich repeats, the
membrane-spanning domain, the leucine zipper, and the P-loop and
other NBS domains).
[0140] On the basis of this discovery, the isolation of virtually
any member of the RPS gene family is made possible using standard
techniques. In particular, using all or a portion of the amino acid
sequence of a conserved RPS motif (for example, the amino acid
sequences defining any RPS P-loop, NBS, leucine-rich repeat,
leucine zipper, or membrane-spanning region), one may readily
design RPS oligonucleotide probes, including RPS degenerate
oligonucleotide probes (i.e., a mixture of all possible coding
sequences for a given amino acid sequence). These oligonucleotides
may be based upon the sequence of either strand of the DNA
comprising the motif. General methods for designing and preparing
such probes are provided, for example, in Ausubel et al., suora and
Guide to Molecular Cloning Techniques, 1987, S. L. Berger and A. R.
Kimmnel, eds., Academic Press, New York. These oligonucleotides are
useful for RPS gene isolation, either through their use as probes
capable of hybridizing to RPS complementary sequences or as primers
for various polymerase chain reaction (PCR) cloning strategies.
[0141] Hybridization techniques and procedures are well known to
those skilled in the art and are described, for example, in Ausubel
et al., supra and Guide to Molecular Cloning Techniques, 1987, S.
L. Berger and A. R. Kimmel, eds., Academic Press, New York. If
desired, a combination of different oligonucleotide probes may be
used for the screening of the recombinant DNA library. The
oligonucleotides are labelled with .sup.32P using methods known in
the art, and the detectably-labelled oligonucleotides are used to
probe filter replicas from a recombinant plant DNA library.
Recombinant DNA libraries may be prepared according to methods well
known in the art, for example, as described in Ausubel et al.,
supra. Positive clones may, if desired, be rescreened with
additional oligonucleotide probes based upon other RPS conserved
regions. Forexample, an RPS clone identified based on hybridization
with a P-loop-derived probe may be confirmed by re-screening with a
leucine-rich repeat-derived oligonucleotide.
[0142] As discussed above, RPS oligonucleotides may also be used as
primers in PCR cloning strategies. Such PCR methods are well known
in the art and described, for example, in PCR Technology, H. A.
Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide
to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J.
Sninsky, and T. J. White, eds., Academic Press, Inc., New York,
1990; and Ausubel et al., supra. If desired, members of the RPS
disease-resistance gene family may be isolated using the PCR "RACE"
technique, or Rapid Amplification of cDNA Ends (see, e.g., Innis et
al., sunra). By this method, oligonucleotide primers based on an
RPS conserved domain are oriented in the 3' and 5' directions and
are used to generate overlapping PCR fragments. These overlapping
3'- and 5'-end RACE products are combined to produce an intact
full-length CDNA. This method is described in Innis et al., supra;
and Frohman et al., Proc. Natl. Acad. Sci. 85:8998, 1988.
[0143] Any number of probes and primers according to the invention
may be designed based on the conserved RPS motifs described herein.
Preferred motifs are boxed in the sequences shown in FIG. 5(A or
B). In particular, oligonucleotides according to the invention may
be based on the conserved P-loop domain, the amino acids of which
are shown below:
TABLE-US-00002 MOTIF 1 L6 G MGGIGKTTTA N G MGGVGKTTIA PrfP G
MPGLGKTTLA RPS2 G PGGVGKTTLM
From these sequences, appropriate oligonucleotides are designed and
prepared using standard methods. Particular examples of RPS
oligonucleotides based on the P-loop domain are as follows (N is A,
C, T, or G).
TABLE-US-00003 Based on MOTIF 1: 5' GGNATGGGNGGNNTNGGNAA (A or G)
ACNAC 3' 5' NCGNG(A/T)NGTNA(T/G) (G/A/T)A(T/A)NCGNA 3' 5' GG(T or
A)NT(T or G or C)GG(T or A)AA(G or A)AC (T or C or A)AC 3' 5'
GGNATGGGNGGNNTNGGNAA (A or G) ACNAC 3' 5' N(G or A) (C or T)N(A or
G) (A or G or T)NGTNGT (C or T)TTNCCNANNCCN(G or L) (G or C)N(G or
A) (T or G)NCC 3' 5' GGN(C or A) (T or C)N(G or C) (G or
C)NGGNNTNGG NAA (A or G)ACNAC 3'
[0144] Other conserved RPS motifs useful for oligonucleotide design
are shown below. These motifs are also depicted in the sequence of
FIG. 5(A or B).
TABLE-US-00004 MOTIF 2 L6 FKILVV LDDVD N KKVLIV LDDID PrfP KRFLIL
IDDVW RPS2 KRFLLL LDDVW MOTIF 3 L6 SRFIIT SR N SRIIIT TR PrfP
SRIILT TR RPS2 CKVMFT TR MOTIF 4 L6 GLPLTLK V N GLPLALK V PrfP
GLPLSVV L RPS2 GLPLALI T MOTIF 5 L6 KISYDAL N KISYDGL PrfP GFSYKNL
RPS2 KFSYDNL
From the above motifs and the sequence motifs designated in FIG. 5A
and B, appropriate oligonucleotides are designed and prepared.
Particular examples of such RPS oligonucleotides are as-follows (N
is A, t, C, or G).
TABLE-US-00005 Based on MOTIF 2: 5' T(T or C)GA(T or C)GA(T or C)
(A or G)T(T or G or C) (T or G) (A or G) (T or G or C) (G or A)A 3'
5' T(T or C)CCA(G or C or A)A(T or C) (G or A)TC (A or G)TCNA 3' 5'
(C or G or A) (T or C) (C or A)NA(T or C) (G or A)TC(G or A)TCNA(G
or A or T)NA(G or A or C) NANNA(G or A)NA 3' 5' (T or A) (T or
A)N(A or C) (A or G) (A or G) (T or G or A)TN(T or C)TNNTN(G or T
or C)TN(A or T or C)TNGA(T or C)GA 3' Based on MOTIF 3: 5' NCGNG(A
or T)NGTNA(T or G) (G or A or T)A(T or A)NCGNGA 3' 5' NCGNG(A or
T)NGTNA(T or G) (G or A or T)A(T or A)NCGNGA 3' 5' NC(G or T)N(G or
C) (A or T)NGTNA(A or G or T) (A or G or T)AT(A or G or T)AATNG 3'
Based on MOTIF 4: 5' NA(G or A)NGGNA(G or A)NCC 3' 5' GG(T or A) (T
or C)T(T or G or C)CC(T or A) (T or C)T(T or G or C)GC(T or C or A)
(T or C)T 3' 5' A(A or G) (T or G or A)GC(G or C or A)A(G or A) (T
or A)GG(G or C or A)A(G or A) (A or G or T or C) CC 3' 5' NA(G or
A)NGGNA(G or A)NCC 3' 5' N(A or G)NN(T or A) (T or C)NA(G or C or
A)N(C or G) (A or T or C)NA(G or A)NGGNA(G or A)NCC 3' 5' GGN(T or
C)TNCCN(T or C)TN(G or A or T) (C or G)N(T or G or C)T 3' Based on
MOTIF 5: 5' A(A or G) (A or G)TT(A or G)TC(A or G)TA(G or A or T)
(G or C) (T or A) (G or A)A(T or A) (C or T)TT 3' 5' A(G or A)N(T
or C) (T or C)NT(C or T) (A or G) TAN(G or C) (A or G)NANN(C or T)
(C or T) 3' 5' (G or A) (G or A)N(A or T)T(A or C or T) (T or A) (G
or C)NTA(T or C) (G or A)AN(A or G) (A or C or G)N(T or C)T 3'
Based on MOTIF 6: 5' GTNTT(T or C) (T or C)TN(T or A) (G or C)NTT(T
or C) (A or C)G(A or G)GG 3' Based on MOTIF 7: 5' CCNAT(A or C or
T)TT(T or C)TA(T or C) (G or A) (T or A) (G or T or C)GTNGA(T or
C)CC 3' Based on MOTIF 8: 5' GTNGGNAT(A or C or T)GA(T or C) (G or
A) (A or C)NCA 3' Based on MOTIF 9: 5' (G or A)AA(G or A)CANGC(A or
G or T)AT(G or A)TCNA(G or A) (G or A)AA 3' 5' TT(T or C) (T or
C)TNGA(T or C)AT(A or C or T)GCNTG(T or C)TT 3' Based on MOTIF 10:
5' CCCAT(G or A)TC(T or C) (T or C) (T or G)NA(T or G or A)N(T or
A) (G or A) (G or A)TC(A or G)TG CAT 3' 5' ATGCA(T or C)GA(T or C)
(T or C) (T or A)N(A or C or T)TN(A or C) (A or G) (A or G)GA(T or
C)A TGGG 3' Based on MOTIF 11: 5' NA(G or A)N(G or C) (A or T) (T
or C)T(T or C) NA(A or G) (C or T)TT 3' 5' (A or T) (G or C)NAA(A
or G) (T or C)TN(A or G)A(A or G) (A or T) (G or C)N(T or C)T 3'
Based on MOTIF 12 5' (A or G or T) (A or T) (A or T) (C or T)TCNA(G
or A)N(G or C) (A or T)N(T or C) (G or T)NA(G or A) NCC 3' 5' GGN(T
or C)TN(A or C) (G or A)N(A or T) (G or L)N(T or C)TNGA 3'
[0145] Once a clone encoding a candidate RPS family gene is
identified, it is then determined whether such gene is capable of
conferring disease-resistance to a plant host using the methods
described herein or other methods well known in the art of
molecular plant pathology.
A Biolistic Transient Exression Assay For Identification of Plant
Resistance Genes
[0146] We have developed a functional transient expression system
capable of providing a rapid and broadly applicable method for
identifying and characterizing virtually any gene for its ability
to confer disease-resistance to a plant cell. In brief, the assay
system involves delivering by biolistic transformation a candidate
plant disease-resistance gene to a plant tissue sample (e.g., a
piece of tissue from a leaf) and then evaluating the expression of
the gene within the tissue by appraising the presence or absence of
a disease-resistance response (e.g., the hypersensitive response).
This assay provides a method for identifying disease-resistance
genes from a wide variety of plant species, including ones that are
not amenable to genetic or transgenic studies.
[0147] The principle of the assay is depicted in the top portion of
FIG. 9. In general, plant cells carrying a mutation in the
resistance gene of interest are utilized. Prior to biolistic
transformation, the plant tissue is infiltrated with a
phytopathogenic bacterium carrying the corresponding avirulence
gene. In addition, a gene to be assayed for its resistance gene
activity is co-introduced by biolistics with a reporter gene. The
expression of the cobombarded reporter gene serves as an indicator
for viability of the transformed cells. Both genes are expressed
under the control of a strong and constitutive promoter. If the
gene to be assayed does not complement the resistance gene
function, the plant cells do not undergo a hypersensitive response
(HR) and, therefore, survive (FIG. 9, top panel, right). In this
case, cells accumulate a large amount of the reporter gene product.
If, on the other hand, a resistance gene is introduced, the plant
cells recognize the signal from the avirulence-gene-carrying
bacterium and undergo the HR because the expressed resistance gene
product complements the function (FIG. 9, top panel, left). In this
case, the plant cells do not have enough time to accumulate a large
amount of reporter gene product before their death. Given the
transformation efficiency estimated by a proper control (such as
the uninfected half of the leaf), measuring the accumulation of
reporter gene product can thus indicate whether the gene to be
assayed complements the resistance gene function.
[0148] In one working example, we now demonstrate the effectiveness
of the transient expression assay, using the bacterial avirulence
gene avrRpt2 and the corresponding Arabidopsis thaliana resistance
gene RPS2 (FIG. 9, bottom panel). In brief, rps2 mutant leaves,
preinfected with P. syringae carrying avrRpt2, were co-bombarded
with two plasmids, one of which contained the RPS2 gene and the
other the Escherichia coli uidA gene encoding .beta.-glucuronidase
(GUS; Jefferson et al., 1986, supra). Both the RPS2 and uidA genes
are located downstream of the strong constitutive 35S promoter from
cauliflower mosaic virus (Odell et al., infra). If the 35S-RPS2
construct complements the rps2 mutation, the transformed cells
rapidly undergo programmed cell death in response to the P.
syringae carrying avrRpt2, and relatively little GUS activity
accumulates. If the rps2 mutation is not complemented, cell death
does not occur and high levels of GUS activity accumulate. These
differences in GUS activity are detected histochemically. Because
the CDNA library used to identify RPS2 was constructed in the
expression vector pKEx4tr, the 35S-RPS2 cDNA construct in pKEx4tr
could be used directly in the transient assay. As shown in FIG. 11,
pKEx4tr is a cDNA expression vector designed for the unidirectional
insertion of cDNA inserts. Inserted cDNA is expressed under the
control of the 355 cauliflower mosaic virus promoter.
[0149] Our results are shown in FIG. 9, lower panel. In this
experiment, we infected one side of a leaf of an rps2 mutant plant
with P. syringae pv. phaseloicola 3121 carrying avrRpt2 (Psp
3121/avrRpt2). Psp 3121 is a weak pathogen of A. thaliana and Psp
3121/avrRpt2 can elicit an HR in a plant carrying the resistance
gene RPS2 (e.g., a wild type plant). Leaves of 5-week-old
Arabidopsis plants were infiltrated with an appropriate bacterial
suspension at a dose of 2.times.10.sup.8/ml by hand infiltration as
described (Dong et al., supra). After an incubation period
(typically 2-4 hours), the leaves were bombarded using a Bio-Rad
PDS-1000/He apparatus (1100 psi) after 2-4 hr of infection. Gold
particles were prepared according to the instructions of the
manufacturer. For each bombardment, 1.4 Ag of pKEx4tr-G, 0.1 .mu.g
of a plasmid to be tested, and 0.5 mg of 1 .mu.m gold particles
were used. After the bombardment, the leaves were leaf,
transformation efficiency (i.e., density of transformed cells) is
similar on both sides of the leaf. If transformed cells on the
infected side are rapidly killed, staining of the cells on the
infected side is weaker than staining on the uninfected side. When
the resistance gene RPS2 was co-introduced, the transformed cells
on the infected side of the leaf showed much weaker staining than
ones on the uninfected side (FIG. 10). In contrast, when an
unrelated gene was co-introduced, the transformed cells on the
infected side showed similar staining intensity to ones on the
uninfected side (FIG. 10).
[0150] Thus, as summarized in the Table 2, 35S-RPS4 (cDNA 4), but
not cDNA-5 or cDNA-6, complemented the HR phenotype of rps2-101C.
(See FIG. 1)
TABLE-US-00006 TABLE 2 Response Gene Tested (Decreased GUS
Activity).sup.a .DELTA.GUS (35S-uidA containing - internal uidA
deletion) cDNA-5 (35S-AB11) cDNA-4 (35S-RPS2) + cDNA-6 (35S-CK1)
.sup.aWhen decreased GUS activity was observed on the infiltrated
side of the leaf, the response was scored as plus (FIG. 10).
Both RPS2 cDNA-4 clones 4 and 11, corresponding to the two RPS2
different transcript sizes, complemented the rps2 mutant phenotype,
indicating that both transcripts encode a functional product.
Moreover, 35S-RPS2 also complemented mutants rps2-102C, rps2-101N,
and rps2-201C, further confirming that the rps2-101C, rps2-102C,
rps2-201C and rps2-10N mutations are all allelic. In short, the
cloned RPS2 gene complemented the rps2 mutation in this transient
expression assay, and complementation by RPS2 was observed in all
four available rps2 mutant stains.
[0151] Next we used the transient assay system to test the
specificity of the cloned RPS2 gene for an avrRpt2-generated signal
(i.e., the "gene-for-gene" specificity of a P. syringae avirulence
gene and a corresponding A. thaliana resistance gene (avrRpm1 and
RPM1, respectively)). This experiment involved the use of an
rps2-101 rpm1 double mutant that cannot mount an HR when challenged
with P. syringae carrying avrRpt2 or the unrelated avirulence gene
avrrpm1 (Debener et al., Plant Journal 1:289-302, 1991). As
summarized in Table 3, complementation of the rps2 mutant phenotype
by 35S-RPS2 was only observed in the presence of a signal generated
by avrRpt2, indicating that RPS2 does not simply sensitize the
plant resistance response in a nonspecific manner.
TABLE-US-00007 TABLE 3 Construct Cobombarded avr Gene with 35S-uidA
Response.sup.a None (vector only) .DELTA.GUS.sup.b - avrRPt2
.DELTA.GUS - avrRpm1 .DELTA.GUS - None (vector only) 35S-RPS2 -
avrRpt2 35S-RPS2 + avrRpm1 35S-RPS2 - .sup.aWhen decreased GUS
activity was observed on the infiltrated side of the leaf, the
response was scored as plus. (FIG. 10, panel B) .sup.b.DELTA.GUS is
35S-uidA containing an internal deletion in the uidA gene.
Also as shown in Table 3, the RPS2 gene complemented the mutant
phenotype when leaves were infected with Psp 3121/avrRpt2 but not
with Psp 3121/avrRpm1. Therefore, the RPS2 gene complemented only
the rps2 mutation; it did not the rpm1 mutation.
