U.S. patent application number 10/697787 was filed with the patent office on 2004-10-28 for generation of plants with improved pathogen resistance and drought tolerance.
Invention is credited to Anderson, Catherine, Bates, Stanley R., Federspiel, Nancy Anne, Fitch, Jonathan R., Lammers, Allan, Lee, Stephen Aaron, Liu, Xing Liang, Powell, Kerrie, Puente, Pilar, Rhein, Jennifer Lee, Timmons, Philip Reid, Westerlund, Christina, Wingate, Vincent Paul Mary.
Application Number | 20040216182 10/697787 |
Document ID | / |
Family ID | 33303345 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040216182 |
Kind Code |
A1 |
Federspiel, Nancy Anne ; et
al. |
October 28, 2004 |
Generation of plants with improved pathogen resistance and drought
tolerance
Abstract
The present invention is directed to plants that display a
pathogen resistance and increased drought tolerance phenotype due
to altered expression of a PRDT1 nucleic acid. The invention is
further directed to methods of generating plants with a pathogen
resistance and increased drought tolerance phenotype.
Inventors: |
Federspiel, Nancy Anne;
(Menlo Park, CA) ; Lammers, Allan; (Portland,
OR) ; Liu, Xing Liang; (Tualatin, OR) ; Bates,
Stanley R.; (Aumsville, OR) ; Westerlund,
Christina; (Portland, OR) ; Fitch, Jonathan R.;
(Portland, OR) ; Anderson, Catherine; (Durham,
NC) ; Lee, Stephen Aaron; (Durham, NC) ;
Powell, Kerrie; (Durham, NC) ; Puente, Pilar;
(Durham, NC) ; Rhein, Jennifer Lee; (Raleigh,
NC) ; Timmons, Philip Reid; (Durham, NC) ;
Wingate, Vincent Paul Mary; (Chapel Hill, NC) |
Correspondence
Address: |
PATENT DEPT
EXELIXIS, INC.
170 HARBOR WAY
P.O. BOX 511
SOUTH SAN FRANCISCO
CA
94083-0511
US
|
Family ID: |
33303345 |
Appl. No.: |
10/697787 |
Filed: |
October 29, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10697787 |
Oct 29, 2003 |
|
|
|
PCT/US03/12981 |
Apr 24, 2003 |
|
|
|
60482076 |
Jun 24, 2003 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/468; 800/279 |
Current CPC
Class: |
C12N 15/8273 20130101;
C07K 14/37 20130101; C12N 15/8282 20130101 |
Class at
Publication: |
800/278 ;
435/468; 800/279 |
International
Class: |
C12N 015/82; C12N
015/87; A01H 005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 24, 2003 |
WO |
PCT/US03/12981 |
Claims
It is claimed:
1. A transgenic plant comprising a plant transformation vector
comprising a nucleotide sequence that encodes or is complementary
to a sequence that encodes a PRDT1 polypeptide comprising the amino
acid sequence of SEQ ID NO:2, or an ortholog thereof, wherein said
transgenic plant has increased resistance to pathogens and/or
increased drought tolerance relative to control plants.
2. The transgenic plant of claim 1 wherein the transformation
vector comprises a constitutive promoter that controls expression
of the PRDT1 polypeptide or ortholog.
3. The transgenic plant of claim 1 wherein the transformation
vector comprises a pathogen-inducible promoter that controls
expression of the PRDT1 polypeptide or ortholog.
4. The transgenic plant of claim 1 which encodes a PRDT1 ortholog
comprising an amino acid sequence selected from any one of SEQ ID
NOs:3-17.
5. A method of producing increased pathogen resistance in a plant,
said method comprising: a) introducing into progenitor cells of the
plant a plant transformation vector comprising a nucleotide
sequence that encodes or is complementary to a sequence that
encodes a PRDT1 polypeptide comprising the amino acid sequence of
SEQ ID NO:2, or an ortholog thereof, and b) growing the transformed
progenitor cells to produce a transgenic plant, wherein said
polynucleotide sequence is expressed, and said transgenic plant
exhibits increased resistance to pathogens relative to control
plants.
6. A plant obtained by a method of claim 5.
7. A plant part obtained from a plant according to claim 6.
8. A method of generating a plant having an increased pathogen
resistance and/or drought tolerance phenotype comprising
identifying a plant that has an allele in its PRDT1 gene that
results in increased pathogen resistance and/or drought tolerance
compared to plants lacking the allele and generating progeny of
said identified plant, wherein the generated progeny inherit the
allele and have the increased pathogen resistance phenotype.
9. The method of claim 8 that employs candidate gene/QTL
methodology.
10. The method of claim 8 that employs TILLING methodology.
Description
RELATED APPLICATIONS
[0001] Priority is claimed under 35 U.S.C. .sctn. 120 to
international application no. PCT/US03/12981 and U.S. provisional
patent application No. 60/482,076, the contents of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Crop production is affected by numerous environmental
factors. Attempts to improve crop yield under stress conditions by
plant breeding have been largely unsuccessful, primarily due to the
multigenic origin of the adaptive responses (Barkla et al. 1999,
Adv Exp Med Biol 464:77-89). Consequently, an increasing amount of
research has been dedicated to developing transgenic plants that
show increased tolerance to various environmental stresses. For
example, transgenic plants have been described having resistance to
a broad range of plant pathogens (Stuiver and Custers, 2001, Nature
411:865-8; Melchers and Stuiver, 2000, Curr Opin Plant Biol
3:147-52; Rommens and Kishore, 2000, Curr Opin Biotechnol 11:120-5;
and Mourgues et al. 1998, Trends Biotechnol 16:203-10). Transgenic
plants displaying increased drought tolerance have also been
described (Laporte et al. 2002, J Exp Bot 53:699-705; Qin et al.
2002, Plant Physiol 128:544-51; and Iuchi et al. 2001, Plant J
27:325-33).
[0003] Activation tagging in plants is a method of generating
random mutations by insertion of a heterologous nucleic acid
construct comprising regulatory sequences (e.g., an enhancer) into
a plant genome. The regulatory sequences can act to enhance
transcription of one or more native plant genes. Accordingly,
activation tagging is a fruitful method for generating
gain-of-function, generally dominant mutants (Hayashi et al.,
Science (1992) 258: 1350-1353; Weigel et al., Plant Physiology
(2000) 122:1003-1013). The inserted construct provides a molecular
tag for rapid identification of a native plant gene whose
mis-expression causes a mutant phenotype of interest (e.g. drought
tolerance or pathogen resistance).
SUMMARY OF THE INVENTION
[0004] The invention provides a transgenic plant comprising a plant
transformation vector comprising a nucleotide sequence that encodes
or is complementary to a sequence that encodes a PRDT1 polypeptide
or an ortholog thereof. The transgenic plant is characterized by
having increased resistance to pathogens and increased drought
tolerance.
[0005] The present invention further provides a method of producing
an altered pathogen resistance and drought tolerance phenotype in a
plant. The method comprises introducing into plant progenitor cells
a vector comprising a nucleotide sequence that encodes or is
complementary to a sequence encoding a PRDT1 polypeptide or an
ortholog thereof and growing a transgenic plant that expresses the
nucleotide sequence. In one embodiment, the PRDT1 polypeptide has
at least 50% sequence identity to the amino acid sequence presented
in SEQ ID NO:2 and comprises a SANT domain. In other embodiments,
the PRDT1 polypeptide has at least 80% or 90% sequence identity to
or has the amino acid sequence presented in SEQ ID NO:2.
[0006] The invention further provides plants and plant parts
obtained by the methods described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0007] Identification of Plants with a PRDT1 Phenotype
[0008] We used activation tagging in Arabidopsis (Weigel et al,
supra) to generate a large collection of transgenic plants, and
tested the plants to identify those that display a phenotype of
interest. Briefly, and as further described in the Examples, a
large number of Arabidopsis plants were mutated with the pSKI015
vector, which comprises a T-DNA from the Ti plasmid of
Agrobacterium tumifaciens, a viral enhancer element, and a
selectable marker gene. When the T-DNA inserts into the genome of
transformed plants, the enhancer element can cause up-regulation
genes in the vicinity, generally within about 10 kilobase (kb) of
the insertion. T1 plants were exposed to the selective agent in
order to specifically recover transformed plants that expressed the
selectable marker and therefore harbored T-DNA insertions. Seed
from transformed plants were planted, and grown under various
environmental stress conditions to identify plants having enhanced
ability to survive the stress condition.
[0009] We discovered a transgenic plant line that exhibits
increased tolerance to drought conditions. Another plant line was
identified that exhibits increased resistance to the plant
pathogen, P. parasitica. An analysis of the genomic DNA sequence
flanking the T-DNA insertions in each of the identified lines
suggested that the same Arabidopsis gene, having TAIR designation
At1g75250 (GenBank Identifier No. 18410812), which encodes a
myb-related protein, was associated with the drought tolerance
phenotype in the one line, and the pathogen resistance phenotype in
the other line. We refer to this gene herein as "PRDT1" (for
Pathogen Resistant Drought Tolerant). Accordingly, in one aspect of
the invention, PRDT1 genes and/or polypeptides may be employed in
the development of transgenic plants having an "altered pathogen
resistance phenotype", meaning that there is a detectable change in
the response of the transgenic plant to pathogenic infection,
compared to the similar, but non-modified plant. The phenotype may
be apparent in the plant itself (e.g., in growth, viability or
particular tissue morphology of the plant) or may be apparent in
the ability of the pathogen to proliferate on and/or infect the
plant. As used herein, "improved pathogen resistance" refers to
increased resistance to a pathogen.
[0010] In another aspect of the invention the PRDT1
gene/polypeptide may be used to generate transgenic plants having
an "altered drought tolerance phenotype", meaning that there is a
detectable change in the ability of the transgenic plant to
withstand low-water conditions compared to the similar, but
non-modified plant. In general, improved (increased) drought
tolerance phenotypes (i.e., ability to a plant to survive in
low-water conditions that would normally be deleterious to a plant)
are of interest.
[0011] A further aspect of the invention is directed to transgenic
plants having a "PRDT1 phenotype" in which the plants exhibit both
an increased pathogen resistance phenotype and a drought tolerance
phenotype, relative to wild type plants, wherein the plants
comprise a plant transformation vector comprising a nucleotide
sequence that encodes or is complementary to a sequence that
encodes a PRDT1 polypeptide.
[0012] As used herein, the term "transgenic plant" refers to a
plant that comprises a heterologous polynucleotide within its
genome. Preferably, the polynucleotide is stably integrated into
the genome, such that it is passed on to successive generations. A
plant cell, tissue, organ, or any other plant part into which the
heterologous polynucleotides have been introduced is considered
"transformed", "transfected", or "transgenic". Direct and indirect
progeny of transformed plants or plant cells that also contain the
heterologous polynucleotide are also considered transgenic. A
"heterologous polynucleotide" is a sequence that is not native to
the plant cell in which it is expressed. When used to describe a
control sequence (e.g. promoter or enhancer), the term
"heterologous" refers to a control sequence (i.e. promoter or
enhancer) that does not function in nature to regulate the same
gene the expression of which it is currently regulating. Generally,
heterologous nucleic acid sequences are not endogenous to the cell
or part of the genome in which they are present, and have been
added to the cell, by infection, transfection, microinjection,
electroporation, or the like. A "heterologous" nucleic acid
construct may contain a control sequence/DNA coding sequence
combination that is the same as, or different from a control
sequence/DNA coding sequence combination found in the native
plant.
[0013] As used herein, the term "plant part" includes any plant
organ or tissue, including, without limitation, seeds, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen, and microspores. Plant cells can
be obtained from any plant organ or tissue and cultures prepared
therefrom. The class of plants which can be used in the methods of
the present invention is generally as broad as the class of higher
plants amenable to transformation techniques, including both
monocotyledenous and dicotyledenous plants.