[0152] We have also discovered that overexpression of an rps gene
family member, e.g., rps2 but not other genes, in the transient
assay leads to apparent cell death, obviating the need to know the
corresponding avirulence gene for a putative resistance gene that
has been cloned.
[0153] Using this assay, any plant disease-resistance gene may be
identified from a cDNA expression library. In one particular
example, a cDNA library is constructed in an expression vector and
then introduced as described herein into a plant cultivar or its
corresponding mutant plant lacking the resistance gene of interest.
Preferably, the cDNA library is divided into small pools, and each
pool co-introduced with a reporter gene. If a pool contains a
resistance gene clone (i.e., the pool "complements" the resistance
gene function), the positive pool is divided into smaller pools and
the same procedure is repeated until identification of a single
positive clone is ultimately achieved. This approach facilitates
the cloning of any resistance gene of interest without genetic
crosses or the creation of transgenics.
[0154] We now describe the cloning of another member of the RPS
gene family, the Prf gene of tomato.
[0155] The initial step for the cloning of the Prf gene came from
classical genetic analysis which showed that Prf was tightly linked
to the tomato Pto gene (Salmeron et al., The Plant Cell 6:511-520,
1994). This prompted construction of a cosmid contig of 200 kb in
length which encompassed the Pto locus. DNA probes from this contig
were used to screen a tomato cDNA library constructed using tomato
leaf tissue that had been infected with Pst expressing the avrPto
avirulence gene as source material. Two classes of cDNAs were
identified based on cross-hybridization of clones to each other.
While one class corresponded to members of the Pto gene family, the
other class displayed no hybridization to Pto family members.
Taking the assumption (based on the aforementioned genetic
analysis) that Prf might reside extremely close to the Pto gene,
cDNAs from the second class were analyzed further as candidate Prf
clones. These clones were hybridized to filters containing DNAs
from six independent prf mutant lines that had been isolated by
diepoxybutane or fast neutron treatment. In one of the fast neutron
mutants, the cDNA probe revealed a 1.1 kb deletion in the genomic
DNA, suggesting that the cDNA clone might in fact represent Prf.
Wild-type DNA corresponding to the deletion was cloned from Prf/Prf
tomato. A 5 kb region was sequenced and found to potentially encode
a protein containing P-loop and leucine-rich repeat motifs,
supporting the hypothesis that this DNA encoded Prf. The
corresponding DNA was cloned and sequenced from the fast neutron
mutant plant. Sequencing this DNA confirmed the mutation to be a
simple 1.1 kb deletion excising DNA between the potential P-loop
and leucine-rich repeat coding regions. The gene is expressed based
on RT-PCR analysis which has shown that an MRNA is transcribed from
this region. The identity of the cloned DNA as the Prf gene is
based on both the existence of the deletion mutation and the
predicted protein sequence, which reveals patches of strong
similarity to other cloned disease resistance gene products
throughout the amino-terminal half (as described herein). A partial
sequence of the Prf gene is shown in FIG. 12.
RPS Expression in Transgenic Plant Cells and Plants
[0156] The expression of the RPS2 genes in plants susceptible to
pathogens carrying avrRpt2 is achieved by introducing into a plant
a DNA sequence containing the RpS2 gene for expression of the Rps2
polypeptide. A number of vectors suitable for stable transfection
of plant cells or for the establishment of transgenic plants are
available to the public; such vectors are described in, e.g.,
Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Supp.
1987); Weissbach and Weissbach, Methods for Plant Molecular
Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular
Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant
expression vectors include (1) one or more cloned plant genes under
the transcriptional control of 5' and 3' regulatory sequences and
(2) a dominant selectable marker. Such plant expression vectors may
also contain, if desired, a promoter regulatory region (e.g., a
regulatory region controlling inducible or constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific expression), a transcription initiation start site,
a ribosome binding site, an RNA processing signal, a transcription
termination site, and/or a polyadenylation signal.
[0157] An example of a useful plant promoter which could be used to
express a plant resistance gene according to the invention is a
caulimovirus promoter, e.g., the cauliflower mosaic virus (CaMV)
35S promoter. These promoters confer high levels of expression in
most plant tissues, and the activity of these promoters is not
dependent on virtually encoded proteins. CaMV is a source for both
the 35S and 19s promoters. In most tissues of transgenic plants,
the CaMV 35S promoter is a strong promoter (see, e.g., Odel et al.,
Nature 313:810, (1985)). The CaMV promoter is also highly active in
monocots (see, e.g., Dekeyser et al., Plant Cell 2:591, (1990);
Terada and Shimamoto, Mol. Gen. Genet. 220:389, (1990)).
[0158] Other useful plant promoters include, without limitation,
the nonpaline synthase promoter (An et al., Plant Physiol. 88:547,
(1988)) and the octopine synthase promoter (Fromm et al., Plant
Cell 1:977, (1989)).
[0159] For certain applications, it may be desirable to produce the
RPS2 gene product or the avrRpt2 gene product in an appropriate
tissue, at an appropriate level, or at an appropriate developmental
time. Thus, there are a variety of gene promoters, each with its
own distinct characteristics embodied in its regulatory sequences,
shown to be regulated in response to the environment, hormones,
and/or developmental cues. These include gene promoters that are
responsible for (1) heat-regulated gene expression (see, e.g.,
Callis et al., Plant Physiol. 88: 965, (1988)), (2) light-regulated
gene expression (e.g., the pea rbcS-3A described by Kuhlemeier et
al., Plant Cell 1: 471, (1989); the maize rbcs promoter described
by Schaffner and Sheen, Plant Cell 3: 997, (1991); or the
chlorophyll a/b-binding protein gene found in pea described by
Simpson et al., EMBO J. 4: 2723, (1985)), (3) hormone-regulated
gene expression (e.g., the abscisic acid responsive sequences from
the Em gene of wheat described Marcotte et al., Plant Cell 1:969,
(1989)), (4) wound induced gene expression (e.g., of wunI described
by Siebertz et al., Plant Cell 1: 961, (1989)), or (5)
organ-specific gene expression (e.g., of the tuber-specific storage
protein gene described by Roshal et al., EMBO J. 6:1155, (1987);
the 23-kDa zein gene from maize described by schernthaner et al.,
EMBO J. 7: 1249, (1988); or the French bean .beta.-phaseolin gene
described by Bustos et al., Plant Cell 1:839, (1989)).
[0160] Plant expression vectors may also optionally include RNA
processing signals, e.g, introns, which have been shown to be
important for efficient RNA synthesis and accumulation (Callis et
al., Genes and Dev. 1: 1183, (1987)). The location of the RNA
splice sequences can influence the level of transgene expression in
plants. In view of this fact, an intron may be positioned upstream
or downstream of an Rps2 polypeptide-encoding sequence in the
transgene to modulate levels of gene expression.
[0161] In addition to the aforementioned 5' regulatory control
sequences, the expression vectors may also include regulatory
control regions which are generally present in the 3' regions of
plant genes (Thornburg et al., Proc. Natl Acad. Sci USA 84: 744,
(1987); An et al., Plant Cell 1: 115, (1989)). For example, the 3'
terminator region may be included in the expression vector to
increase stability of the mRNA. One such terminator region may be
derived from the PI-II terminator region of potato. In addition,
other commonly used terminators are derived from the octopine or
nopaline synthase signals.
[0162] The plant expression vector also typically contains a
dominant selectable marker gene used to identify the cells that
have become transformed. Useful selectable marker genes for plant
systems include genes encoding antibiotic resistance genes, for
example, those encoding resistance to hygromycin, kanamycin,
bleomycin, G418, streptomycin or spectinomycin. Genes required for
photosynthesis may also be used as selectable markers in
photosynthetic-deficient strains. Finally, genes encoding herbicide
resistance may be used as selectable markers; useful herbicide
resistance genes include the bar gene encoding the enzyme
phosphinothricin acetyltransferase, which confers resistance to the
broad spectrum herbicide Basta.RTM. (Hoechst A G, Frankfurt,
Germany).
[0163] Efficient use of selectable markers is facilitated by a
determination of the susceptibility of a plant cell to a particular
selectable agent and a determination of the concentration of this
agent which effectively kills most, if not all, of the transformed
cells. Some useful concentrations of antibiotics for tobacco
transformation include, e.g., 75-100 .mu.g/ml (kanamycin), 20-50
.mu.g/ml (hygromycin), or 5-10 .mu.g/ml (bleomycin). A useful
strategy for selection of transformants for herbicide resistance is
described, e.g., in Vasil I. K., Cell Culture and Somatic Cell
Genetics of Plants, Vol I, II, III Laboratory Procedures and Their
Applications Academic Press, New York, 1984.
[0164] It should be readily apparent to one skilled in the field of
plant molecular biology that the level of gene expression is
dependent not only on the combination of promoters, RNA processing
signals and terminator elements, but also on how these elements are
used to increase the levels of gene expression.
[0165] The above exemplary techniques may be used for the
expression of any gene in the RPS family.
Plant Transformation
[0166] Upon construction of the plant expression vector, several
standard methods are known for introduction of the recombinant
genetic material into the host plant for the generation of a
transgenic plant. These methods include (1) Agrobacterium-mediated
transformation (A. tumefaciens or A. rhizogenes) (see, e.g.,
Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby,
ed, London, Academic Press, 1987; and Lichtenstein, C. P., and
Draper, J,. In: DNA Cloning, Vol II, D. M. Glover, ed, Oxford, IRI
Press, 1985), (2) the particle delivery system (see, e.g.,
Gordon-Kamm et al., Plant Cell 2:603, (1990); or BioRad Technical
Bulletin 1687, supra), (3) microinjection protocols (see, e.g.,
Green et al., Plant Tissue and Cell Culture, Academic Press, New
York, 1987), (4) polyethylene glycol (PEG) procedures (see, e.g.,
Draper et al., Plant Cell Physiol 23:451, (1982); or e.g., Zhang
and Wu, Theor. Appl. Genet. 76:835, (1988)), (5) liposome-mediated
DNA uptake (see, e.g., Freeman et al., Plant Cell Physiol 25: 1353,
(1984)), (6) electroporation protocols (see, e.g., Gelvin et al
supra; Dekeyser et al. supra; or Fromm et al Nature 319: 791,
(1986)), and (7) the vortexing method (see, e.g., Kindle, K., Proc.
Natl. Acad. Sci., USA 87:1228, (1990)).
[0167] The following is an example outlining an
Agrobacterium-mediated plant transformation. The general process
for manipulating genes to be transferred into the genome of plant
cells is carried out in two phases. First, all the cloning and DNA
modification steps are done in E. coil, and the plasmid containing
the gene construct of interest is transferred by conjugation into
Agrobacterium. Second, the resulting Agrobacterium strain is used
to transform plant cells. Thus, for the generalized plant
expression vector, the plasmid contains an origin of replication
that allows it to replicate in Agrobacterium and a high copy number
origin of replication functional in E. coli. This permits facile
production and testing of transgenes in E.coli prior to transfer to
Agrobacterium for subsequent introduction into plants. Resistance
genes can be carried on the vector, one for selection in bacteria,
e.g., streptomycin, and the other that will express in plants,
e.g., a gene encoding for kanamycin resistance or an herbicide
resistance gene. Also present are restriction endonuclease sites
for the addition of one or more transgenes operably linked to
appropriate regulatory sequences and directional T-DNA border
sequences which, when recognized by the transfer functions of
Agrobacterium, delimit the region that will be transferred to the
plant.
[0168] In another example, plant cells may be transformed by
shooting into the cell tungsten microprojectiles on which cloned
DNA is precipitated. In the Biolistic Apparatus (Bio-Rad, Hercules,
Calif.) used for the shooting, a gunpowder charge (22 caliber Power
Piston Tool Charge) or an air-driven blast drives a plastic
macroprojectile through a gun barrel. An aliquot of a suspension of
tungsten particles on which DNA has been precipitated is placed on
the front of the plastic macroprojectile. The latter is fired at an
acrylic stopping plate that has a hole through it that is too small
for the macroprojectile to go through. As a result, the plastic
macroprojectile smashes against the stopping plate and the tungsten
microprojectiles continue toward their target through the hole in
the plate. For the instant invention the target can be any plant
cell, tissue, seed, or embryo. The DNA introduced into the cell on
the microprojectiles becomes integrated into either the nucleus or
the chloroplast.
[0169] Transfer and expression of transgenes in plant cells is now
routine practice to those skilled in the art. It has become a major
tool to carry out gene expression studies and to attempt to obtain
improved plant varieties of agricultural or commercial
interest.
Transgenic Plant Regeneration
[0170] Plant cells transformed with a plant expression vector can
be regenerated, e.g., from single cells, callus tissue or leaf
discs according to standard plant tissue culture techniques. It is
well known in the art that various cells, tissues and organs from
almost any plant can be successfully cultured to regenerate an
entire plant; such techniques are described, e.g., in Vasil supra;
Green et al., supra; Weissbach and Weissbach, supra; and Gelvin et
al., supra.
[0171] In one possible example, a vector carrying a selectable
marker gene (e.g., kanamycin resistance), a cloned RPS2 gene under
the control of its own promoter and terminator or, if desired,
under the control of exogenous regulatory sequences such as the 35S
CaMV promoter and the nopaline synthase terminator is transformed
into Agrobacterium. Transformation of leaf tissue with
vector-containing Agrobacterium is carried out as described by
Horsch et al. (Science 227: 1229, (1985)). Putative transformants
are selected after a few weeks (e.g., 3 to 5 weeks) on plant tissue
culture media containing kanamycin (e.g. 100 .mu.g/ml).
Kanamycin-resistant shoots are then placed on plant tissue culture
media without hormones for root initiation. Kanamycin-resistant
plants are then selected for greenhouse growth. If desired, seeds
from self-fertilized transgenic plants can then be sowed in a
soil-less media and grown in a greenhouse. Kanamycin-resistant
progeny are selected by sowing surfaced sterilized seeds on
hormone-free kanamycin-containing media. Analysis for the
integration of the transgene is accomplished by standard techniques
(see, e.g., Ausubel et al. supra; Gelvin et al. supra).
[0172] Transgenic plants expressing the selectable marker are then
screened for transmission of the transgene DNA by standard
immunoblot and DNA and,RNA detection techniques. Each positive
transgenic plant and its transgenic progeny are unique in
comparison to other transgenic plants established with the same
transgene. Integration of the transgene DNA into the plant genomic
DNA is in most cases random and the site of integration can
profoundly effect the levels, and the tissue and developmental
patterns of transgene expression. Consequently, a number of
transgenic lines are usually screened for each transgene to
identify and select plants with the most appropriate expression
profiles.
[0173] Transgenic lines are evaluated for levels of transgene
expression. Expression at the RNA level is determined initially to
identify and quantitate expression-positive plants. Standard
techniques for RNA analysis are employed and include PCR
amplification assays using oligonucleotide primers designed to
amplify only transgene RNA templates and solution hybridization
assays using transgene-specific probes (see, e.g., Ausubel et al.,
supra). The RNA-positive plants are then analyzed for protein
expression by Western immunoblot analysis using Rps2
polypeptide-specific antibodies (see, e.g., Ausubel et al., supra).
In addition, in situ hybridization and immunocytochemistry
according to standard protocols can be done using
transgene-specific nucleotide probes and antibodies, respectively,
to localize sites of expression within transgenic tissue.
[0174] Once the Rps2 polypeptide has been expressed in any cell or
in a transgenic plant (e.g., as described above), it can be
isolated using any standard technique, e.g., affinity
chromatography. In one example, an anti-Rps2 antibody (e.g.,
produced as described in Ausubel et al., supra, or by any standard
technique) may be attached to a column and used to isolate the
polypeptide. Lysis and fractionation of Rps2-producing cells prior
to affinity chromatography may be performed by standard methods
(see, e.g., Ausubel et al., supra). Once isolated, the recombinant
polypeptide can, if desired, be further purified, e.g., by high
performance liquid chromatography (see, e.g., Fisher, Laboratory
Techniques In Biochemistry And Molecular Biology, Work and Burdon,
eds., Elsevier, 1980).
[0175] These general techniques of polypeptide expression and
purification can also be used to produce and isolate useful Rps2
fragments or analogs.
Antibody Production
[0176] Using a polypeptide described above (e.g., the recombinant
protein or a chemically synthesized RPS peptide based on its
deduced amino acid sequence), polyclonal antibodies which bind
specifically to an RPS polypeptide may be produced by standard
techniques (see, e.g., Ausubel et al., ra) and isolated, e.g.,
following peptide antigen affinity chromatography. Monoclonal
antibodies can also be prepared using standard hybridoma technology
(see, e.g., Kohler et al., Nature 256: 495, 1975; Kohler et al.,
Eur. J. Immunol. 6: 292, 1976; Hammerling et al., in Monoclonal
Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; and Ausubel
et al., supra).