[0014] PRDT1 genes may be used in the generation of crops and/or
other plant species that have improved ability to survive in
low-water conditions, and improved resistance to infection by P.
parasitica and other oomycetes and may also be useful the
generation of plants with improved resistance to fungal, bacterial,
and/or other pathogens. Mis-expression of PRDT1 genes may thus
reduce the need for fungicides and/or pesticides.
[0015] PRDT1 NUCLEIC ACIDS AND POLYPEPTIDES
[0016] Arabidopsis PRDT1 nucleic acid (coding) sequence is provided
in SEQ ID NO:1 and in Genbank entry GI 12331602, nucleotides
20955-21335 (designated F22H5.3 and At1g75250). The corresponding
protein sequence is provided in SEQ ID NO:2 and in GI 10092271.
[0017] As used herein, the term "PRDT1 polypeptide" refers to a
full-length PRDT1 protein or a fragment, derivative (variant), or
ortholog thereof that is "functionally active," meaning that the
protein fragment, derivative, or ortholog exhibits one or more or
the functional activities associated with the polypeptide of SEQ ID
NO:2. In one preferred embodiment, a functionally active PRDT1
polypeptide causes an altered pathogen resistance and drought
tolerance phenotype when mis-expressed in a plant. In a further
preferred embodiment, mis-expression of the functionally active
PRDT1 polypeptide causes increased resistance to P. parasitica
and/or other oomycetes and increased drought tolerance. In another
embodiment, a functionally active PRDT1 polypeptide is capable of
rescuing defective (including deficient) endogenous PRDT1 activity
when expressed in a plant or in plant cells; the rescuing
polypeptide may be from the same or from a different species as
that with defective activity. In another embodiment, a functionally
active fragment of a full length PRDT1 polypeptide (i.e., a native
polypeptide having the sequence of SEQ ID NO:2 or a naturally
occurring ortholog thereof) retains one of more of the biological
properties associated with the full-length PRDT1 polypeptide, such
as signaling activity, binding activity, catalytic activity, or
cellular or extra-cellular localizing activity. Some preferred
PRDT1 polypeptides display DNA binding activity. A PRDT1 fragment
preferably comprises a PRDT1 domain, such as a C- or N-terminal or
catalytic domain, among others, and preferably comprises at least
10, preferably at least 20, more preferably at least 25, and most
preferably at least 50 contiguous amino acids of a PRDT1 protein.
Functional domains can be identified using the PFAM program
(Bateman A et al., 1999 Nucleic Acids Res 27:260-262; website at
pfam.wustl.edu). A preferred PRDT1 fragment comprises a SANT domain
(SM00395) identified by PFAM, at approximately amino acids 8-60.
Functionally active variants of full-length PRDT1 polypeptides or
fragments thereof include polypeptides with amino acid insertions,
deletions, or substitutions that retain one of more of the
biological properties associated with the full-length PRDT1
polypeptide. In some cases, variants are generated that change the
post-translational processing of a PRDT1 polypeptide. For instance,
variants may have altered protein transport or protein localization
characteristics or altered protein half-life compared to the native
polypeptide.
[0018] As used herein, the term "PRDT1 nucleic acid" encompasses
nucleic acids with the sequence provided in or complementary to the
sequence provided in SEQ ID NO:1, as well as functionally active
fragments, derivatives, or orthologs thereof. A PRDT1 nucleic acid
of this invention may be DNA, derived from genomic DNA or cDNA, or
RNA.
[0019] In one embodiment, a functionally active PRDT1 nucleic acid
encodes or is complementary to a nucleic acid that encodes a
functionally active PRDT1 polypeptide. Included within this
definition is genomic DNA that serves as a template for a primary
RNA transcript (i.e., an mRNA precursor) that requires processing,
such as splicing, before encoding the functionally active PRDT1
polypeptide. A PRDT1 nucleic acid can include other non-coding
sequences, which may or may not be transcribed; such sequences
include 5' and 3' UTRs, polyadenylation signals and regulatory
sequences that control gene expression, among others, as are known
in the art. Some polypeptides require processing events, such as
proteolytic cleavage, covalent modification, etc., in order to
become fully active. Accordingly, functionally active nucleic acids
may encode the mature or the pre-processed PRDT1 polypeptide, or an
intermediate form. A PRDT1 polynucleotide can also include
heterologous coding sequences, for example, sequences that encode a
marker included to facilitate the purification of the fused
polypeptide, or a transformation marker.
[0020] In another embodiment, a functionally active PRDT1 nucleic
acid is capable of being used in the generation of loss-of-function
pathogen resistance or drought tolerance phenotypes, for instance,
via antisense suppression, co-suppression, etc.
[0021] In one preferred embodiment, a PRDT1 nucleic acid used in
the methods of this invention comprises a nucleic acid sequence
that encodes or is complementary to a sequence that encodes a PRDT1
polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%
or more sequence identity to the polypeptide sequence presented in
SEQ ID NO:2.
[0022] In another embodiment a PRDT1 polypeptide of the invention
comprises a polypeptide sequence with at least 50% or 60% identity
to the PRDT1 polypeptide sequence of SEQ ID NO:2, and may have at
least 70%, 80%, 85%, 90% or 95% or more sequence identity to the
PRDT1 polypeptide sequence of SEQ ID NO:2. In another embodiment, a
PRDT1 polypeptide comprises a polypeptide sequence with at least
50%, 60%, 70%, 80%, 85%, 90% or 95% or more sequence identity to a
functionally active fragment of the polypeptide presented in SEQ ID
NO:2, such as a SANT domain. In yet another embodiment, a PRDT1
polypeptide comprises a polypeptide sequence with at least 50%,
60%, 70%, 80%, or 90% identity to the polypeptide sequence of SEQ
ID NO:2 over its entire length and comprises a SANT domain.
[0023] In another aspect, a PRDT1 polynucleotide sequence is at
least 50% to 60% identical over its entire length to the PRDT1
nucleic acid sequence presented as SEQ ID NO:1, or nucleic acid
sequences that are complementary to such a PRDT1 sequence, and may
comprise at least 70%, 80%, 85%, 90% or 95% or more sequence
identity to the PRDT1 sequence presented as SEQ ID NO:1 or a
functionally active fragment thereof, or complementary
sequences.
[0024] As used herein, "percent (%) sequence identity" with respect
to a specified subject sequence, or a specified portion thereof, is
defined as the percentage of nucleotides or amino acids in the
candidate derivative sequence identical with the nucleotides or
amino acids in the subject sequence (or specified portion thereof),
after aligning the sequences and introducing gaps, if necessary to
achieve the maximum percent sequence identity, as generated by the
program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1990)
215:403410; website at blast.wustl.edu/blast/README.- html) with
search parameters set to default values. The HSP S and HSP S2
parameters are dynamic values and are established by the program
itself depending upon the composition of the particular sequence
and composition of the particular database against which the
sequence of interest is being searched. A "% identity value" is
determined by the number of matching identical nucleotides or amino
acids divided by the sequence length for which the percent identity
is being reported. "Percent (%) amino acid sequence similarity" is
determined by doing the same calculation as for determining % amino
acid sequence identity, but including conservative amino acid
substitutions in addition to identical amino acids in the
computation. A conservative amino acid substitution is one in which
an amino acid is substituted for another amino acid having similar
properties such that the folding or activity of the protein is not
significantly affected. Aromatic amino acids that can be
substituted for each other are phenylalanine, tryptophan, and
tyrosine; interchangeable hydrophobic amino acids are leucine,
isoleucine, methionine, and valine; interchangeable polar amino
acids are glutamine and asparagine; interchangeable basic amino
acids are arginine, lysine and histidine; interchangeable acidic
amino acids are aspartic acid and glutamic acid; and
interchangeable small amino acids are alanine, serine, threonine,
cysteine and glycine.
[0025] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of SEQ ID NO:1. The stringency of hybridization can be
controlled by temperature, ionic strength, pH, and the presence of
denaturing agents such as formamide during hybridization and
washing. Conditions routinely used are well known (see, e.g.,
Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John
Wiley & Sons, Publishers (1994); Sambrook et al., supra). In
some embodiments, a nucleic acid molecule of the invention is
capable of hybridizing to a nucleic acid molecule containing the
nucleotide sequence of SEQ ID NO:1 under stringent hybridization
conditions that comprise: prehybridization of filters containing
nucleic acid for 8 hours to overnight at 65.degree. C. in a
solution comprising 6.times. single strength citrate (SSC)
(1.times.SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5.times.
Denhardt's solution, 0.05% sodium pyrophosphate and 100 .mu.g/ml
herring sperm DNA; hybridization for 18-20 hours at 65.degree. C.
in a solution containing 6.times.SSC, 1.times. Denhardt's solution,
100 .mu.g/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing
of filters at 65.degree. C. for 1 h in a solution containing
0.2.times.SSC and 0.1% SDS (sodium dodecyl sulfate). In other
embodiments, moderately stringent hybridization conditions are used
that comprise: pretreatment of filters containing nucleic acid for
6 h at 40.degree. C. in a solution containing 35% formamide,
5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%
Ficoll, 1% BSA, and 500 .mu.g/ml denatured salmon sperm DNA;
hybridization for 18-20 h at 40.degree. C. in a solution containing
35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA,
0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA,
and 10% (wt/vol) dextran sulfate; followed by washing twice for 1
hour at 55.degree. C. in a solution containing 2.times.SSC and 0.1%
SDS. Alternatively, low stringency conditions can be used that
comprise: incubation for 8 hours to overnight at 37 .degree. C. in
a solution comprising 20% formamide, 5 .times.SSC, 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured sheared salmon sperm DNA;
hybridization in the same buffer for 18 to 20 hours; and washing of
filters in 1 .times.SSC at about 37.degree. C. for 1 hour.
[0026] As a result of the degeneracy of the genetic code, a number
of polynucleotide sequences encoding a PRDT1 polypeptide can be
produced. For example, codons may be selected to increase the rate
at which expression of the polypeptide occurs in a particular host
species, in accordance with the optimum codon usage dictated by the
particular host organism (see, e.g., Nakamura Y et al, Nucleic
Acids Res (1999) 27:292). Such sequence variants may be used in the
methods of this invention.
[0027] The methods of the invention may use orthologs of the
Arabidopsis PRDT1. Methods of identifying the orthologs in other
plant species are known in the art. Normally, orthologs in
different species retain the same function, due to presence of one
or more protein motifs and/or 3-dimensional structures. In
evolution, when a gene duplication event follows speciation, a
single gene in one species, such as Arabidopsis, may correspond to
multiple genes (paralogs) in another. As used herein, the term
"orthologs" encompasses paralogs. When sequence data is available
for a particular plant species, orthologs are generally identified
by sequence homology analysis, such as BLAST analysis, usually
using protein bait sequences. Sequences are assigned as a potential
ortholog if the best hit sequence from the forward BLAST result
retrieves the original query sequence in the reverse BLAST (Huynen
M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A
et al., Genome Research (2000) 10:1204-1210). Programs for multiple
sequence alignment, such as CLUSTAL (Thompson J D et al, 1994,
Nucleic Acids Res 22:4673-4680) may be used to highlight conserved
regions and/or residues of orthologous proteins and to generate
phylogenetic trees. In a phylogenetic tree representing multiple
homologous sequences from diverse species (e.g., retrieved through
BLAST analysis), orthologous sequences from two species generally
appear closest on the tree with respect to all other sequences from
these two species. Structural threading or other analysis of
protein folding (e.g., using software by ProCeryon, Biosciences,
Salzburg, Austria) may also identify potential orthologs. Nucleic
acid hybridization methods may also be used to find orthologous
genes and are preferred when sequence data are not available.