[0177] Once produced, polyclonal or monoclonal antibodies are
tested for specific RSP polypeptide recognition by Western blot or
immunoprecipitation analysis (by methods described in Ausubel et
al., supra). Antibodies which specifically recognize a RPS
polypeptide are considered to be useful in the invention; such
antibodies may be used, e.g., for screening recombinant expression
libraries as described in Ausubel et al., supra. Exemplary peptides
(derived from Rps2) for antibody production include:
TABLE-US-00008 LKFSYDNLESDLL GVYGPGGVGKTTLMQS GGLPLALITLGGAM
Use
[0178] Introduction of RPS2 into a transformed plant cell provides
for resistance to bacterial pathogens carrying the avrRpt2
avirulence gene. For example, transgenic plants of the instant
invention expressing RPS2 might be used to alter, simply and
inexpensively, the disease resistance of plants normally
susceptible to plant pathogens carrying the avirulence gene,
avrRpt2.
[0179] The invention also provides for broad-spectrum pathogen
resistance by mimicking the natural mechanism of host resistance.
First, the RPS2 transgene is expressed in plant cells at a
sufficiently high level to initiate the plant defense response
constitutively in the absence of signals from the pathogen. The
level of expression associated with plant defense response
initiation is determined by measuring the levels of defense
response gene expression as described in Dong et al., supra.
Second, the RPS2 transgene is expressed by a controllable promoter
such as a tissue-specific promoter, cell-type specific promoter or
by a promoter that is induced by an external signal or agent thus
limiting the temporal and tissue expression of a defense response.
Finally, the RPS2 gene product is co-expressed with the avrRpt2
gene product. The RPS2 gene is expressed by its natural promoter,
by a constitutively expressed promoter such as the CaMV 358
promoter, by a tissue-specific or cell-type specific promoter, or
by a promoter that is activated by an external signal or agent.
Co-expression of RPS2 and avrRpt2 will mimic the production of gene
products associated with the initiation of the plant defense
response and provide resistance to pathogens in the absence of
specific resistance gene-avirulence gene corresponding pairs in the
host plant and pathogen.
[0180] The invention also provides for expression in plant cells of
a nucleic acid having the sequence of FIG. 2 or the expression of a
degenerate variant thereof encoding the amino acid sequence of open
reading frame "a" of FIG. 2.
[0181] The invention further provides for the isolation of nucleic
acid sequences having about 50% or greater sequence identity to
RPS2 by using the RPS2 sequence of FIG. 2 or a portion thereof
greater than 9 nucleic acids in length, and preferably greater than
about 18 nucleic acids in length as a probe. Appropriate reduced
hybridization stringency conditions are utilized to isolate DNA
sequences having about 50% or greater sequence identity to the RPS2
sequence of FIG. 2.
[0182] Also provided by the invention are short conserved regions
characteristic of RPS disease resistance genes. These conserved
regions provide oligonucleotide sequences useful for the production
of hybridization probes and PCR primers for the isolation of other
plant disease-resistance genes.
[0183] Both the RPS2 gene and related RPS family genes provide
disease resistance to plants, especially crop plants, most
especially important crop plants such as tomato, pepper, maize,
wheat, rice and legumes-such as soybean and bean, or any plant
which is susceptible to pathogens carrying an avirulence gene,
e.g., the avrfpt2 avirulence gene. Such pathogens include, but are
not limited to, Pseudomonas syringae strains.
[0184] The invention also includes any biologically active fragment
or analog of an Rps2 polypeptide. By "biologically active" is meant
possessing any in vivo activity which is characteristic of the Rps2
polypeptide shown in FIG. 2. A useful Rps2 fragment or Rps2 analog
is one which exhibits a biological activity in any biological assay
for disease resistance gene product activity, for example, those
assays described by Dong et al. (1991), supra; Yu et al. (1993)
supra; Kunkel et al. (1993) supra; and Whalen et al. (1991). In
particular, a biologically active Rps2 polypeptide fragment or
analog is capable of providing substantial resistance to plant
pathogens carrying the avrRpt2 avirulence gene. By substantial
resistance is meant at least partial reduction in susceptibility to
plant pathogens carrying the avrRpt2 gene.
[0185] Preferred analogs include Rps2 polypeptides (or biologically
active fragments thereof) whose sequences differ from the wild-type
sequence only by conservative amino acid substitutions, for
example, substitution of one amino acid for another with similar
characteristics (e.g., valine for glycine, arginine for lysine,
etc.) or by one or more non-conservative amino acid substitutions,
deletions, or insertions which do not abolish the polypeptiders
biological activity.
[0186] Analogs can differ from naturally occurring Rps2 polypeptide
in amino acid sequence or can be modified in ways that do not
involve sequence, or both. Analogs of the invention will generally
exhibit at least 70%, preferably 80%, more preferably 90%, and most
preferably 95% or even 99%, homology with a segment of 20 amino
acid residues, preferably 40 amino acid residues, or more
preferably the entire sequence of a naturally occurring Rps2
polypeptide sequence.
[0187] Alterations in primary sequence include genetic variants,
both natural and induced. Also included are analogs that include
residues other than naturally occurring L-amino acids, e.g.,
D-amino acids or non-naturally occurring or synthetic amino acids,
e.g., .beta. or .gamma. amino acids. Also included in the invention
are Rps2 polypeptides modified by in vivo chemical derivatization
of polypeptides, including acetylation, methylation,
phosphorylation, carboxylation, or glycosylation.
[0188] In addition to substantially full-length polypeptides, the
invention also includes biologically active fragments of the
polypeptides. As used herein, the term "fragment", as applied to a
polypeptide, will ordinarily be at least 20 residues, more
typically at least 40 residues, and preferably at least 60 residues
in length. Fragments of Rps2 polypeptide can be generated by
methods known to those skilled in the art. The ability of a
candidate fragment to exhibit a biological activity of Rps2 can be
assessed by those methods described herein. Also included in the
invention are Rps2 polypeptides containing residues that are not
required for biological activity of the peptide, e.g., those added
by alternative mRNA splicing or alternative protein processing
events.
[0189] Other embodiments are within the following claims.
Sequence CWU 1
1
21212903DNAArabidopsis thaliana 1aagtaaaaga aagagcgaga aatcatcgaa
atggatttca tctcatctct tatcgttggc 60tgtgctcagg tgttgtgtga atctatgaat
atggcggaga gaagaggaca taagactgat 120cttagacaag ccatcactga
tcttgaaaca gccatcggtg acttgaaggc catacgtgat 180gacctgactt
tacggatcca acaagacggt ctagagggac gaagctgctc aaatcgtgcc
240agagagtggc ttagtgcggt gcaagtaacg gagactaaaa cagccctact
tttagtgagg 300tttaggcgtc gggaacagag gacgcgaatg aggaggagat
acctcagttg tttcggttgt 360gccgactaca aactgtgcaa gaaggtttct
gccatattga agagcattgg tgagctgaga 420gaacgctctg aagctatcaa
aacagatggc gggtcaattc aagtaacttg tagagagata 480cccatcaagt
ccgttgtcgg aaataccacg atgatggaac aggttttgga atttctcagt
540gaagaagaag aaagaggaat cattggtgtt tatggacctg gtggggttgg
gaagacaacg 600ttaatgcaga gcattaacaa cgagctgatc acaaaaggac
atcagtatga tgtactgatt 660tgggttcaaa tgtccagaga attcggcgag
tgtacaattc agcaagccgt tggagcacgg 720ttgggtttat cttgggacga
gaaggagacc ggcgaaaaca gagctttgaa gatatacaga 780gctttgagac
agaaacgttt cttgttgttg ctagatgatg tctgggaaga gatagacttg
840gagaaaactg gagttcctcg acctgacagg gaaaacaaat gcaaggtgat
gttcacgaca 900cggtctatag cattatgcaa caatatgggt gcggaataca
agttgagagt ggagtttctg 960gagaagaaac acgcgtggga gctgttctgt
agtaaggtat ggagaaaaga tcttttagag 1020tcatcatcaa ttcgccggct
cgcggagatt atagtgagta aatgtggagg attgccacta 1080gcgttgatca
ctttaggagg agccatggct catagagaga cagaagaaga gtggatccat
1140gctagtgaag ttctgactag atttccagca gagatgaagg gtatgaacta
tgtatttgcc 1200cttttgaaat tcagctacga caacctcgag agtgatctgc
ttcggtcttg tttcttgtac 1260tgcgctttat tcccagaaga acattctata
gagatcgagc agcttgttga gtactgggtc 1320ggcgaagggt ttctcaccag
ctcccatggc gttaacacca tttacaaggg atattttctc 1380attggggatc
tgaaagcggc atgtttgttg gaaaccggag atgagaaaac acaggtgaag
1440atgcataatg tggtcagaag ctttgcattg tggatggcat ctgaacaggg
gacttataag 1500gagctgatcc tagttgagcc tagcatggga catactgaag
ctcctaaagc agaaaactgg 1560cgacaagcgt tggtgatctc attgttagat
aacagaatcc agaccttgcc tgaaaaactc 1620atatgcccga aactgacaac
actgatgctc caacagaaca gctctttgaa gaagattcca 1680acagggtttt
tcatgcatat gcctgttctc agagtcttgg acttgtcgtt cacaagtatc
1740actgagattc cgttgtctat caagtatttg gtggagttgt atcatctgtc
tatgtcagga 1800acaaagataa gtgtattgcc acaggagctt gggaatctta
gaaaactgaa gcatctggac 1860ctacaaagaa ctcagtttct tcagacgatc
ccacgagatg ccatatgttg gctgagcaag 1920ctcgaggttc tgaacttgta
ctacagttac gccggttggg aactgcagag ctttggagaa 1980gatgaagcag
aagaactcgg attcgctgac ttggaatact tggaaaacct aaccacactc
2040ggtatcactg ttctctcatt ggagacccta aaaactctct tcgagttcgg
tgctttgcat 2100aaacatatac agcatctcca cgttgaagag tgcaatgaac
tcctctactt caatctccca 2160tcactcacta accatggcag gaacctgaga
agacttagca ttaaaagttg ccatgacttg 2220gagtacctgg tcacacccgc
agattttgaa aatgattggc ttccgagtct agaggttctg 2280acgttacaca
gccttcacaa cttaaccaga gtgtggggaa attctgtaag ccaagattgt
2340ctgcggaata tccgttgcat aaacatttca cactgcaaca agctgaagaa
tgtctcatgg 2400gttcagaaac tcccaaagct agaggtgatt gaactgttcg
actgcagaga gatagaggaa 2460ttgataagcg aacacgagag tccatccgtc
gaagatccaa cattgttccc aagcctgaag 2520accttgagaa ctagggatct
gccagaacta aacagcatcc tcccatctcg attttcattc 2580caaaaagttg
aaacattagt catcacaaat tgccccagag ttaagaaact gccgtttcag
2640gagaggagga cccagatgaa cttgccaaca gtttattgtg aggagaaatg
gtggaaagca 2700ctggaaaaag atcaaccaaa cgaagagctt tgttatttac
cgcgctttgt tccaaattga 2760tataagagct aagagcactc tgtacaaata
tgtccattca taagtagcag gaagccagga 2820aggttgttcc agtgaagtca
tcaactttcc acatagccac aaaactagag attatgtaat 2880cataaaaacc
aaactatccg cga 29032885PRTArabidopsis thaliana 2Lys Lys Glu Arg Glu
Ile Ile Glu Met Asp Phe Ile Ser Ser Leu Ile1 5 10 15Val Gly Cys Ala
Gln Val Leu Cys Glu Ser Met Asn Met Ala Glu Arg 20 25 30Arg Gly His
Lys Thr Asp Leu Arg Gln Ala Ile Thr Asp Leu Arg Ile35 40 45Gln Gln
Asp Gly Leu Glu Gly Arg Ser Cys Ser Asn Arg Ala Arg Glu50 55 60Trp
Leu Ser Ala Val Gln Val Thr Glu Thr Lys Thr Ala Leu Leu Leu65 70 75
80Val Arg Phe Arg Arg Arg Glu Gln Arg Thr Arg Met Arg Arg Arg Tyr
85 90 95Leu Ser Cys Phe Gly Cys Ala Asp Tyr Lys Leu Cys Lys Lys Val
Ser 100 105 110Ala Ile Leu Lys Ser Ile Gly Glu Leu Arg Glu Arg Ser
Glu Ala Ile115 120 125Lys Thr Asp Gly Gly Ser Ile Gln Val Thr Cys