Degenerate PCR and screening of cDNA or genomic DNA libraries are
common methods for finding related gene sequences and are well
known in the art (see, e.g., Sambrook, supra; Dieffenbach and
Dveksler (Eds.) PCR Primer: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, NY, 1989). For instance, methods for generating a
cDNA library from the plant species of interest and probing the
library with partially homologous gene probes are described in
Sambrook et al. A highly conserved portion of the Arabidopsis PRDT1
coding sequence may be used as a probe. PRDT1 ortholog nucleic
acids may hybridize to the nucleic acid of SEQ ID NO:1 under high,
moderate, or low stringency conditions. After amplification or
isolation of a segment of a putative ortholog, that segment may be
cloned and sequenced by standard techniques and utilized as a probe
to isolate a complete cDNA or genomic clone. Alternatively, it is
possible to initiate an EST project to generate a database of
sequence information for the plant species of interest. In another
approach, antibodies that specifically bind known PRDT1
polypeptides are used for ortholog isolation. Western blot analysis
can determine that a PRDT1 ortholog (i.e., an orthologous protein)
is present in a crude extract of a particular plant species. When
reactivity is observed, the sequence encoding the candidate
ortholog may be isolated by screening expression libraries
representing the particular plant species. Expression libraries can
be constructed in a variety of commercially available vectors,
including lambda gt11, as described in Sambrook, et al., supra.
Once the candidate ortholog(s) are identified by any of these
means, candidate orthologous sequence are used as bait (the
"query") for the reverse BLAST against sequences from Arabidopsis
or other species in which PRDT1 nucleic acid and/or polypeptide
sequences have been identified.
[0028] PRDT1 nucleic acids and polypeptides may be obtained using
any available method. For instance, techniques for isolating cDNA
or genomic DNA sequences of interest by screening DNA libraries or
by using polymerase chain reaction (PCR), as previously described,
are well known in the art. Alternatively, nucleic acid sequence may
be synthesized. Any known method, such as site directed mutagenesis
(Kunkel T A et al., Methods Enzymol. (1991) 204:125-39), may be
used to introduce desired changes into a cloned nucleic acid.
[0029] In general, the methods of the invention involve
incorporating the desired form of the PRDT1 nucleic acid into a
plant expression vector for transformation of in plant cells, and
the PRDT1 polypeptide is expressed in the host plant.
[0030] An isolated PRDT1 nucleic acid molecule is other than in the
form or setting in which it is found in nature and is identified
and separated from least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
PRDT1 nucleic acid. However, an isolated PRDT1 nucleic acid
molecule includes PRDT1 nucleic acid molecules contained in cells
that ordinarily express PRDT1 where, for example, the nucleic acid
molecule is in a chromosomal location different from that of
natural cells.
[0031] Generation of Transgenic Plants with a PRDT1 Phenotype
[0032] PRDT1 nucleic acids and polypeptides may be used in the
generation of transgenic plants having a modified pathogen
resistance phenotype; in general, improved resistance phenotypes
are of interest. Pathogenic infection may affect seeds, fruits,
blossoms, foliage, stems, tubers, roots, etc. Accordingly,
resistance may be observed in any part of the plant. In a preferred
embodiment, altered expression of the PRDT1 gene in a plant is used
to generate plants with increased resistance to P. parasitica. In a
further preferred embodiment, plants that mis-express PRDT1 may
also display altered resistance to other pathogens. Other oomycete
pathogens of interest include Pythium spp, Phytophthora spp, Bremia
lactucae, Peronosclerospora spp., Pseudoperonospora. Sclerophthora
macrospora, Sclerospora graminicola, Plasmopara viticola, and
Albugo candidia. Fungal pathogens of interest include Altemaria
brassicicola, Botrytis cinerea, Erysiphe cichoracearum, Fusarium
oxysporum, Plasmodiophora brassica, Rhizoctonia solani,
Colletotrichum coccode, Sclerotinia spp., Aspergillus spp.,
Penicillium spp., Ustilago spp., and Tilletia spp. Bacterial
pathogens of interest include Agrobacterium tumefaciens, Erwinia
tracheiphila, Erwinia stewartii, Xanthomonas phaseoli , Erwinia
amylovora, Erwinia carotovora, Pseudomonas syringae, Pelargonium
spp, Pseudomonas cichorii, Xanthomonasfragariae, Pseudomonas
morsprunorum, Xanthomonas campestris.
[0033] PRDT1 nucleic acids and polypeptides may also be used in the
generation of transgenic plants having a modified, preferably an
improved drought tolerance phenotype. Such plants may further
display increased tolerance to other abiotic stresses, particular
salt-stress and freezing, as responses to these stresses and
drought stress are mediated by ABA (Thomashow, 1999 Annu. Revl
Plant Physiol. Plant Mol. Biol 50: 571; Cushman and Bohnert, 2000,
Curr. Opin. Plant Biol. 3: 117; Kang et al. 2002, Plant Cell
14:343-357; Quesada et al. 2000, Genetics 154: 421; Kasuga et al.
1999, Nature Biotech. 17: 287-291).
[0034] The methods described herein are generally applicable to all
plants. Although activation tagging and gene identification is
carried out in Arabidopsis, the PRDT1 gene (or an ortholog, variant
or fragment thereof) may be expressed in any type of plant. In
preferred embodiments, the invention is directed to crops including
maize, soybean, cotton, rice, wheat, barley, tomato, canola,
turfgrass, sugar beets, onions, beans, and flax. Other crops
include alfalfa, tobacco, and other forage crops. The invention may
also be directed to fruit- and vegetable-bearing plants, plants
used in the cut flower industry, grain-producing plants,
oil-producing plants, and nut-producing plants, among others.
[0035] The skilled artisan will recognize that a wide variety of
transformation techniques exist in the art, and new techniques are
continually becoming available. Any technique that is suitable for
the target host plant can be employed within the scope of the
present invention. For example, the constructs can be introduced in
a variety of forms including, but not limited to as a strand of
DNA, in a plasmid, or in an artificial chromosome. The introduction
of the constructs into the target plant cells can be accomplished
by a variety of techniques, including, but not limited to
Agrobacterium-mediated transformation, electroporation,
microinjection, microprojectile bombardment calcium-phosphate-DNA
co-precipitation or liposome-mediated transformation of a
heterologous nucleic acid. The transformation of the plant is
preferably permanent, i.e. by integration of the introduced
expression constructs into the host plant genome, so that the
introduced constructs are passed onto successive plant generations.
Depending upon the intended use, a heterologous nucleic acid
construct comprising a PRDT1 polynucleotide may encode the entire
protein or a biologically active portion thereof.
[0036] In one embodiment, binary Ti-based vector systems may be
used to transfer polynucleotides. Standard Agrobacterium binary
vectors are known to those of skill in the art, and many are
commercially available (e.g., pBI121 Clontech Laboratories, Palo
Alto, Calif.).
[0037] The optimal procedure for transformation of plants with
Agrobacterium vectors will vary with the type of plant being
transformed. Exemplary methods for Agrobacterium-mediated
transformation include transformation of explants of hypocotyl,
shoot tip, stem or leaf tissue, derived from sterile seedlings
and/or plantlets. Such transformed plants may be reproduced
sexually, or by cell or tissue culture. Agrobacterium
transformation has been previously described for a large number of
different types of plants and methods for such transformation may
be found in the scientific literature.
[0038] Expression (including transcription and translation) of
PRDT1 may be regulated with respect to the level of expression, the
tissue type(s) where expression takes place and/or developmental
stage of expression. A number of heterologous regulatory sequences
(e.g., promoters and enhancers) are available for controlling the
expression of a PRDT1 nucleic acid. These include constitutive,
inducible and regulatable promoters, as well as promoters and
enhancers that control expression in a tissue- or temporal-specific
manner. Exemplary constitutive promoters include the raspberry E4
promoter (U.S. Pat. Nos. 5,783,393 and 5,783,394), the 35S CaMV
(Jones J D et al, Transgenic Res (1992) 1:285-297), the CsVMV
promoter (Verdaguer B et al., Plant Mol Biol (1998) 37:1055-1067)
and the melon actin promoter (published PCT application WO0056863).
Exemplary tissue-specific promoters include the tomato E4 and E8
promoters (U.S. Pat. No. 5,859,330) and the tomato 2AII gene
promoter (Van Haaren M J J et al., Plant Mol Bio (1993)
21:625-640). In one preferred embodiment, PRDT1 expression is under
the control of a pathogen-inducible promoter (Rushton et al., The
Plant Cell (2002) 14:749-762). In another preferred embodiment,
PRDT1 expression is under control of regulatory sequences from
genes whose expression is associated with drought stress. For
example, when the promoter of the drought stress responsive
Arabidopsis rd29A gene was used to drive expression of DREB1A,
Arabidopsis plants were more tolerant to drought, salt and freezing
stress and did not have the stunted stature associated with plants
over-expressing the DREB1A gene from the CaMV 35S promoter (Kasuga
et al, 1999 Nature Biotech 17: 287). Promoters from other
Arabidopsis genes that are responsive to drought stress, such as
COR47 (Welin et al. 1995, Plant Mol. Biol. 29: 391), KIN1 (Kurkela
and Franck, 1990, Plant Mol. Biol. 15: 137), RD22BP (Abe et al.
1997, Plant Cell 9, 1859), ABA1 (Accession Number AAG17703), and
ABA3 (Xiong et al. 2001, Plant Cell 13: 2063), could be used.
Promoters from drought stress inducible genes in other species
could be used also. Examples are the rab17, ZmFer1 and ZmFer2 genes
from maize (Bush et al, 1997 Plant J 11:1285; Fobis-Loisy, 1995 Eur
J Biochem 231:609), the tdi-65 gene from tomato (Harrak, 2001
Genome 44:368), the His1 gene of tobacco (Wei and O'Connell, 1996
Plant Mol Biol 30:255), the Vupat1 gene from cowpea (Matos, 2001
FEBS Lett 491:188), and CDSP34 from Solanum tuberosum (Gillet et
al, 1998 Plant J 16:257).
[0039] In yet another aspect, in some cases it may be desirable to
inhibit the expression of endogenous PRDT1 in a host cell.
Exemplary methods for practicing this aspect of the invention
include, but are not limited to antisense suppression (Smith, et
al., Nature (1988) 334:724-726; van der Krol et al., Biotechniques
(1988) 6:958-976); co-suppression (Napoli, et al., Plant Cell
(1990) 2:279-289); ribozymes (PCT Publication WO 97/10328); and
combinations of sense and antisense (Waterhouse, et al., Proc.
Natl. Acad. Sci. USA (1998) 95:13959-13964). Methods for the
suppression of endogenous sequences in a host cell typically employ
the transcription or transcription and translation of at least a
portion of the sequence to be suppressed. Such sequences may be
homologous to coding as well as non-coding regions of the
endogenous sequence. Antisense inhibition may use the entire cDNA
sequence (Sheehy et al., Proc. Natl. Acad. Sci. USA (1988)
85:8805-8809), a partial cDNA sequence including fragments of 5'
coding sequence, (Cannon et al., Plant Molec. Biol. (1990)
15:3947), or 3' non-coding sequences (Ch'ng et al., Proc. Natl.
Acad. Sci. USA (1989) 86:10006-10010). Cosuppression techniques may
use the entire cDNA sequence (Napoli et al., supra; van der Krol et
al., The Plant Cell (1990) 2:291-299), or a partial cDNA sequence
(Smith et al., Mol. Gen. Genetics (1990) 224:477481).
[0040] Standard molecular and genetic tests may be performed to
further analyze the association between a gene and an observed
phenotype. Exemplary techniques are described below.
[0041] 1. DNA/RNA Analysis
[0042] The stage- and tissue-specific gene expression patterns in
mutant versus wild-type lines may be determined, for instance, by
in situ hybridization. Analysis of the methylation status of the
gene, especially flanking regulatory regions, may be performed.
Other suitable techniques include overexpression, ectopic
expression, expression in other plant species and gene knock-out
(reverse genetics, targeted knock-out, viral induced gene silencing
[VIGS, see Baulcombe D, (1999) Arch Virol Suppl 15:189-201]).