Arg Glu Ile Pro Ile130 135 140Lys Ser Val Val Gly Asn Thr Thr Met
Met Glu Gln Val Leu Glu Phe145 150 155 160Leu Ser Glu Glu Glu Glu
Arg Gly Ile Ile Gly Val Tyr Gly Pro Gly 165 170 175Gly Val Gly Lys
Thr Thr Leu Met Gln Ser Ile Asn Asn Glu Leu Ile 180 185 190Thr Lys
Gly His Gln Tyr Asp Val Leu Ile Trp Val Gln Met Ser Arg195 200
205Glu Phe Gly Glu Cys Thr Ile Gln Gln Ala Val Gly Ala Arg Leu
Gly210 215 220Leu Ser Trp Asp Glu Lys Glu Thr Gly Glu Asn Arg Ala
Leu Lys Ile225 230 235 240Tyr Arg Ala Leu Arg Gln Lys Arg Phe Leu
Leu Leu Leu Asp Asp Val 245 250 255Trp Glu Glu Ile Asp Leu Glu Lys
Thr Gly Val Pro Arg Pro Asp Arg 260 265 270Glu Asn Lys Cys Lys Val
Met Phe Thr Thr Arg Ser Ile Ala Leu Cys275 280 285Asn Asn Met Gly
Ala Glu Tyr Lys Leu Arg Val Glu Phe Leu Glu Lys290 295 300Lys His
Ala Trp Glu Leu Phe Cys Ser Lys Val Trp Arg Lys Asp Leu305 310 315
320Leu Glu Ser Ser Ser Ile Arg Arg Leu Ala Glu Ile Ile Val Ser Lys
325 330 335Cys Gly Gly Leu Pro Leu Ala Leu Ile Thr Leu Gly Gly Ala
Met Ala 340 345 350His Arg Glu Thr Glu Glu Glu Trp Ile His Ala Ser
Glu Val Leu Thr355 360 365Arg Phe Pro Ala Glu Met Lys Gly Met Asn
Tyr Val Phe Ala Leu Leu370 375 380Lys Phe Ser Tyr Asp Asn Leu Glu
Ser Asp Leu Leu Arg Ser Cys Phe385 390 395 400Leu Tyr Cys Ala Leu
Phe Pro Glu Glu His Ser Ile Glu Ile Glu Gln 405 410 415Leu Val Glu
Tyr Trp Val Gly Glu Gly Phe Leu Thr Ser Ser His Gly 420 425 430Val
Asn Thr Ile Tyr Lys Gly Tyr Phe Leu Ile Gly Asp Leu Lys Ala435 440
445Ala Cys Leu Leu Glu Thr Gly Asp Glu Lys Thr Gln Val Lys Met
His450 455 460Asn Val Val Arg Ser Phe Ala Leu Trp Met Ala Ser Glu
Gln Gly Thr465 470 475 480Tyr Lys Glu Leu Ile Leu Val Glu Pro Ser
Met Gly His Thr Glu Ala 485 490 495Pro Lys Ala Glu Asn Trp Arg Gln
Ala Leu Val Ile Ser Leu Leu Asp 500 505 510Asn Arg Ile Gln Thr Leu
Pro Glu Lys Leu Ile Cys Pro Lys Leu Thr515 520 525Thr Leu Met Leu
Gln Gln Asn Ser Ser Leu Lys Lys Ile Pro Thr Gly530 535 540Phe Phe
Met His Met Pro Val Leu Arg Val Leu Asp Leu Ser Phe Thr545 550 555
560Ser Ile Thr Glu Ile Pro Leu Ser Ile Lys Tyr Leu Val Glu Leu Tyr
565 570 575His Leu Ser Met Ser Gly Thr Lys Ile Ser Val Leu Pro Gln
Glu Leu 580 585 590Gly Asn Leu Arg Lys Leu Lys His Leu Asp Leu Gln
Arg Thr Gln Phe595 600 605Leu Gln Thr Ile Pro Arg Asp Ala Ile Cys
Trp Leu Ser Lys Leu Glu610 615 620Val Leu Asn Leu Tyr Tyr Ser Tyr
Ala Gly Trp Glu Leu Gln Ser Phe625 630 635 640Gly Glu Asp Glu Ala
Glu Glu Leu Gly Phe Ala Asp Leu Glu Tyr Leu 645 650 655Glu Asn Leu
Thr Thr Leu Gly Ile Thr Val Leu Ser Leu Glu Thr Leu 660 665 670Lys
Thr Leu Phe Glu Phe Gly Ala Leu His Lys His Ile Gln His Leu675 680
685His Val Glu Glu Cys Asn Glu Leu Leu Tyr Phe Asn Leu Pro Ser
Leu690 695 700Thr Asn His Gly Arg Asn Leu Arg Arg Leu Ser Ile Lys
Ser Cys His705 710 715 720Asp Leu Glu Tyr Leu Val Thr Pro Ala Asp
Phe Glu Asn Asp Trp Leu 725 730 735Pro Ser Leu Glu Val Leu Thr Leu
His Ser Leu His Asn Leu Arg Cys 740 745 750Ile Asn Ile Ser His Cys
Asn Lys Leu Lys Asn Val Ser Trp Val Gln755 760 765Lys Leu Pro Lys
Leu Glu Val Ile Glu Leu Phe Asp Cys Arg Glu Ile770 775 780Glu Glu
Leu Ile Ser Glu His Glu Ser Pro Ser Val Glu Asp Pro Thr785 790 795
800Leu Phe Pro Ser Leu Lys Thr Leu Arg Thr Arg Asp Leu Pro Glu Leu
805 810 815Asn Ser Ile Leu Pro Ser Arg Phe Ser Phe Gln Lys Val Glu
Thr Leu 820 825 830Val Ile Thr Asn Cys Pro Arg Val Lys Lys Leu Pro
Phe Gln Glu Arg835 840 845Arg Thr Gln Met Asn Leu Pro Thr Val Tyr
Cys Glu Glu Lys Trp Trp850 855 860Lys Ala Leu Glu Lys Asp Gln Pro
Asn Glu Glu Leu Cys Tyr Leu Pro865 870 875 880Arg Phe Val Pro Asn
885320PRTArabidopsis thaliana 3Glu His Ser Val Gln Ile Cys Pro Phe
Ile Ser Ser Arg Lys Pro Gly1 5 10 15Arg Leu Phe Gln
2046PRTArabidopsis thaliana 4Ser His Gln Leu Ser Thr1
5511PRTArabidopsis thaliana 5Arg Leu Cys Asn His Lys Asn Gln Thr
Ile Arg1 5 10628PRTArabidopsis thaliana 6Ser Lys Arg Lys Ser Glu
Lys Ser Ser Lys Trp Ile Ser Ser His Leu1 5 10 15Leu Ser Leu Ala Val
Leu Arg Cys Cys Val Asn Leu 20 25725PRTArabidopsis thaliana 7Ile
Trp Arg Arg Glu Glu Asp Ile Arg Leu Ile Leu Asp Lys Pro Ser1 5 10
15Leu Ile Leu Lys Gln Pro Ser Val Thr 20 2586PRTArabidopsis
thaliana 8Arg Pro Tyr Val Met Thr1 598PRTArabidopsis thaliana 9Leu
Tyr Gly Ser Asn Lys Thr Val1 51017PRTArabidopsis thaliana 10Arg Asp
Glu Ala Ala Gln Ile Val Pro Glu Ser Gly Leu Val Arg Cys1 5 10
15Lys118PRTArabidopsis thaliana 11Arg Arg Leu Lys Gln Pro Tyr Phe1
51210PRTArabidopsis thaliana 12Gly Leu Gly Val Gly Asn Arg Gly Arg
Glu1 5 101322PRTArabidopsis thaliana 13Gly Gly Asp Thr Ser Val Val
Ser Val Val Pro Thr Thr Asn Cys Ala1 5 10 15Arg Arg Phe Leu Pro Tyr
20145PRTArabidopsis thaliana 14Arg Ala Leu Val Ser1
51515PRTArabidopsis thaliana 15Glu Asn Ala Leu Lys Leu Ser Lys Gln
Met Ala Gly Gln Phe Lys1 5 10 151615PRTArabidopsis thaliana 16Leu
Val Glu Arg Tyr Pro Ser Ser Pro Leu Ser Glu Ile Pro Arg1 5 10
151729PRTArabidopsis thaliana 17Trp Asn Arg Phe Trp Asn Phe Ser Val
Lys Lys Lys Lys Glu Glu Ser1 5 10 15Leu Val Phe Met Asp Leu Val Gly
Leu Gly Arg Gln Arg 20 25187PRTArabidopsis thaliana 18Cys Arg Ala
Leu Thr Thr Ser1 5199PRTArabidopsis thaliana 19Ser Gln Lys Asp Ile
Ser Met Met Tyr1 52036PRTArabidopsis thaliana 20Phe Gly Phe Lys Cys
Pro Glu Asn Ser Ala Ser Val Gln Phe Ser Lys1 5 10 15Pro Leu Glu His
Gly Trp Val Tyr Leu Gly Thr Arg Arg Arg Pro Ala 20 25 30Lys Thr Glu
Leu35215PRTArabidopsis thaliana 21Arg Tyr Thr Glu Leu1
5228PRTArabidopsis thaliana 22Asp Arg Asn Val Ser Cys Cys Cys1
5236PRTArabidopsis thaliana 23Met Met Ser Gly Lys Arg1
52417PRTArabidopsis thaliana 24Thr Trp Arg Lys Leu Glu Phe Leu Asp
Leu Thr Gly Lys Thr Asn Ala1 5 10 15Arg256PRTArabidopsis thaliana
25Cys Ser Arg His Gly Leu1 52611PRTArabidopsis thaliana 26His Tyr
Ala Thr Ile Trp Val Arg Asn Thr Ser1 5 102723PRTArabidopsis
thaliana 27Glu Trp Ser Phe Trp Arg Arg Asn Thr Arg Gly Ser Cys Ser
Val Val1 5 10 15Arg Tyr Gly Glu Lys Ile Phe 202811PRTArabidopsis
thaliana 28Ser His His Gln Phe Ala Gly Ser Arg Arg Leu1 5
10297PRTArabidopsis thaliana 29Val Asn Val Glu Asp Cys His1
53019PRTArabidopsis thaliana 30Glu Glu Pro Trp Leu Ile Glu Arg Gln
Lys Lys Ser Gly Ser Met Leu1 5 10 15Val Lys Phe316PRTArabidopsis
thaliana 31Leu Asp Phe Gln Gln Arg1 5326PRTArabidopsis thaliana
32Thr Met Tyr Leu Pro Phe1 53327PRTArabidopsis thaliana 33Asn Ser
Ala Thr Thr Thr Ser Arg Val Ile Cys Phe Gly Leu Val Ser1 5 10 15Cys
Thr Ala Leu Tyr Ser Gln Lys Asn Ile Leu 20 253433PRTArabidopsis
thaliana 34Arg Ser Ser Ser Leu Leu Ser Thr Gly Ser Ala Lys Gly Phe
Ser Pro1 5 10 15Ala Pro Met Ala Leu Thr Pro Phe Thr Arg Asp Ile Phe
Ser Leu Gly 20 25 30Ile3514PRTArabidopsis thaliana 35Lys Arg His
Val Cys Trp Lys Pro Glu Met Arg Lys His Arg1 5 103622PRTArabidopsis
thaliana 36Arg Cys Ile Met Trp Ser Glu Ala Leu His Cys Gly Trp His
Leu Asn1 5 10 15Arg Gly Leu Ile Arg Ser 203720PRTArabidopsis
thaliana 37Leu Ser Leu Ala Trp Asp Ile Leu Lys Leu Leu Lys Gln Lys
Thr Gly1 5 10 15Asp Lys Arg Trp 203815PRTArabidopsis thaliana 38Ile
Thr Glu Ser Arg Pro Cys Leu Lys Asn Ser Tyr Ala Arg Asn1 5 10
15397PRTArabidopsis thaliana 39Cys Ser Asn Arg Thr Ala Leu1
54046PRTArabidopsis thaliana 40Arg Arg Phe Gln Gln Gly Phe Ser Cys
Ile Cys Leu Phe Ser Glu Ser1 5 10 15Trp Thr Cys Arg Ser Gln Val Ser
Leu Arg Phe Arg Cys Leu Ser Ser 20 25 30Ile Trp Trp Ser Cys Ile Ile
Cys Leu Cys Gln Glu Gln Arg35 40 454112PRTArabidopsis thaliana
41Val Tyr Cys His Arg Ser Leu Gly Ile Leu Glu Asn1 5
104221PRTArabidopsis thaliana 42Ser Ile Trp Thr Tyr Lys Glu Leu Ser
Phe Phe Arg Arg Ser His Glu1 5 10 15Met Pro Tyr Val Gly
20435PRTArabidopsis thaliana 43Ala Ser Ser Arg Phe1
54432PRTArabidopsis thaliana 44Thr Cys Thr Thr Val Thr Pro Val Gly
Asn Cys Arg Ala Leu Glu Lys1 5 10 15Met Lys Gln Lys Asn Ser Asp Ser
Leu Thr Trp Asn Thr Trp Lys Thr 20 25 304512PRTArabidopsis thaliana
45Pro His Ser Val Ser Leu Phe Ser His Trp Arg Pro1 5
104638PRTArabidopsis thaliana 46Lys Leu Ser Ser Ser Ser Val Leu Cys
Ile Asn Ile Tyr Ser Ile Ser1 5 10 15Thr Leu Lys Ser Ala Met Asn Ser
Ser Thr Ser Ile Ser His His Ser 20 25 30Leu Thr Met Ala Gly
Thr354727PRTArabidopsis thaliana 47Glu Asp Leu Ala Leu Lys Val Ala
Met Thr Trp Ser Thr Trp Ser His1 5 10 15Pro Gln Ile Leu Lys Met Ile
Gly Phe Arg Val 20 25487PRTArabidopsis thaliana 48Arg Tyr Thr Ala
Phe Thr Thr1 5497PRTArabidopsis thaliana 49Pro Glu Cys Gly Glu Ile
Leu1 55010PRTArabidopsis thaliana 50Ala Lys Ile Val Cys Gly Ile Ser
Val Ala1 5 10517PRTArabidopsis thaliana 51Thr Phe His Thr Ala Thr
Ser1 5526PRTArabidopsis thaliana 52Phe Arg Asn Ser Gln Ser1
5538PRTArabidopsis thaliana 53Leu Asn Cys Ser Thr Ala Glu Arg1
55416PRTArabidopsis thaliana 54Ala Asn Thr Arg Val His Pro Ser Lys
Ile Gln His Cys Ser Gln Ala1 5 10 15557PRTArabidopsis thaliana
55Glu Leu Gly Ile Cys Gln Asn1 55615PRTArabidopsis thaliana 56Thr
Ala Ser Ser His Leu Asp Phe His Ser Lys Lys Leu Lys His1 5 10
155719PRTArabidopsis thaliana 57Ser Ser Gln Ile Ala Pro Glu Leu Arg
Asn Cys Arg Phe Arg Arg Gly1 5 10 15Gly Pro Arg5847PRTArabidopsis
thaliana 58Thr Cys Gln Gln Phe Ile Val Arg Arg Asn Gly Gly Lys His
Trp Lys1 5 10 15Lys Ile Asn Gln Thr Lys Ser Phe Val Ile Tyr Arg Ala
Leu Phe Gln 20 25 30Ile Asp Ile Arg Ala Lys Ser Thr Leu Tyr Lys Tyr
Val His Ser35 40 455933PRTArabidopsis thaliana 59Asp Ala Gly Ser
Gln Glu Gly Cys Ser Ser Glu Val Ile Asn Phe Pro1 5 10 15His Ser His
Lys Thr Arg Asp Tyr Val Ile Ile Lys Thr Lys Leu Ser 20 25
30Ala6025PRTArabidopsis thaliana 60Val Lys Glu Arg Ala Arg Asn His
Arg Asn Gly Phe His Leu Ile Ser1 5 10 15Tyr Arg Trp Leu Cys Ser Gly
Val Val 20
256110PRTArabidopsis thaliana 61Ile Tyr Glu Tyr Gly Gly Glu Lys Arg
Thr1 5 10625PRTArabidopsis thaliana 62Leu Glu Gly His Thr1
56323PRTArabidopsis thaliana 63Pro Asp Phe Thr Asp Pro Thr Arg Arg
Ser Arg Gly Thr Lys Leu Leu1 5 10 15Lys Ser Cys Gln Arg Val Ala
20647PRTArabidopsis thaliana 64Cys Gly Ala Ser Asn Gly Asp1
5658PRTArabidopsis thaliana 65Asn Ser Pro Thr Phe Ser Glu Val1
56635PRTArabidopsis thaliana 66Ala Ser Gly Thr Glu Asp Ala Asn Glu
Glu Glu Ile Pro Gln Leu Phe1 5 10 15Arg Leu Cys Arg Leu Gln Thr Val
Gln Glu Gly Phe Cys His Ile Glu 20 25 30Glu His
Trp35675PRTArabidopsis thaliana 67Ala Glu Arg Thr Leu1
56813PRTArabidopsis thaliana 68Ser Tyr Gln Asn Arg Trp Arg Val Asn
Ser Ser Asn Leu1 5 106922PRTArabidopsis thaliana 69Arg Asp Thr His
Gln Val Arg Cys Arg Lys Tyr His Asp Asp Gly Thr1 5 10 15Gly Phe Gly
Ile Ser Gln 207024PRTArabidopsis thaliana 70Arg Arg Arg Lys Arg Asn
His Trp Cys Leu Trp Thr Trp Trp Gly Trp1 5 10 15Glu Asp Asn Val Asn
Ala Glu His 207110PRTArabidopsis thaliana 71Gln Arg Ala Asp His Lys
Arg Thr Ser Val1 5 107255PRTArabidopsis thaliana 72Cys Thr Asp Leu
Gly Ser Asn Val Gln Arg Ile Arg Arg Val Tyr Asn1 5 10 15Ser Ala Ser
Arg Trp Ser Thr Val Gly Phe Ile Leu Gly Arg Glu Gly 20 25 30Asp Arg
Arg Lys Gln Ser Phe Glu Asp Ile Gln Ser Phe Glu Thr Glu35 40 45Thr
Phe Leu Val Val Ala Arg50 557315PRTArabidopsis thaliana 73Cys Leu
Gly Arg Asp Arg Leu Gly Glu Asn Trp Ser Ser Ser Thr1 5 10
15749PRTArabidopsis thaliana 74Arg Asp Arg Arg Arg Val Asp Pro Cys1
57541PRTArabidopsis thaliana 75Gln Gly Lys Gln Met Gln Gly Asp Val
His Asp Thr Val Tyr Ser Ile1 5 10 15Met Gln Gln Tyr Gly Cys Gly Ile