[0043] In a preferred application expression profiling, generally
by microarray analysis, is used to simultaneously measure
differences or induced changes in the expression of many different
genes. Techniques for microarray analysis are well known in the art
(Schena M et al., Science (1995) 270:467-470; Baldwin D et al., Cur
Opin Plant Biol. (1999) 2(2):96-103; Dangond F, Physiol Genomics
(2000) 2:53-58; van Hal N L et al., J Biotechnol (2000) 78:271-280;
Richmond T and Somerville S, Curr Opin Plant Biol (2000)
3:108-116). Expression profiling of individual tagged lines may be
performed. Such analysis can identify other genes that are
coordinately regulated as a consequence of the overexpression of
the gene of interest, which may help to place an unknown gene in a
particular pathway.
[0044] 2. Gene Product Analysis
[0045] Analysis of gene products may include recombinant protein
expression, antisera production, immunolocalization, biochemical
assays for catalytic or other activity, analysis of phosphorylation
status, and analysis of interaction with other proteins via yeast
two-hybrid assays.
[0046] 3. Pathway Analysis
[0047] Pathway analysis may include placing a gene or gene product
within a particular biochemical, metabolic or signaling pathway
based on its mis-expression phenotype or by sequence homology with
related genes. Alternatively, analysis may comprise genetic crosses
with wild-type lines and other mutant lines (creating double
mutants) to order the gene in a pathway, or determining the effect
of a mutation on expression of downstream "reporter" genes in a
pathway.
[0048] Generation of Mutated Plants with a PRDT1 Phenotype
[0049] The invention further provides a method of identifying
plants that have mutations in endogenous PRDT1 that confer
increased pathogen resistance and drought tolerance, and generating
pathogen-resistant and drought-tolerant progeny of these plants
that are not genetically modified. In one method, called "TILLING"
(for targeting induced local lesions in genomes), mutations are
induced in the seed of a plant of interest, for example, using EMS
treatment. The resulting plants are grown and self-fertilized, and
the progeny are used to prepare DNA samples. PRDT 1-specific PCR
are used to identify whether a mutated plant has a PRDT1 mutation.
Plants having PRDT1 mutations may then be tested for the PRDT1
phenotype, or alternatively, plants may be tested for the PRDT1
phenotype, and then PRDT1-specific PCR is used to determine whether
a plant having increased pathogen resistance and drought tolerance
has a mutated PRDT1 gene. TILLING can identify mutations that may
alter the expression of specific genes or the activity of proteins
encoded by these genes (see Colbert et al (2001) Plant Physiol
126:480-484; McCallum et al (2000) Nature Biotechnology
18:455-457).
[0050] In another method, a candidate gene/Quantitative Trait Locus
(QTLs) approach can be used in a marker-assisted breeding program
to identify alleles of or mutations in the PRDT1 gene or orthologs
of PRDT1 that may confer the PRDT1 phenotype (see Foolad et al.,
Theor Appl Genet. (2002) 104(6-7):945-958; Rothan et al., Theor
Appl Genet (2002) 105(1):145-159); Dekkers and Hospital, Nat Rev
Genet. (2002) Jan;3(1):22-32). Thus, in a further aspect of the
invention, a PRDT1 nucleic acid is used to identify whether a plant
having increased pathogen resistance and/or drought tolerance has a
mutation in endogenous PRDT1 or has a particular allele that causes
the increased pathogen resistance and/or drought tolerance.
[0051] All publications cited herein are expressly incorporated
herein by reference for the purpose of describing and disclosing
compositions and methodologies that might be used in connection
with the invention. All cited patents, patent applications, and
sequence information in referenced websites and public databases
are also incorporated by reference.
EXAMPLES
Example 1
[0052] Activation Tagging in Arabidopsis
[0053] Mutants were generated using the activation tagging "ACTTAG"
vector, pSKI015 (GI 6537289; Weigel D et al., 2000). Standard
methods were used for the generation of Arabidopsis transgenic
plants, and were essentially as described in published application
PCT WO0183697. Briefly, T0 Arabidopsis (Col-0) plants were
transformed with Agrobacterium carrying the pSKI015 vector, which
comprises T-DNA derived from the Agrobacterium Ti plasmid, an
herbicide resistance selectable marker gene, and the 4.times.CaMV
35S enhancer element. Transgenic plants were selected at the T1
generation based on herbicide resistance. T2 seed was collected
from T1 plants and stored in an indexed collection, and a portion
of the T2 seed was accessed for the screen.
Example 2
[0054] Pathogen Resistance Screen
[0055] Approximately 18 T2 seeds from each of the greater than
40,00 lines generated in Example 1, were planted in soil. The seed
were stratified for three days and then grown in the greenhouse for
seven days. The seedlings were inoculated with approximately
1.times.10.sup.5 conidia per ml P. parasitica spores and incubated
in a dew room at 18.degree. C. and 100% humidity for 24 hours. The
plants were then moved to a growth room at 20.degree. C. and 60%
relative humidity with ten-hour long light period for six days.
Individual plants were evaluated for the presence or absence of
conidiophores on cotyledons. Lines in which at least a single plant
showed no conidiophore growth were re-tested in a secondary screen
by releasing three sets of 18 seed and screening for resistance to
P. parasitica growth as before.
[0056] Lines in which a significant number of plants showed no
conidiophores after infection were subjected to a tertiary screen.
Approximately 54 T2 seed were released, planted individually and
infected with P. parasitica as before. The plants were evaluated
for the number of conidiophores growing on a single cotyledon and
ranked by the following scoring system: a score of 0 indicates 0
conidiophores per cotyledon, 1 indicates 1-5 conidiophores per
cotyledon, 2 indicates 6-10 conidiophores per cotyledon, 3
indicates 11-20 conidiophores per cotyledon, and 4 indicates
greater than 20 conidiophores per cotyledon
[0057] The ACTTAG line designated W000058335 was identified as
having an increased resistance phenotype. Specifically, 15.2% of
individual plants showed no conidiophores in the secondary screen.