Gln Val Glu Ser Gly Val Ser Gly 20 25 30Glu Glu Thr Arg Val Gly Ala
Val Leu35 407621PRTArabidopsis thaliana 76Gly Met Glu Lys Arg Ser
Phe Arg Val Ile Ile Asn Ser Pro Ala Arg1 5 10 15Gly Asp Tyr Ser Glu
207717PRTArabidopsis thaliana 77Met Trp Arg Ile Ala Thr Ser Val Asp
His Phe Arg Arg Ser His Gly1 5 10 15Ser7824PRTArabidopsis thaliana
78Ile Ser Ser Arg Asp Glu Gly Tyr Glu Leu Cys Ile Cys Pro Phe Glu1
5 10 15Ile Gln Leu Arg Gln Pro Arg Glu 207924PRTArabidopsis
thaliana 79Ser Ala Ser Val Leu Phe Leu Val Leu Arg Phe Ile Pro Arg
Arg Thr1 5 10 15Phe Tyr Arg Asp Arg Ala Ala Cys
208014PRTArabidopsis thaliana 80Val Leu Gly Arg Arg Arg Val Ser His
Gln Leu Pro Trp Arg1 5 108122PRTArabidopsis thaliana 81His His Leu
Gln Gly Ile Phe Ser His Trp Gly Ser Glu Ser Gly Met1 5 10 15Phe Val
Gly Asn Arg Arg 20827PRTArabidopsis thaliana 82Glu Asn Thr Gly Glu
Asp Ala1 58343PRTArabidopsis thaliana 83Lys Thr His Met Pro Glu Thr
Asp Asn Thr Asp Ala Pro Thr Glu Gly1 5 10 15Leu Phe Glu Glu Asp Ser
Asn Arg Val Phe His Ala Tyr Ala Cys Ser 20 25 30Gln Ser Leu Gly Leu
Val Val His Lys Tyr His35 408411PRTArabidopsis thaliana 84Cys Gly
Gln Lys Leu Cys Ile Val Asp Gly Ile1 5 10855PRTArabidopsis thaliana
85Gly Ala Asp Pro Ser1 58614PRTArabidopsis thaliana 86Ser Arg Lys
Leu Ala Thr Ser Val Gly Asp Leu Ile Val Arg1 5 10876PRTArabidopsis
thaliana 87Gln Asn Pro Asp Leu Ala1 58831PRTArabidopsis thaliana
88Asp Ser Val Val Tyr Gln Val Phe Gly Gly Val Val Ser Ser Val Tyr1
5 10 15Val Arg Asn Lys Asp Lys Cys Ile Ala Thr Gly Ala Trp Glu Ser
20 25 308947PRTArabidopsis thaliana 89Lys Thr Glu Ala Ser Gly Pro
Thr Lys Asn Ser Val Ser Ser Asp Asp1 5 10 15Pro Thr Arg Cys His Met
Leu Ala Glu Gln Ala Arg Gly Ser Glu Leu 20 25 30Val Leu Gln Leu Arg
Arg Leu Gly Thr Ala Glu Leu Trp Arg Arg35 40 45907PRTArabidopsis
thaliana 90Ser Arg Arg Thr Arg Ile Arg1 59130PRTArabidopsis
thaliana 91Leu Gly Ile Leu Gly Lys Pro Asn His Thr Arg Tyr His Cys
Ser Leu1 5 10 15Ile Gly Asp Pro Lys Asn Ser Leu Arg Val Arg Cys Phe
Ala 20 25 30927PRTArabidopsis thaliana 92Thr Tyr Thr Ala Ser Pro
Arg1 59310PRTArabidopsis thaliana 93Thr Pro Leu Leu Gln Ser Pro Ile
Thr His1 5 10948PRTArabidopsis thaliana 94Pro Trp Gln Glu Pro Glu
Lys Thr1 59510PRTArabidopsis thaliana 95Leu Gly Val Pro Gly His Thr
Arg Arg Phe1 5 109658PRTArabidopsis thaliana 96Leu Ala Ser Glu Ser
Arg Gly Ser Asp Val Thr Gln Pro Ser Gln Leu1 5 10 15Asn Gln Ser Val
Gly Lys Phe Cys Lys Pro Arg Leu Ser Ala Glu Tyr 20 25 30Pro Leu His
Lys His Phe Thr Leu Gln Gln Ala Glu Glu Cys Leu Met35 40 45Gly Ser
Glu Thr Pro Lys Ala Arg Gly Asp50 559733PRTArabidopsis thaliana
97Thr Val Arg Leu Gln Arg Asp Arg Gly Ile Asp Lys Arg Thr Arg Glu1
5 10 15Ser Ile Arg Arg Arg Ser Asn Ile Val Pro Lys Pro Glu Asp Leu
Glu 20 25 30Asn9818PRTArabidopsis thaliana 98Gly Ser Ala Arg Thr
Lys Gln His Pro Pro Ile Ser Ile Phe Ile Pro1 5 10 15Lys
Ser9910PRTArabidopsis thaliana 99Asn Ile Ser His His Lys Leu Pro
Gln Ser1 5 1010018PRTArabidopsis thaliana 100Glu Thr Ala Val Ser
Gly Glu Glu Asp Pro Asp Glu Leu Ala Asn Ser1 5 10 15Leu
Leu1014PRTArabidopsis thaliana 101Thr Ser His
His110214PRTArabidopsis thaliana 102Glu Leu Arg Ala Leu Cys Thr Asn
Met Ser Ile His Lys Met1 5 1010323PRTArabidopsis thaliana 103Gln
Glu Ala Arg Lys Val Val Pro Val Lys Ser Ser Thr Phe His Ile1 5 10
15Ala Thr Lys Leu Glu Ile Met 201046PRTArabidopsis thaliana 104Lys
Pro Asn Tyr Pro Arg1 51051491DNAArabidopsis thaliana 105atcgattgat
ctctggctca gtgcgagtag tccatttgag agcagtcgta gccccgcgtg 60gcgcatcatg
gagctatttg gaattttcgc agggttatcg attcgtagtg ggaacccatt
120cattgtttgg aaccaccaac ggacgactta acaagctccc cgaggtgcat
gatgaaaatt 180gctccagttg ccataaatca cagcccgctc agcagggagg
tcccgtcaca cgcggcaccc 240actcaggcaa agcaaaccaa ccttcaatct
gaagctggcg atttagatgc aagaaaaagt 300agcgcttcaa gcccggaaac
ccgcgcatta ctcgctacta agacagtact cgggagacac 360aagatagagg
ttccggcctt tggagggtgg ttcaaaaaga aatcatctaa gcacgagacg
420ggcggttcaa gtgccaacgc agatagttcg agcgtggctt ccgattccac
cgaaaaacct 480ttgttccgtc tcacgcacgt tccttacgta tcccaaggta
atgagcgaat gggatgttgg 540tatgcctgcg caagaatggt tggccattct
gtcgaagctg ggcctcgcct agggctgccg 600gagctctatg agggaaggga
ggcgccagct gggctacaag atttttcaga tgtagaaagg 660tttattcaca
atgaaggatt aactcgggta gaccttccag acaatgagag atttacacac
720gaagagttgg gtgcactgtt gtataagcac gggccgatta tatttgggtg
gaaaactccg 780aatgacagct ggcacatgtc ggtcctcact ggtgtcgata
aagagacgtc gtccattact 840tttcacgatc cccgacaggg gccggaccta
gcaatgccgc tcgattactt taatcagcga 900ttggcatggc aggttccaca
cgcaatgctc taccgctaag tagcagggta tcttcacgtg 960gcggcatcat
gacaagccca tgatgccgcc agcagctacc tgaatgccgt ctggcttttt
1020ggtccctatt gtcgtatccg gaagatgacg tcaaagaatc tcggcaagag
ctttcttgct 1080cgactcctca gcttccggat cgatcaggtc gcttgccaga
gcgcgcttgt ccatgagcat 1140ctgccacagc tgctggtcga tggtgtcctc
agctaaaggg attttgacga caaccatgcg 1200caactgcccg ttgcgatacg
ctcgatcctg aagccccggt gtccatggca gccccaagaa 1260aaagacatag
ttcgccgctg tgaggttgta gcctgtgccg gcggccgacc tggtcccgat
1320aaacaccctg cagtccggat cctgctggaa agcatcaatc gccttctgcc
gcttcttggg 1380cgagtcactg cccaccaacg tcacgcaccc gacgccaagc
ttgaggcagt gctcccgcaa 1440cgtggccacg gattcctgat actcgcagaa
gaggatcacc ttgtcgtcga c 1491106255PRTArabidopsis thaliana 106Met
Lys Ile Ala Pro Val Ala Ile Asn His Ser Pro Leu Ser Arg Glu1 5 10
15Val Pro Ser His Ala Ala Pro Thr Gln Ala Lys Gln Thr Asn Leu Gln
20 25 30Ser Glu Ala Gly Asp Leu Asp Ala Arg Lys Ser Ser Ala Ser Ser
Pro35 40 45Glu Thr Arg Ala Leu Leu Ala Thr Lys Thr Val Leu Gly Arg
His Lys50 55 60Ile Glu Val Pro Ala Phe Gly Gly Trp Phe Lys Lys Lys
Ser Ser Lys65 70 75 80His Glu Thr Gly Gly Ser Ser Ala Asn Ala Asp
Ser Ser Ser Val Ala 85 90 95Ser Asp Ser Thr Glu Lys Pro Leu Phe Arg
Leu Thr His Val Pro Tyr 100 105 110Val Ser Gln Gly Asn Glu Arg Met
Gly Cys Trp Tyr Ala Cys Ala Arg115 120 125Met Val Gly His Ser Val
Glu Ala Gly Pro Arg Leu Gly Leu Pro Glu130 135 140Leu Tyr Glu Gly
Arg Glu Ala Pro Ala Gly Leu Gln Asp Phe Ser Asp145 150 155 160Val
Glu Arg Phe Ile His Asn Glu Gly Leu Thr Arg Val Asp Leu Pro 165 170
175Asp Asn Glu Arg Phe Thr His Glu Glu Leu Gly Ala Leu Leu Tyr Lys
180 185 190His Gly Pro Ile Ile Phe Gly Trp Lys Thr Pro Asn Asp Ser
Trp His195 200 205Met Ser Val Leu Thr Gly Val Asp Lys Glu Thr Ser
Ser Ile Thr Phe210 215 220His Asp Pro Arg Gln Gly Pro Asp Leu Ala
Met Pro Leu Asp Tyr Phe225 230 235 240Asn Gln Arg Leu Ala Trp Gln
Val Pro His Ala Met Leu Tyr Arg 245 250 2551071258PRTArabidopsis
thaliana 107Met Ser Tyr Leu Arg Glu Val Ala Thr Ala Val Ala Leu Leu
Leu Pro1 5 10 15Phe Ile Leu Leu Asn Lys Phe Asn Arg Pro Asn Ser Lys
Asp Ser Ile 20 25 30Val Asn Asp Asp Asp Asp Ser Thr Ser Glu Val Asp
Ala Ile Ser Asp35 40 45Ser Thr Asn Pro Ser Gly Ser Phe Pro Ser Val
Glu Tyr Glu Val Phe50 55 60Leu Ser Phe Arg Gly Pro Asp Thr Arg Glu
Gln Phe Thr Asp Phe Leu65 70 75 80Tyr Gln Ser Leu Arg Arg Tyr Lys
Ile His Thr Phe Arg Asp Asp Asp 85 90 95Glu Leu Leu Lys Gly Lys Glu
Ile Gly Pro Asn Leu Leu Arg Ala Ile 100 105 110Asp Gln Ser Lys Ile
Tyr Val Pro Ile Ile Ser Ser Gly Tyr Ala Asp115 120 125Ser Lys Trp
Cys Leu Met Glu Leu Ala Glu Ile Val Arg Arg Gln Glu130 135 140Glu
Asp Pro Arg Arg Ile Ile Leu Pro Ile Phe Tyr Met Val Asp Pro145 150
155 160Ser Asp Val Arg His Gln Thr Gly Cys Tyr Lys Lys Ala Phe Arg
Lys 165 170 175His Ala Asn Lys Phe Asp Gly Gln Thr Ile Gln Asn Trp
Lys Asp Ala 180 185 190Leu Lys Lys Val Gly Asp Leu Lys Gly Trp His
Ile Gly Lys Asn Asp195 200 205Lys Gln Gly Ala Ile Ala Asp Lys Val
Ser Ala Asp Ile Trp Ser His210 215 220Ile Ser Lys Glu Asn Leu Ile
Leu Glu Thr Asp Glu Leu Val Gly Ile225 230 235 240Asp Asp His Ile
Thr Ala Val Leu Glu Lys Leu Ser Leu Asp Ser Glu 245 250 255Asn Val
Thr Met Val Gly Leu Tyr Gly Met Gly Gly Ile Gly Lys Thr 260 265
270Thr Thr Ala Lys Ala Val Tyr Asn Lys Ile Ser Ser Cys Phe Asp
Cys275 280 285Cys Cys Phe Ile Asp Asn Ile Arg Glu Thr Gln Glu Lys
Asp Gly Val290 295 300Val Val Leu Gln Lys Lys Leu Val Ser Glu Ile
Leu Arg Ile Asp Ser305 310 315 320Gly Ser Val Gly Phe Asn Asn Asp
Ser Gly Gly Arg Lys Thr Ile Lys 325 330 335Glu Arg Val Ser Arg Phe
Lys Ile Leu Val Val Leu Asp Asp Val Asp 340 345 350Glu Lys Phe Lys
Phe Glu Asp Met Leu Gly Ser Pro Lys Asp Phe Ile355 360 365Ser Gln
Ser Arg Phe Ile Ile Thr Ser Arg Ser Met Arg Val Leu Gly370 375
380Thr Leu Asn Glu Asn Gln Cys Lys Leu Tyr Glu Val Gly Ser Met
Ser385 390 395 400Lys Pro Arg Ser Leu Glu Leu Phe Ser Lys His Ala
Phe Lys Lys Asn 405 410 415Thr Pro Pro Ser Ser Tyr Tyr Glu Thr Leu
Ala Asn Asp Val Val Asp 420 425 430Thr Thr Ala Gly Leu Pro Leu Thr
Leu Lys Val Ile Gly Ser Leu Leu435 440 445Phe Lys Gln Glu Ile Ala
Val Trp Glu Asp Thr Leu Glu Gln Leu Arg450 455 460Arg Thr Leu Asn
Leu Asp Glu Val Tyr Asp Arg Leu Lys Ile Ser Tyr465 470 475 480Asp
Ala Leu Asn Pro Glu Ala Lys Glu Ile Phe Leu Asp Ile Ala Cys 485 490
495Phe Phe Ile Gly Gln Asn Lys Glu Glu Pro Tyr Tyr Met Trp Thr Asp
500 505 510Cys Asn Phe Tyr Pro Ala Ser Asn Ile Ile Phe Leu Ile Gln
Arg Cys515 520 525Met Ile Gln Val Gly Asp Asp Asp Glu Phe Lys Met
His Asp Gln Leu530 535 540Arg Asp Met Gly Arg Glu Ile Val Arg Arg
Glu Asp Val Leu Pro Trp545 550 555 560Lys Ser Arg Ile Trp Ser Ala
Glu Glu Gly Ile Asp Leu Leu Leu Asn 565 570 575Lys Arg Lys Gly Ser
Ser Lys Val Lys Ala Ile Ser Ile Pro Trp Gly 580 585 590Val Lys Tyr
Glu Phe Lys Ser Glu Cys Phe Leu Asn Leu Ser Glu Leu595 600 605Arg
Tyr Leu His Ala Arg Glu Ala Met Leu Thr Gly Asp Phe Asn Asn610 615
620Leu Leu Pro Asn Leu Lys Trp Leu Glu Leu Pro Phe Tyr Lys His
Gly625 630 635 640Glu Asp Asp Pro Pro Leu Thr Asn Tyr Thr Met Lys
Asn Leu Ile Ile 645 650 655Val Ile Leu Glu His Ser His Ile Thr Ala
Asp Asp Trp Gly Gly Trp 660 665 670Arg His Met Met Lys Met Ala Glu
Arg Leu Lys Val Val Arg Leu Ala675 680 685Ser Asn Tyr Ser Leu Tyr
Gly Arg Arg Val Arg Leu Ser Asp Cys Trp690 695 700Arg Phe Pro Lys
Ser Ile Glu Val Leu Ser Met Thr Ala Ile Glu Met705 710 715 720Asp
Glu Val Asp Ile Gly Glu Leu Lys Lys Leu Lys Thr Leu Val Leu 725 730
735Lys Pro Cys Pro Ile Gln Lys Ile Ser Gly Gly Thr Phe Gly Met Leu
740 745 750Lys Gly Leu Arg Glu Leu Cys Leu Glu Phe Asn Trp Gly Thr
Asn Leu755 760 765Arg Glu Val Val Ala Asp Ile Gly Gln Leu Ser Ser
Leu Lys Val Leu770 775 780Lys Thr Gly Ala Lys Glu Val Glu Ile Asn
Glu Phe Pro Leu Gly Leu785 790 795 800Lys Thr Glu Leu Ser Thr Ser
Ser Arg Ile Pro Asn Asn Leu Ser Gln 805 810 815Leu Leu Asp Leu Glu
Val Leu Lys Val Tyr Asp Cys Lys Asp Gly Phe 820 825 830Asp Met Pro
Pro Ala Ser Pro Ser Glu Asp Glu Ser Ser Val Trp Trp835 840 845Lys
Val Ser Lys Leu Lys Ser Leu Gln Leu Glu Lys Thr Arg Ile Asn850 855
860Val Asn Val Val Asp Asp Ala Ser Ser Gly Gly His Leu Pro Arg
Tyr865 870 875 880Leu Leu Pro Thr Ser Leu Thr Tyr Leu Lys Ile Tyr
Gln Cys Thr Glu 885 890 895Pro Thr Trp Leu Pro Gly Ile Glu Asn Leu
Glu Asn Leu Thr Ser Leu 900 905 910Glu Val Asn Asp Ile Phe Gln Thr
Leu Gly Gly Asp Leu Asp Gly Leu915 920 925Gln Gly Leu Arg Ser Leu
Glu Ile Leu Arg Ile Arg Lys Val Asn Gly930 935 940Leu Ala Arg Ile
Lys Gly Leu Lys Asp Leu Leu Cys Ser Ser Thr Cys945 950 955 960Lys
Leu Arg Lys Phe Tyr Ile Thr Glu Cys Pro Asp Leu Ile Glu Leu 965 970
975Leu Pro Cys Glu Leu Gly Val Gln Thr Val Val Val Pro Ser Met Ala
980 985 990Glu Leu Thr Ile Arg Asp Cys Pro Arg Leu Glu Val Gly Pro
Met Ile995 1000 1005Arg Ser Leu Pro Lys Phe Pro Met Leu Lys Lys Leu
Asp Leu Ala1010 1015 1020Val Ala Asn Ile Thr Lys Glu Glu Asp Leu
Asp Ala Ile Gly Ser1025 1030
1035Leu Glu Glu Leu Val Ser Leu Glu Leu Glu Leu Asp Asp Thr Ser1040
1045 1050Ser Gly Ile Glu Arg Ile Val Ser Ser Ser Lys Leu Gln Lys
Leu1055 1060 1065Thr Thr Leu Val Val Lys Val Pro Ser Leu Arg Glu
Ile Glu Gly1070 1075 1080Leu Glu Glu Leu Lys Ser Leu Gln Asp Leu
Tyr Leu Glu Gly Cys1085 1090 1095Thr Ser Leu Gly Arg Leu Pro Leu
Glu Lys Leu Lys Glu Leu Asp1100 1105 1110Ile Gly Gly Cys Pro Asp
Leu Thr Glu Leu Val Gln Thr Val Val1115 1120 1125Ala Val Pro Ser
Leu Arg Gly Leu Thr Ile Arg Asp Cys Pro Arg1130 1135 1140Leu Glu
Val Gly Pro Met Ile Gln Ser Leu Pro Lys Phe Pro Met1145 1150
1155Leu Asn Glu Leu Thr Leu Ser Met Val Asn Ile Thr Lys Glu Asp1160
1165 1170Glu Leu Glu Val Leu Gly Ser Leu Glu Glu Leu Asp Ser Leu
Glu1175 1180 1185Leu Thr Leu Asp Asp Thr Cys Ser Ser Ile Glu Arg
Ile Ser Phe1190 1195 1200Leu Ser Lys Leu Gln Lys Leu Thr Thr Leu
Ile Val Glu Val Pro1205 1210 1215Ser Leu Arg Glu Ile Glu Gly Leu
Ala Glu Leu Lys Ser Leu Arg1220 1225 1230Ile Leu Tyr Leu Glu Gly
Cys Thr Ser Leu Glu Arg Leu Trp Pro1235 1240 1245Asp Gln Gln Gln
Leu Gly Ser Leu Lys Asn1250 12551081143PRTArabidopsis thaliana
108Met Ala Ser Ser Ser Ser Ser Ser Arg Trp Ser Tyr Asp Val Phe Leu1
5 10 15Ser Phe Arg Gly Glu Asp Thr Arg Lys Thr Phe Thr Ser His Leu
Tyr 20 25 30Glu Val Leu Asn Asp Lys Gly Ile Lys Thr Phe Gln Asp Asp
Lys Arg35 40 45Leu Glu Tyr Gly Ala Thr Ile Pro Gly Glu Leu Cys Lys
Ala Ile Glu50 55 60Glu Ser Gln Phe Ala Ile Val Val Phe Ser Glu Asn
Tyr Ala Thr Ser65 70 75 80Arg Trp Cys Leu Asn Glu Leu Val Lys Ile
Met Glu Cys Lys Thr Arg 85 90 95Phe Lys Gln Thr Val Ile Pro Ile Phe
Tyr Asp Val Asp Pro Ser His 100 105 110Val Arg Asn Gln Lys Glu Ser
Phe Ala Lys Ala Phe Glu Glu His Glu115 120 125Thr Lys Tyr Lys Asp
Asp Val Glu Gly Ile Gln Arg Trp Arg Ile Ala130 135 140Leu Asn Glu
Ala Ala Asn Leu Lys Gly Ser Cys Asp Asn Arg Asp Lys145 150 155
160Thr Asp Ala Asp Cys Ile Arg Gln Ile Val Asp Gln Ile Ser Ser Lys
165 170 175Leu Cys Lys Ile Ser Leu Ser Tyr Leu Gln Asn Ile Val Gly
Ile Asp 180 185 190Thr His Leu Glu Lys Ile Glu Ser Leu Leu Glu Ile
Gly Ile Asn Gly195 200 205Val Arg Ile Met Gly Ile Trp Gly Met Gly
Gly Val Gly Lys Thr Thr210 215 220Ile Ala Arg Ala Ile Phe Asp Thr
Leu Leu Gly Arg Met Asp Ser Ser225 230 235 240Tyr Gln Phe Asp Gly
Ala Cys Phe Leu Lys Asp Ile Lys Glu Asn Lys 245 250 255Arg Gly Met
His Ser Leu Gln Asn Ala Leu Leu Ser Glu Leu Leu Arg 260 265 270Glu
Lys Ala Asn Tyr Asn Asn Glu Glu Asp Gly Lys His Gln Met Ala275 280
285Ser Arg Leu Arg Ser Lys Lys Val Leu Ile Val Leu Asp Asp Ile
Asp290 295 300Asn Lys Asp His Tyr Leu Glu Tyr Leu Ala Gly Asp Leu
Asp Trp Phe305 310 315 320Gly Asn Gly Ser Arg Ile Ile Ile Thr Thr
Arg Asp Lys His Leu Ile 325 330 335Glu Lys Asn Asp Ile Ile Tyr Glu
Val Thr Ala Leu Pro Asp His Glu 340 345 350Ser Ile Gln Leu Phe Lys
Gln His Ala Phe Gly Lys Glu Val Pro Asn355 360 365Glu Asn Phe Glu
Lys Leu Ser Leu Glu Val Val Asn Tyr Ala Lys Gly370 375 380Leu Pro
Leu Ala Leu Lys Val Trp Gly Ser Leu Leu His Asn Leu Arg385 390 395
400Leu Thr Glu Trp Lys Ser Ala Ile Glu His Met Lys Asn Asn Ser Tyr
405 410 415Ser Gly Ile Ile Asp Lys Leu Lys Ile Ser Tyr Asp Gly Leu
Glu Pro 420 425 430Lys Gln Gln Glu Met Phe Leu Asp Ile Ala Cys Phe
Leu Arg Gly Glu435 440 445Glu Lys Asp Tyr Ile Leu Gln Ile Leu Glu
Ser Cys His Ile Gly Ala450 455 460Glu Tyr Gly Leu Arg Ile Leu Ile
Asp Lys Ser Leu Val Phe Ile Ser465 470 475 480Glu Tyr Asn Gln Val
Gln Met His Asp Leu Ile Gln Asp Met Gly Lys 485 490 495Tyr Ile Val
Asn Phe Gln Lys Asp Pro Gly Glu Arg Ser Arg Leu Trp 500 505 510Leu
Ala Lys Glu Val Glu Glu Val Met Ser Asn Asn Thr Gly Thr Met515 520
525Ala Met Glu Ala Ile Trp Val Ser Ser Tyr Ser Ser Thr Leu Arg
Phe530 535 540Ser Asn Gln Ala Val Lys Asn Met Lys Arg Leu Arg Val
Phe Asn Met545 550 555 560Gly Arg Ser Ser Thr His Tyr Ala Ile Asp
Tyr Leu Pro Asn Asn Leu 565 570 575Arg Cys Phe Val Cys Thr Asn Tyr
Pro Trp Glu Ser Phe Pro Ser Thr 580 585 590Phe Glu Leu Lys Met Leu
Val His Leu Gln Leu Arg His Asn Ser Leu595 600 605Arg His Leu Trp
Thr Glu Thr Lys His Leu Pro Ser Leu Arg Arg Ile610 615 620Asp Leu
Ser Trp Ser Lys Arg Leu Thr Arg Thr Pro Asp Phe Thr Gly625 630 635
640Met Pro Asn Leu Glu Tyr Val Asn Leu Tyr Gln Cys Ser Asn Leu Glu
645 650 655Glu Val His His Ser Leu Gly Cys Cys Ser Lys Val Ile Gly
Leu Tyr 660 665 670Leu Asn Asp Cys Lys Ser Leu Lys Arg Phe Pro Cys
Val Asn Val Glu675 680 685Ser Leu Glu Tyr Leu Gly Leu Arg Ser Cys
Asp Ser Leu Glu Lys Leu690 695 700Pro Glu Ile Tyr Gly Arg Met Lys
Pro Glu Ile Gln Ile His Met Gln705 710 715 720Gly Ser Gly Ile Arg
Glu Leu Pro Ser Ser Ile Phe Gln Tyr Lys Thr 725 730 735His Val Thr
Lys Leu Leu Leu Trp Asn Met Lys Asn Leu Val Ala Leu 740 745 750Pro
Ser Ser Ile Cys Arg Leu Lys Ser Leu Val Ser Leu Ser Val Ser755 760
765Gly Cys Ser Lys Leu Glu Ser Leu Pro Glu Glu Ile Gly Asp Leu
Asp770 775 780Asn Leu Arg Val Phe Asp Ala Ser Asp Thr Leu Ile Leu
Arg Pro Pro785 790 795 800Ser Ser Ile Ile Arg Leu Asn Lys Leu Ile
Ile Leu Met Phe Arg Gly 805 810 815Phe Lys Asp Gly Val His Phe Glu
Phe Pro Pro Val Ala Glu Gly Leu 820 825 830His Ser Leu Glu Tyr Leu
Asn Leu Ser Tyr Cys Asn Leu Ile Asp Gly835 840 845Gly Leu Pro Glu
Glu Ile Gly Ser Leu Ser Ser Leu Lys Lys Leu Asp850 855 860Leu Ser
Arg Asn Asn Phe Glu His Leu Pro Ser Ser Ile Ala Gln Leu865 870 875
880Gly Ala Leu Gln Ser Leu Asp Leu Lys Asp Cys Gln Arg Leu Thr Gln
885 890 895Leu Pro Glu Leu Pro Pro Glu Leu Asn Glu Leu His Val Asp
Cys His 900 905 910Met Ala Leu Lys Phe Ile His Tyr Leu Val Thr Lys
Arg Lys Lys Leu915 920 925His Arg Val Lys Leu Asp Asp Ala His Asn
Asp Thr Met Tyr Asn Leu930 935 940Phe Ala Tyr Thr Met Phe Gln Asn
Ile Ser Ser Met Arg His Asp Ile945 950 955 960Ser Ala Ser Asp Ser
Leu Ser Leu Thr Val Phe Thr Gly Gln Pro Tyr 965 970 975Pro Glu Lys
Ile Pro Ser Trp Phe His His Gln Gly Trp Asp Ser Ser 980 985 990Val
Ser Val Asn Leu Pro Glu Asn Trp Tyr Ile Pro Asp Lys Phe Leu995 1000
1005Gly Phe Ala Val Cys Tyr Ser Arg Ser Leu Ile Asp Thr Thr Ala1010
1015 1020His Leu Ile Pro Val Cys Asp Asp Lys Met Ser Arg Met Thr
Gln1025 1030 1035Lys Leu Ala Leu Ser Glu Cys Asp Thr Glu Ser Ser
Asn Tyr Ser1040 1045 1050Glu Trp Asp Ile His Phe Phe Phe Val Pro
Phe Ala Gly Leu Trp1055 1060 1065Asp Thr Ser Lys Ala Asn Gly Lys
Thr Pro Asn Asp Tyr Gly Ile1070 1075 1080Ile Arg Leu Ser Phe Ser
Gly Glu Glu Lys Met Tyr Gly Arg Leu1085 1090 1095Arg Leu Tyr Lys
Glu Gly Pro Glu Val Asn Ala Leu Leu Gln Met1100 1105 1110Arg Glu
Asn Ser Asn Glu Pro Thr Glu His Ser Thr Gly Ile Arg1115 1120
1125Arg Thr Gln Tyr Asn Asn Arg Thr Ser Phe Tyr Glu Leu Ile Asn1130
1135 1140109429PRTArabidopsis thaliana 109Leu Arg Ser Lys Leu Asp
Leu Ile Ile Asp Leu Lys His Gln Ile Glu1 5 10 15Ser Val Lys Glu Gly
Leu Leu Cys Leu Arg Ser Phe Ile Asp His Phe 20 25 30Ser Glu Ser Tyr
Val Glu His Asp Glu Ala Cys Gly Leu Ile Ala Arg35 40 45Val Ser Val
Met Ala Tyr Lys Ala Glu Tyr Val Ile Asp Ser Cys Leu50 55 60Ala Tyr
Ser His Pro Leu Trp Tyr Lys Val Leu Trp Ile Ser Glu Val65 70 75
80Leu Glu Asn Ile Lys Leu Val Asn Lys Val Val Gly Glu Thr Cys Glu
85 90 95Arg Arg Asn Thr Glu Val Thr Val His Glu Val Ala Lys Thr Thr
Thr 100 105 110Asn Val Ala Pro Ser Phe Ser Ala Tyr Thr Gln Arg Ala
Asn Glu Glu115 120 125Met Glu Gly Phe Gln Asp Thr Ile Asp Glu Leu
Lys Asp Lys Leu Leu130 135 140Gly Gly Ser Pro Glu Leu Asp Val Ile
Ser Ile Val Gly Met Pro Gly145 150 155 160Leu Gly Lys Thr Thr Leu
Ala Lys Lys Ile Tyr Asn Asp Pro Glu Val 165 170 175Thr Ser Arg Phe
Asp Val His Ala Gln Cys Val Val Thr Gln Leu Tyr 180 185 190Ser Trp
Arg Glu Leu Leu Leu Thr Ile Leu Asn Asp Val Leu Glu Pro195 200
205Ser Asp Arg Asn Glu Lys Glu Asp Gly Glu Ile Ala Asp Glu Leu
Arg210 215 220Arg Phe Leu Leu Thr Lys Arg Phe Leu Ile Leu Ile Asp
Asp Val Trp225 230 235 240Asp Tyr Lys Val Trp Asp Asn Leu Cys Met
Cys Phe Ser Asp Val Ser 245 250 255Asn Arg Ser Arg Ile Ile Leu Thr
Thr Arg Leu Asn Asp Val Ala Glu 260 265 270Tyr Val Lys Cys Glu Ser
Asp Pro His His Leu Arg Leu Phe Arg Asp275 280 285Asp Glu Ser Trp
Thr Leu Leu Gln Lys Glu Val Phe Gln Gly Glu Ser290 295 300Cys Pro
Pro Glu Leu Glu Asp Val Gly Phe Glu Ile Ser Lys Ser Cys305 310 315
320Arg Gly Leu Pro Leu Ser Val Val Leu Val Ala Gly Val Leu Lys Gln
325 330 335Lys Lys Lys Thr Leu Asp Ser Trp Lys Val Val Glu Gln Ser
Leu Ser 340 345 350Ser Gln Arg Ile Gly Ser Leu Glu Glu Ser Ile Ser
Ile Ile Gly Phe355 360 365Ser Tyr Lys Asn Leu Pro His Tyr Leu Lys
Pro Cys Phe Leu Tyr Phe370 375 380Gly Gly Phe Leu Gln Gly Lys Asp
Ile His Asp Ser Lys Met Thr Lys385 390 395 400Leu Trp Val Ala Glu
Glu Phe Val Gln Ala Asn Asn Glu Lys Gly Gln 405 410 415Glu Asp Thr
Arg Thr Arg Phe Leu Gly Arg Ser Tyr Trp 420 42511011PRTArabidopsis
thaliana 110Gly Met Gly Gly Ile Gly Lys Thr Thr Thr Ala1 5
1011111PRTArabidopsis thaliana 111Gly Met Gly Gly Val Gly Lys Thr
Thr Ile Ala1 5 1011211PRTArabidopsis thaliana 112Gly Met Pro Gly
Leu Gly Lys Thr Thr Leu Ala1 5 1011311PRTArabidopsis thaliana
113Gly Pro Gly Gly Val Gly Lys Thr Thr Leu Met1 5
1011411PRTArabidopsis thaliana 114Phe Lys Ile Leu Val Val Leu Asp
Asp Val Asp1 5 1011511PRTArabidopsis thaliana 115Lys Lys Val Leu
Ile Val Leu Asp Asp Ile Asp1 5 1011611PRTArabidopsis thaliana
116Lys Arg Phe Leu Ile Leu Ile Asp Asp Val Trp1 5
1011711PRTArabidopsis thaliana 117Lys Arg Phe Leu Leu Leu Leu Asp
Asp Val Trp1 5 101188PRTArabidopsis thaliana 118Ser Arg Phe Ile Ile
Thr Ser Arg1 51198PRTArabidopsis thaliana 119Ser Arg Ile Ile Ile
Thr Thr Arg1 51208PRTArabidopsis thaliana 120Ser Arg Ile Ile Leu
Thr Thr Arg1 51218PRTArabidopsis thaliana 121Cys Lys Val Met Phe
Thr Thr Arg1 51228PRTArabidopsis thaliana 122Gly Leu Pro Leu Thr
Leu Lys Val1 51238PRTArabidopsis thaliana 123Gly Leu Pro Leu Ala
Leu Lys Val1 51248PRTArabidopsis thaliana 124Gly Leu Pro Leu Ser
Val Val Leu1 51258PRTArabidopsis thaliana 125Gly Leu Pro Leu Ala
Leu Ile Thr1 51267PRTArabidopsis thaliana 126Lys Ile Ser Tyr Asp
Ala Leu1 51277PRTArabidopsis thaliana 127Lys Ile Ser Tyr Asp Gly
Leu1 51287PRTArabidopsis thaliana 128Gly Phe Ser Tyr Lys Asn Leu1
51297PRTArabidopsis thaliana 129Val Phe Leu Ser Phe Arg Gly1
51309PRTArabidopsis thaliana 130Pro Ile Phe Tyr Met Val Asp Pro
Ser1 51319PRTArabidopsis thaliana 131Pro Ile Phe Tyr Asp Val Asp
Pro Ser1 51326PRTArabidopsis thaliana 132Val Gly Ile Asp Asp His1
51336PRTArabidopsis thaliana 133Val Gly Ile Asp Thr His1
51347PRTArabidopsis thaliana 134Phe Leu Asp Ile Ala Cys Phe1
51359PRTArabidopsis thaliana 135Met His Asp Gln Leu Arg Asp Met
Gly1 51369PRTArabidopsis thaliana 136Met His Asp Leu Ile Gln Asp
Met Gly1 51376PRTArabidopsis thaliana 137Ser Lys Leu Glu Ser Leu1
51388PRTArabidopsis thaliana 138Gly Leu His Ser Leu Glu Tyr Leu1
51398PRTArabidopsis thaliana 139Gly Leu Arg Ser Leu Glu Ile Leu1
51403432DNAArabidopsis thaliana 140acaagtaaaa gaaagagcga gaaatcatcg
aaatggattt catctcatct cttatcgttg 60gctgtgctca ggtgttgtgt gaatctatga
atatggcgga gagaagagga cataagactg 120atcttagaca agccatcact
gatcttgaaa cagccatcgg tgacttgaag gccatacgtg 180atgacctgac
tttacggatc caacaagacg gtctagaggg acgaagctgc tcaaatcgtg
240ccagagagtg gcttagtgcg gtgcaagtaa cggagactaa aacagcccta
cttttagtga 300ggtttaggcg tcgggaacag aggacgcgaa tgaggaggag
atacctcagt tgtttcggtt 360gtgccgacta caaactgtgc aagaaggttt
ctgccatatt gaagagcatt ggtgagctga 420gagaacgctc tgaagctatc
aaaacagatg gcgggtcaat tcaagtaact tgtagagaga 480tacccatcaa
gtccgttgtc ggaaatacca cgatgatgga acaggttttg gaatttctca
540gtgaagaaga agaaagagga atcattggtg tttatggacc tggtggggtt
gggaagacaa 600cgttaatgca gagcattaac aacgagctga tcacaaaagg
acatcagtat gatgtactga 660tttgggttca aatgtccaga gaattcggcg
agtgtacaat tcagcaagcc gttggagcac 720ggttgggttt atcttgggac
gagaaggaga ccggcgaaaa cagagctttg aagatataca 780gagctttgag
acagaaacgt ttcttgttgt tgctagatga gtctgggaag agatagactt
840ggagaaaact ggagttcctc gaccttgaca gggaaaacaa atgcaaggtg
atgttcacga 900cacggtctat agcattatgc aacaatatgg gtgcggaata
caagttgaga gtggagtttc 960tggagaagaa acacgcgtgg gagctgttct
gtagtaaggt atggagaaaa gatcttttag 1020agtcatcatc aattcgccgg
ctcgcggaga ttatagtgag taaatgtgga ggattgccac 1080tagcgttgat
cactttagga ggagccatgg ctcatagaga gacagaagaa gagtggatcc
1140atgctagtga agttctgact agatttccag cagagatgaa gggtatgaac
tatgtatttg 1200cccttttgaa attcagctac gacaacctcg agagtgatct
gcttcggtct tgtttcttgt 1260actgcgcttt attcccagaa gaacattgta
tagagatcga gcagcttgtt cagtactggg 1320tcggcgaagg gtttctcacc
agctcccatg gcgttaacac catttacaag ggatattttc 1380tcattgggga
tctgaaagcg gcatgtttgt tggaaaccgg agatgagaaa acacaggtga
1440agatgcataa tgtggtcaga agctttgcat tgtggatggc atctgaacag
gggacttata 1500aggagctgat cctagttgag cctagcatgg gacatactga
agctcctaaa gcagaaaact 1560ggcgacaagc ttggtgatct cattgttaga
taacagaatc cagaccttgc ctgaaaaact 1620catatgcccg aaactgacaa
cactgatgct ccaacagaac agctctttga agaagattcc 1680aacagggttt
ttcatgcata tgcctgttct cagagtcttg gacttgtcgt tcacaagtat
1740cactgagatt ccgttgtcta tcaagtattt ggtggagttg tatcatctgt
ctatgtcagg 1800aacaaagata agtgtattgc cacaggagct tgggaatctt
agaaaactga agcatctgga 1860cctacaaaga actcagtttc ttcagacgat
cccacgagat gccatatgtt ggctgagcaa 1920gctcgaggtt ctgaacttgt
actacagtta cgccggttgg gaactgcaga gctttggaga 1980agatgaagca
gaagaactcg gattcgctga cttggaatac ttggaaaacc taaccacact
2040cggtatcact gttctctcat tggagaccct aaaaactctc ttcgagttcg
gtgctttgca 2100taaacatata cagcatctcc acgttgaaga gtgcaatgaa
ctcctctact tcaatctccc 2160atcactcact aaccatggca ggaacctgag
aagacttagc attaaaagtt gccatgactt 2220ggagtacctg gtcacacccg
cagattttga aaatgattgg cttccgagtc tagaggttct 2280gacgttacac
agccttcaca acttaaccag
agtgtgggga aattctgtaa gccaagattg 2340tctgcggaat atccgttgca
taaacatttc acactgcaac aagctgaaga atgtctcatg 2400ggttcagaaa
ctcccaaagc tagaggtgat tgaactgttc gactgcagag agatagagga
2460attgataagc gaacacgaga gtccatccgt cgaagatcca acattgttcc
caagcctgaa 2520gaccttgaga actagggatc tgccagaact aaacagcatc
ctcccatctc gattttcatt 2580ccaaaaagtt gaaacattag tcatcacaaa
ttgccccaga gttaagaaac tgccgtttca 2640ggagaggagg acccagatga
acttgccaac agtttattgt gaggagaaat ggtggaaagc 2700actggaaaaa
gttgaaacat tagtcatcac aaattgcccc agagttaaga aactgccgtt
2760tcaggagagg aggacccaga tgaacttgcc aacagtttat tgtgaggaga
aatggtggaa 2820agcactggaa aaagatcaac caaacgaaga gctttgttat
ttaccgcgct ttgttccaaa 2880ttgatataag agctaagagc actctgtaca
aatatgtcca ttcataagta gcaggaagcc 2940aggaaggttg ttccagtgaa
gtcatcaact ttccactaga ccacaaaact agagattatg 3000taatcataaa
aaccaaacta tccgcgatca aatagatctc acgactatga ggacgaagac
3060tcaccgagta tcgtcgatat agaaactcca agctccagtt ccgatcagtg
aagacgaaca 3120agtttatcag atctctgcaa caattctggg aatcgtcacc
tcagattaga cctccagtaa 3180gaagtgagaa agcatggacg acgactgtga
agaattgagc taatgagctg aaccggatcc 3240ggtgaaattg cagaaccgga
tcggagaaga agaattttgc atttgtgcat ctttattttt 3300aattgttacg
tttgagcccc aataatcata gatattgtag tgaagaccaa atttcatggt
3360ggatcaatca aattgtattt tcaaattttc gtagtgtaat aacggaaaaa
ggaataaaaa 3420ggtcactgag ta 3432141909PRTArabidopsis thaliana
141Met Asp Phe Ile Ser Ser Leu Ile Val Gly Cys Ala Gln Val Leu Cys1
5 10 15Glu Ser Met Asn Met Ala Glu Arg Arg Gly His Lys Thr Asp Leu
Arg 20 25 30Gln Ala Ile Thr Asp Leu Glu Thr Ala Ile Gly Asp Leu Lys
Ala Ile35 40 45Arg Asp Asp Leu Thr Leu Arg Ile Gln Gln Asp Gly Leu
Glu Gly Arg50 55 60Ser Cys Ser Asn Arg Ala Arg Glu Trp Leu Ser Ala
Val Gln Val Thr65 70 75 80Glu Thr Lys Thr Ala Leu Leu Leu Val Arg
Phe Arg Arg Arg Glu Gln 85 90 95Arg Thr Arg Met Arg Arg Arg Tyr Leu
Ser Cys Phe Gly Cys Ala Asp 100 105 110Tyr Lys Leu Cys Lys Lys Val
Ser Ala Ile Leu Lys Ser Ile Gly Glu115 120 125Leu Arg Glu Arg Ser
Glu Ala Ile Lys Thr Asp Gly Gly Ser Ile Gln130 135 140Val Thr Cys
Arg Glu Ile Pro Ile Lys Ser Val Val Gly Asn Thr Thr145 150 155
160Met Met Glu Gln Val Leu Glu Phe Leu Ser Glu Glu Glu Glu Arg Gly
165 170 175Ile Ile Gly Val Tyr Gly Pro Gly Gly Val Gly Lys Thr Thr
Leu Met 180 185 190Gln Ser Ile Asn Asn Glu Leu Ile Thr Lys Gly His
Gln Tyr Asp Val195 200 205Leu Ile Trp Val Gln Met Ser Arg Glu Phe
Gly Glu Cys Thr Ile Gln210 215 220Gln Ala Val Gly Ala Arg Leu Gly
Leu Ser Trp Asp Glu Lys Glu Thr225 230 235 240Gly Glu Asn Arg Ala
Leu Lys Ile Tyr Arg Ala Leu Arg Gln Lys Arg 245 250 255Phe Leu Leu
Leu Leu Asp Asp Val Trp Glu Glu Ile Asp Leu Glu Lys 260 265 270Thr
Gly Val Pro Arg Pro Asp Arg Glu Asn Lys Cys Lys Val Met Phe275 280
285Thr Thr Arg Ser Ile Ala Leu Cys Asn Asn Met Gly Ala Glu Tyr
Lys290 295 300Leu Arg Val Glu Phe Leu Glu Lys Lys His Ala Trp Glu
Leu Phe Cys305 310 315 320Ser Lys Val Trp Arg Lys Asp Leu Leu Glu
Ser Ser Ser Ile Arg Arg 325 330 335Leu Ala Glu Ile Ile Val Ser Lys
Cys Gly Gly Leu Pro Leu Ala Leu 340 345 350Ile Thr Leu Gly Gly Ala
Met Ala His Arg Glu Thr Glu Glu Glu Trp355 360 365Ile His Ala Ser
Glu Val Leu Thr Arg Phe Pro Ala Glu Met Lys Gly370 375 380Met Asn
Tyr Val Phe Ala Leu Leu Lys Phe Ser Tyr Asp Asn Leu Glu385 390 395
400Ser Asp Leu Leu Arg Ser Cys Phe Leu Tyr Cys Ala Leu Phe Pro Glu
405 410 415Glu His Ser Ile Glu Ile Glu Gln Leu Val Glu Tyr Trp Val
Gly Glu 420 425 430Gly Phe Leu Thr Ser Ser His Gly Val Asn Thr Ile
Tyr Lys Gly Tyr435 440 445Phe Leu Ile Gly Asp Leu Lys Ala Ala Cys
Leu Leu Glu Thr Gly Asp450 455 460Glu Lys Thr Gln Val Lys Met His
Asn Val Val Arg Ser Phe Ala Leu465 470 475 480Trp Met Ala Ser Glu
Gln Gly Thr Tyr Lys Glu Leu Ile Leu Val Glu 485 490 495Pro Ser Met
Gly His Thr Glu Ala Pro Lys Ala Glu Asn Trp Arg Gln 500 505 510Ala
Leu Val Ile Ser Leu Leu Asp Asn Arg Ile Gln Thr Leu Pro Glu515 520
525Lys Leu Ile Cys Pro Lys Leu Thr Thr Leu Met Leu Gln Gln Asn
Ser530 535 540Ser Leu Lys Lys Ile Pro Thr Gly Phe Phe Met His Met
Pro Val Leu545 550 555 560Arg Val Leu Asp Leu Ser Phe Thr Ser Ile
Thr Glu Ile Pro Leu Ser 565 570 575Ile Lys Tyr Leu Val Glu Leu Tyr
His Leu Ser Met Ser Gly Thr Lys 580 585 590Ile Ser Val Leu Pro Gln
Glu Leu Gly Asn Leu Arg Lys Leu Lys His595 600 605Leu Asp Leu Gln
Arg Thr Gln Phe Leu Gln Thr Ile Pro Arg Asp Ala610 615 620Ile Cys
Trp Leu Ser Lys Leu Glu Val Leu Asn Leu Tyr Tyr Ser Tyr625 630 635
640Ala Gly Trp Glu Leu Gln Ser Phe Gly Glu Asp Glu Ala Glu Glu Leu
645 650 655Gly Phe Ala Asp Leu Glu Tyr Leu Glu Asn Leu Thr Thr Leu
Gly Ile 660 665 670Thr Val Leu Ser Leu Glu Thr Leu Lys Thr Leu Phe
Glu Phe Gly Ala675 680 685Leu His Lys His Ile Gln His Leu His Val
Glu Glu Cys Asn Glu Leu690 695 700Leu Tyr Phe Asn Leu Pro Ser Leu
Thr Asn His Gly Arg Asn Leu Arg705 710 715 720Arg Leu Ser Ile Lys
Ser Cys His Asp Leu Glu Tyr Leu Val Thr Pro 725 730 735Ala Asp Phe
Glu Asn Asp Trp Leu Pro Ser Leu Glu Val Leu Thr Leu 740 745 750His
Ser Leu His Asn Leu Thr Arg Val Trp Gly Asn Ser Val Ser Gln755 760
765Asp Cys Leu Arg Asn Ile Arg Cys Ile Asn Ile Ser His Cys Asn
Lys770 775 780Leu Lys Asn Val Ser Trp Val Gln Lys Leu Pro Lys Leu
Glu Val Ile785 790 795 800Glu Leu Phe Asp Cys Arg Glu Ile Glu Glu
Leu Ile Ser Glu His Glu 805 810 815Ser Pro Ser Val Glu Asp Pro Thr
Leu Phe Pro Ser Leu Lys Thr Leu 820 825 830Arg Thr Arg Asp Leu Pro
Glu Leu Asn Ser Ile Leu Pro Ser Arg Phe835 840 845Ser Phe Gln Lys
Val Glu Thr Leu Val Ile Thr Asn Cys Pro Arg Val850 855 860Lys Lys
Leu Pro Phe Gln Glu Arg Arg Thr Gln Met Asn Leu Pro Thr865 870 875
880Val Tyr Cys Glu Glu Lys Trp Trp Lys Ala Leu Glu Lys Asp Gln Pro
885 890 895Asn Glu Glu Leu Cys Tyr Leu Pro Arg Phe Val Pro Asn 900
90514222PRTArabidopsis thaliana 142Pro Lys Ala Glu Asn Trp Arg Gln
Ala Leu Val Ile Ser Leu Leu Asp1 5 10 15Asn Arg Ile Gln Thr Leu
2014323PRTArabidopsis thaliana 143Pro Glu Lys Leu Ile Cys Pro Lys
Leu Thr Thr Leu Met Leu Gln Gln1 5 10 15Asn Ser Ser Leu Lys Lys Ile
2014424PRTArabidopsis thaliana 144Pro Thr Gly Phe Phe Met His Met
Pro Val Leu Arg Val Leu Asp Leu1 5 10 15Ser Phe Thr Ser Ile Thr Glu
Ile 2014523PRTArabidopsis thaliana 145Pro Leu Ser Ile Lys Tyr Leu
Val Glu Leu Tyr His Leu Ser Met Ser1 5 10 15Gly Thr Lys Ile Ser Val
Leu 2014624PRTArabidopsis thaliana 146Pro Gln Glu Leu Gly Asn Leu
Arg Lys Leu Lys His Leu Asp Leu Gln1 5 10 15Arg Thr Gln Phe Leu Gln
Thr Ile 2014737PRTArabidopsis thaliana 147Pro Arg Asp Ala Ile Cys
Trp Leu Ser Lys Leu Glu Val Leu Asn Leu1 5 10 15Tyr Tyr Ser Tyr Ala
Gly Trp Glu Leu Gln Ser Phe Gly Glu Asp Glu 20 25 30Ala Glu Glu Leu
Gly3514825PRTArabidopsis thaliana 148Phe Ala Asp Leu Glu Tyr Leu
Glu Asn Leu Thr Thr Leu Gly Ile Thr1 5 10 15Val Leu Ser Leu Glu Thr
Leu Lys Thr 20 2514927PRTArabidopsis thaliana 149Leu Phe Glu Phe
Gly Ala Leu His Lys His Ile Gln His Leu His Val1 5 10 15Glu Glu Cys
Asn Glu Leu Leu Tyr Phe Asn Leu 20 2515026PRTArabidopsis thaliana
150Pro Ser Leu Thr Asn His Gly Arg Asn Leu Arg Arg Leu Ser Ile Lys1
5 10 15Ser Cys His Asp Leu Glu Tyr Leu Val Thr 20
2515129PRTArabidopsis thaliana 151Pro Ala Asp Phe Glu Asn Asp Trp
Leu Pro Ser Leu Glu Val Leu Thr1 5 10 15Leu His Ser Leu His Asn Leu
Thr Arg Val Trp Gly Asn 20 2515230PRTArabidopsis thaliana 152Ser
Val Ser Gln Asp Cys Leu Arg Asn Ile Arg Cys Ile Asn Ile Ser1 5 10
15His Cys Asn Lys Leu Lys Asn Val Ser Trp Val Gln Lys Leu 20 25
3015328PRTArabidopsis thaliana 153Pro Lys Leu Glu Val Ile Glu Leu
Phe Asp Cys Arg Glu Ile Glu Glu1 5 10 15Leu Ile Ser Glu His Glu Ser
Pro Ser Val Glu Asp 20 2515422PRTArabidopsis thaliana 154Pro Thr
Leu Phe Pro Ser Leu Lys Thr Leu Arg Thr Arg Asp Leu Pro1 5 10 15Glu
Leu Asn Ser Ile Leu 2015523PRTArabidopsis thaliana 155Pro Ser Arg
Phe Ser Phe Gln Lys Val Glu Thr Leu Val Ile Thr Asn1 5 10 15Cys Pro
Arg Val Lys Lys Leu 201565134DNAArabidopsis thaliana 156aagctttaca
gattggatga tctcttaatg catgctgaag tgactgcaaa aaggttagca 60atattcagtg
gttctcgtta tgaatatttc atgaacggaa gcagcactga gaaaatgagg
120cccttgttat ctgattttct gcaagagatt gagtctgtca aggtagagtt
cagaaatgtt 180tgcttgcaag ttctggatat atcacctttt tccctgacag
atggagaagg ccttgttaat 240ttcttattaa aaaaccaggc caaggtgccg
aatgatgatg ctgtttcttc tgatggaagt 300ttagaggatg caagcagcac
tgagaaaatg ggacttccat ctgattttct ccgagagatt 360gagtctgttg
agataaagga ggccagaaaa ttatatgatc aagttttgga tgcaacacat
420tgtgagacga gtaagcacga tggaaaaagc tttatcaaca ttatgttaac
ccaacaggac 480aaggtgctgg actatgatgc tggttcagtg tcttatcttc
ttaaccaaat ctcagtagtt 540aaagacaaaa tattgcacat tggctcttta
cttgtagata ttgtacagta ccggaatatg 600catatagaac ttacagatct
cgctgaacgt gttcaagata aaaactacat tcgtttcttc 660tctgtcaagg
gttatattcc tgcttggtat tacacactat atctctctga tgtcaagcaa
720ttgcttaagt ttgttgaggc agaggtaaag attatttgtc tgaaagtacc
agattcttca 780agttatagct tccctaagac aaatggatta ggatatctca
attgcttttt aggcaaattg 840gaggagcttt tacgttctaa gctcgatttg
ataatcgact taaaacatca gattgaatca 900gtcaaggagg gcttattgtg
cctaagatca ttcattgatc atttttcaga aagctatgtt 960gagcatgatg
aagcttgtgg tcttatagca agagtttctg taatggcata caaggctgag
1020tatgtcattg actcatgctt ggcctattct catccactct ggtacaaagt
tctttggatt 1080tctgaagttc ttgagaatat taagcttgta aataaagttg
ttggggagac atgtgaaaga 1140aggaacactg aagttactgt gcatgaagtt
gcaaagacta ccactaatgt agcaccatct 1200ttttcagctt atactcaaag
agcaaacgaa gaaatggagg gttttcagga tacaatagat 1260gaattaaagg
ataaactact tggaggatca cctgagcttg atgtcatctc aatcgttggc
1320atgccaggat tgggcaagac tacactagca aagaagattt acaatgatcc
agaagtcacc 1380tctcgcttcg atgtccatgc tcaatgtgtt gtgactcaat
tatattcatg gagagagttg 1440ttgctcacca ttttgaatga tgtgcttgag
ccttctgatc gcaatgaaaa agaagatgga 1500gaaatagctg atgatctacg
ccgatttttg ttgaccaaga gattcttgat tctcattgat 1560gatgtgtggg
actataaagt gtgggacaat ctatgtatgt gcttcagtga tgtttcaaat
1620aggagtagaa ttatcctaac aacccgcttg aatgatgtcg ccgaatatgt
caaatgtgaa 1680agtgatcccc atcatcttcg tttattcaga gatgacgaga
gttggacatt attacagaaa 1740gaagtctttc aaggagagag ctgtccacct
gaacttgaag atgtgggatt tgaaatatca 1800aaaagttgta gagggttgcc
tctctcagtt gtgttagtag ctggtgttct gaaacagaaa 1860aagaagacac
tagattcatg gaaagtagta gaacaaagtc taagttccca gaggattggc
1920agcttggaag agagcatatc tataattgga ttcagttaca agaatttacc
acactatctt 1980aagccttgtt ttctctattt tggaggattt ttgcagggaa
aggatattca tgactcaaaa 2040atgaccaagt tgtgggtagc tgaagagttt
gtacaagcaa acaacgaaaa aggacaagaa 2100gatacccgca caaggtttct
tggacgatct tattggtagg aatctggtga tggccatgga 2160gaagagacct
aatgccaagg tgaaaacgtg ccgcattcat gatttgttgc ataaattctg
2220catggaaaag gccaaacaag aggatttcct tctccagatc aataggtaaa
aaaaactgta 2280ttaattttac attacaaaaa aaaagaactg tattaatttt
actgtattat gtttatgcca 2340actctcattt ccatgtgttc tcttttattc
aattcagtgg agaaggtgta tttcctgaac 2400gattggaaga ataccgattg
ttcgttcatt cttaccaaga tgaaattgat ctgtggcgcc 2460catctcgctc
taatgtccgc tctttactat tcaatgcaat tgatccagat aacttgttat
2520ggccgcgtga tatctccttc atttttgaga gcttcaagct tgttaaagtg
ttggatttgg 2580aatcattcaa cattggtggt acttttccca ttgaaacaca
atatctaatt cagatgaagt 2640actttgcggc ccaaactgat gcaaattcaa
ttccttcatc tatagctaag cttgaaaatc 2700ttgagacttt tgtcgtaaga
ggattgggag gagagatgat attaccttgt tcacttctga 2760agatggtgaa
attgaggcat atacatgtaa atgatcgggt ttcttttggt ttgcgtgaga
2820acatggatgt tttaactggt aactcacaat aacctaattt ggaaaccttt
tctactccgc 2880gtctctttta tggtaaagac gcagagaaga ttttgaggaa
gatgccaaaa ttgagaaaat 2940tgagttgcat attttcaggg acatttggtt
attcaaggaa attgaagggt aggtgtgttc 3000gttttcccag attagatttt
ctaagtcacc ttgagtccct caagctggtt tcgaacagct 3060atccagccaa
acttcctcac aagttcaatt tcccctcgca actaagggaa ctgactttat
3120caaagttccg tctaccttgg acccaaattt cgatcattgc agaactgccc
aacttggtga 3180ttcttaagtt attgctcaga gcctttgaag gggatcactg
ggaagtgaaa gattcagagt 3240tcctagaact caaatactta aaactggaca
acctcaaagt tgtacaatgg tccatctctg 3300atgatgcttt tcctaagctt
gaacatttgg ttttaacgaa atgtaagcat cttgagaaaa 3360tcccttctcg
ttttgaagat gctgtttgtc taaatagagt tgaggtgaac tggtgcaact
3420ggaatgttgc caattcagcc caagatattc aaactatgca acatgaagtt
atagcaaatg 3480attcattcac agttactata cagcctccag attggtctaa
agaacagccc cttgactctt 3540agcaaaggtt tgttcttgct gtgttcatcc
aagtgcattt aacatttatt cattttgttt 3600tacaccagaa catgtttatt
ttgctagtat tacttgatac attaaaagaa atcgaactca 3660tatttctgct
acagtcttaa cttttcttgg gcttacttga ggtctagatt agatcaatgg
3720ttcatgtaat ttttaattca ctgtttcatt caactgtctt atgatagttg
tgaaatgaca 3780atattgttat ccctagccaa atttattatg ttcaaatgaa
aactgatgtc acaactactt 3840ttttgtgaaa tgtttttgaa ttttttgcta
taaaattgac gaattgacag cttctatatt 3900tgtcagctaa actctttgtc
accagaagtg tatttagaat tactgtggtt ttatgaaaga 3960gttctgtaga
attttatgct tttgcagaat atagtttaaa acaacaacac ttctctgttt
4020cagagatagc agaagctaaa gttcaaggca ttttgtttat ttctagaaca
agtggagttc 4080ttatgttgaa ttcttgaaaa gaagaagaat caggagcagg
taaagttatc tctttttatg 4140tttttcttct tttagatgtt atttcttcat
cttgaacgtg aacaccgctg aaagcatttt 4200aataaaaccg gagagaaaaa
taagatcttt ttatataaag cattatcatg taaatatgcc 4260taaatccata
tggtacaact gtttgacaaa atgatagaga ggggagtttt atagtataag
4320taaaacagga ttgagaaaaa aatccttgca cgattttcaa tttctggcca
catcacaatg 4380tgtgtcaaag ttcccctctt taagtggaac aagcaatcag
aaaagctcat tcttatcggt 4440gacataccaa taccagctga ctgtctcatc
ttggttaact tagccttgct tacttagact 4500attagattag ttactaatga
actggtaaat tggaaccaaa tgtagttagc ttgatgagct 4560ggtagacatg
tatatatgaa gatacacgcg taactttagt cgatggttaa tttttcattt
4620ttgatttttt ttcttcacag agtatatatg aacttggcct aaaagttttg
cttcactaat 4680ttaactatta ccgtggatga aacaagcatg gcaacatttt
caacaactat cactcaagca 4740atgtaaaaaa tggaggttct acgagcggta
catgtaagag ttttgtgcac acaagaggtt 4800ctgagacttg aaccatccat
gtccaaggca gttgagatgc tagtaaagaa agaagaagat 4860gagcctgcac
taattaatct ccctgtatga atgagagaat gagaaaaaga tggagcttca
4920tgaaccaaaa gttacctttt ttttttcttc ttaatggcat tactttgaag
cacatgtttg 4980ttagttgtaa attgtaatgg tgaagtgttt gtaaatatag
ggagtgatat ttgaaagaat 5040ggttgtgtta tctttacaaa ccggaatcat
ttctgtataa ttttcttctg taatttttgg 5100tttcggttta ttcattactc
atttcagtaa gctt 513415726DNAArabidopsis
thalianamisc_feature(3)..(3)n is a, c, g, or t 157ggnatgggng
gnntnggnaa racnac 2615820DNAArabidopsis
thalianamisc_feature(1)..(1)n is a, c, g, or t 158ncgngwngtn
akdawncgna 2015917DNAArabidopsis thalianamisc_feature(4)..(4)n is
a, c, g, or t 159ggwntbggwa arachac 1716033DNAArabidopsis
thalianamisc_feature(1)..(1)n is a, c, g, or t 160nrynrdngtn
gtyttnccna nnccnssnrk ncc 3316126DNAArabidopsis
thalianamisc_feature(3)..(3)n is a, c, g, or t 161ggnmynssng
gnntnggnaa racnac 2616216DNAArabidopsis thaliana 162tygaygayrt
bkrbra 1616316DNAArabidopsis thalianamisc_feature(15)..(15)n is a,
c, g, or t 163tyccavayrt crtcna 1616426DNAArabidopsis
thalianamisc_feature(4)..(4)n is a, c, g, or t 164vymnayrtcr
tcnadnavna
nnarna 2616526DNAArabidopsis thalianamisc_feature(3)..(3)n is a, c,
g, or t 165wwnmrrdtny tnntnbtnht ngayga 2616621DNAArabidopsis
thalianamisc_feature(1)..(1)n is a, c, g, or t 166ncgngwngtn
akdawncgng a 2116721DNAArabidopsis thalianamisc_feature(1)..(1)n is
a, c, g, or t 167ncknswngtn addatdaatn g 2116812DNAArabidopsis
thalianamisc_feature(1)..(1)n is a, c, g, or t 168narnggnarn cc
1216917DNAArabidopsis thaliana 169ggwytbccwy tbgchyt
1717017DNAArabidopsis thalianamisc_feature(15)..(15)n is a, c, g,
or t 170ardgcvarwg gvarncc 1717124DNAArabidopsis
thalianamisc_feature(1)..(1)n is a, c, g, or t 171nrnnwynavn
shnarnggna rncc 2417217DNAArabidopsis thalianamisc_feature(3)..(3)n
is a, c, g, or t 172ggnytnccny tndsnbt 1717320DNAArabidopsis
thaliana 173arrttrtcrt adswrawytt 2017420DNAArabidopsis
thalianamisc_feature(3)..(3)n is a, c, g, or t 174arnyyntyrt
ansrnannyy 2017520DNAArabidopsis thalianamisc_feature(3)..(3)n is
a, c, g, or t 175rrnwthwsnt ayranrvnyt 2017620DNAArabidopsis
thalianamisc_feature(3)..(3)n is a, c, g, or t 176gtnttyytnw
snttymgrgg 2017723DNAArabidopsis thalianamisc_feature(3)..(3)n is
a, c, g, or t 177ccnathttyt ayrwbgtnga ycc 2317817DNAArabidopsis
thalianamisc_feature(3)..(3)n is a, c, g, or t 178gtnggnathg
ayrmnca 1717921DNAArabidopsis thalianamisc_feature(7)..(7)n is a,
c, g, or t 179raarcangcd atrtcnarra a 2118020DNAArabidopsis
thalianamisc_feature(6)..(6)n is a, c, g, or t 180ttyytngaya
thgcntgytt 2018126DNAArabidopsis thalianamisc_feature(12)..(12)n is
a, c, g, or t 181cccatrtcyy knadnwrrtc rtgcat 2618226DNAArabidopsis
thalianamisc_feature(12)..(12)n is a, c, g, or t 182atgcaygayy
wnhtnmrrga yatggg 2618315DNAArabidopsis
thalianamisc_feature(1)..(1)n is a, c, g, or t 183narnswytyn arytt
1518417DNAArabidopsis thalianamisc_feature(3)..(3)n is a, c, g, or
t 184wsnaarytnr arwsnyt 1718521DNAArabidopsis
thalianamisc_feature(7)..(7)n is a, c, g, or t 185dwwytcnarn
swnyknarnc c 2118617DNAArabidopsis thalianamisc_feature(3)..(3)n is
a, c, g, or t 186ggnytnmrnw snytnga 1718713PRTArabidopsis thaliana
187Leu Lys Phe Ser Tyr Asp Asn Leu Glu Ser Asp Leu Leu1 5
1018816PRTArabidopsis thaliana 188Gly Val Tyr Gly Pro Gly Gly Val
Gly Lys Thr Thr Leu Met Gln Ser1 5 10 1518914PRTArabidopsis
thaliana 189Gly Gly Leu Pro Leu Ala Leu Ile Thr Leu Gly Gly Ala
Met1 5 1019011PRTArabidopsis thalianamisc_feature(2)..(3)Xaa can be
any naturally occurring amino acid 190Gly Xaa Xaa Gly Xaa Gly Lys
Thr Thr Xaa Xaa1 5 1019111PRTArabidopsis
thalianamisc_feature(1)..(3)Xaa can be any naturally occurring
amino acid 191Xaa Xaa Xaa Leu Xaa Xaa Xaa Asp Asp Xaa Xaa1 5
101928PRTArabidopsis thalianamisc_feature(1)..(5)Xaa can be any
naturally occurring amino acid 192Xaa Xaa Xaa Xaa Xaa Thr Xaa Arg1
51938PRTArabidopsis thalianamisc_feature(5)..(8)Xaa can be any
naturally occurring amino acid 193Gly Leu Pro Leu Xaa Xaa Xaa Xaa1
51947PRTArabidopsis thalianamisc_feature(1)..(2)Xaa can be any
naturally occurring amino acid 194Xaa Xaa Ser Tyr Xaa Xaa Leu1
51954PRTArabidopsis thaliana 195Asn Ser His Arg11964PRTArabidopsis
thaliana 196Thr Gly Asp Leu11974PRTArabidopsis thaliana 197His Gly
Thr Tyr119811PRTArabidopsis thaliana 198Arg Met Ser His Gly Phe Arg
Asn Ser Gln Ser1 5 1019927PRTArabidopsis thaliana 199Gly Glu Met
Val Glu Ser Thr Gly Lys Arg Ser Thr Lys Arg Arg Ala1 5 10 15Leu Leu
Phe Thr Ala Leu Cys Ser Lys Leu Ile 20 252009PRTArabidopsis
thalianamisc_feature(5)..(5)Xaa can be any naturally occurring
amino acid 200Pro Ile Phe Tyr Xaa Val Asp Pro Ser1
52016PRTArabidopsis thalianamisc_feature(5)..(5)Xaa can be any
naturally occurring amino acid 201Val Gly Ile Asp Xaa His1
52029PRTArabidopsis thalianamisc_feature(4)..(6)Xaa can be any
naturally occurring amino acid 202Met His Asp Xaa Xaa Xaa Asp Met
Gly1 52036PRTArabidopsis thaliana 203Ser Lys Leu Lys Ser Leu1
52048PRTArabidopsis thalianamisc_feature(3)..(3)Xaa can be any
naturally occurring amino acid 204Gly Leu Xaa Ser Leu Glu Xaa Leu1
52056PRTArabidopsis thaliana 205Ser Lys Leu Lys Ser Leu1
52067PRTArabidopsis thaliana 206Lys Phe Ser Tyr Asp Asn Leu1
520723PRTArabidopsis Thaliamisc_feature(2)..(6)Xaa can be any
naturally occurring amino acid 207Pro Xaa Xaa Xaa Xaa Xaa Leu Xaa
Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa
2020823PRTYeastmisc_feature(2)..(6)Xaa can be any naturally
occurring amino acid 208Pro Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa
Xaa Leu Xaa Leu Xaa1 5 10 15Xaa Asn Xaa Xaa Xaa Xaa Xaa
2020912PRTArabidopsis thalianamisc_feature(1)..(3)Xaa can be any
naturally occurring amino acid 209Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa
Xaa Xaa Xaa Leu1 5 102107PRTArabidopsis
thalianamisc_feature(1)..(2)Xaa can be any naturally occurring
amino acid 210Xaa Xaa Asp Leu Xaa Xaa Xaa1 52118PRTArabidopsis
thaliana 211Gly Pro Gly Gly Val Gly Lys Thr1 521216PRTArabidopsis
thaliana 212Thr Tyr Gly Ala Tyr Gly Ala Tyr Arg Thr Asx Tyr Arg Asx
Arg Ala1 5 10 15
* * * * *