In the tertiary screen, 31 plants scored as 0 (39.2%), 31 as 1
(39.2%), 4 as 2 (5.1%), 10 as 3 (12.7%)and 3 as 4 (3.8%). Control
wild-type Col-0 plants were more susceptible; 36 plants scored 0
(7.6%), 21 as 1 (4.4%), 79 as 2 (16.6%), 250 as 3 (52.5%) and 90 as
4 (18.9%).
[0058] We performed standard molecular analyses, essentially as
described in patent application PCT WO0183697, to determine the
site of the T-DNA insertion associated with the increased pathogen
resistance phenotype. Briefly, genomic DNA was extracted from
plants exhibiting increased pathogen resistance. PCR, using primers
specific to the pSKI015 vector, confirmed the presence of the 35S
enhancer in plants from line W000058335, and Southern blot analysis
verified the genomic integration of the ACTTAG T-DNA and showed the
presence of a single T-DNA insertion in the transgenic line.
[0059] Plasmid rescue and inverse PCR were used to recover genomic
DNA flanking the T-DNA insertion, which was then subjected to
sequence analysis.
[0060] The sequence flanking the right T-DNA border was subjected
to a basic BLASTN search and/or a search of the Arabidopsis
Information Resource (TAIR) database (available at the
arabidopsis.org website), which revealed sequence identity to BAC
F22H5, (GI 12331602), mapped to chromosome 1. The junction of the
left border of the T-DNA is at nt 20167 of F22H5, and the right
border junction is at nt 20229. Sequence analysis revealed that the
T-DNA had inserted in the vicinity (ie., within about 10 kb) of the
gene whose nucleotide sequence is presented as SEQ ID NO: 1 (GI
18410812 and GI 12331602, nucleotides 20955-21335) and which we
designated PRDT1. Specifically, the right border was approximately
500 bp upstream of the start codon of SEQ ID NO:1.
Example 3
[0061] Drought Tolerance Screen
[0062] Drought stress was imposed on transgenic plants generated in
Example 1 by withholding water for 21-25 days. At this time the
plants were beginning to bolt. From 6 to 10 days after the
initiation of drought stress, observations of the plants were taken
daily. Transgenic plants were compared to each other and wild-type
control plants. Putative drought tolerant lines were identified as
containing at least 2 plants that remained green and viable after
wild-type plants had died or as plants that contained a soil
moisture content of at least 50 mV (using a Delt-T Devices HH2 Soil
Moisture Meter with a ML2x Theta Probe).
[0063] After drought tolerant lines were identified, water was
applied to the plants to allow them to recover. When possible, T3
seed was collected from the plants. This T3 seed was then grown and
the plants assessed for drought tolerant phenotype as described
above.
[0064] The drought tolerant plants were then subjected to an
"excised leaf transpiration test" in which seeds were planted,
stratified, and grown for three weeks as described above. Then,
either the entire rosette or a single leaf was excised and placed
on a pre-weighed plastic weigh dish and left on the bench at room
temperature. The mass of the plant material was recorded
immediately after excision and at 30 min intervals afterward. The
mass of drought tolerant plants often decreased less rapidly
indicating that they were transpiring less rapidly.
[0065] To detect lines containing or lacking the insert, PCR
analysis was performed using a set of DNA oligonucleotide primers;
one that hybridizes to sequences in pSKI015, the other that
hybridizes to sequences flanking the insert. Genotyping of
individuals analyzed drought tolerance experiments indicated that
plants containing the insert identified in plant line W000114956
were more tolerant of drought stress than plants without the
insert.
[0066] The same methods described in Example 2 were used to
determine the site of the T-DNA insertion associated with the
increased pathogen resistance phenotype. Sequence analysis revealed
that the T-DNA had inserted 423 base pairs from the start codon in
At1g75250, which is the same gene that was responsible for the
pathogen resistance phenotype of plant line W000058335, as
described in Example 2.
Example 4
[0067] Analysis of Arabidopsis PRDT1 Sequence
[0068] The amino acid sequence predicted from the PRDT1 nucleic
acid sequence is presented in SEQ ID NO:2 and GI 10092271. Sequence
analyses were performed with BLAST (Altschul et al., 1997, J. Mol.
Biol. 215:403-410), PFAM (Bateman et al., 1999), PSORT (Nakai K,
and Horton P, 1999, Trends Biochem Sci 24:34-6), and CLUSTALW
(Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680), among
others. The PRDT1 protein has been characterized as a myb-related
protein. PFAM analysis indicated a SANT DNA-binding domain at
approximately amino acids 8-60.
[0069] The retroviral oncogene v-myb, and its cellular counterpart
c-myb, encode nuclear DNA-binding proteins (Klempnauer and Sippel,
1987, EMBO J. 6: 2719-2725; Biednkapp et al. 1988, Nature 335:
835-837). These belong to the SANT domain family that specifically
recognize the sequence YAAC(G/T)G (Aasland et al. 1996, Trends
Biochem. Sci. 21:87-88). In myb, one of the most conserved regions
consisting of three tandem repeats has been shown to be involved in
DNA-binding.
[0070] Analysis using BLASTP or TBLASTN identified a number of
related proteins and proteins predicted from nucleic acid
(generally EST) sequences in other plant species. Related
sequences, which are candidate orthologs, are presented in SEQ ID
NOs 3-14 and descriptions from GenBank are provided below:
[0071] SEQ ID NO:3 translation,
gi.vertline.887283.vertline.gb.vertline.L3- 8243.1.vertline.L38243
BNAF0581E Mustard flower buds Brassica rapa cDNA-ORF 98aa Brassica
rapa
[0072] SEQ ID NO:4 translation,
gi.vertline.18459015.vertline.gb.vertline.-
BM437293.1.vertline.BM437293 VVA017C08.sub.--54081 An expressed
sequence tag database for abiotic st 75aa Vitis vinifera
[0073] SEQ ID NO:5 translation,
gi.vertline.15288211.vertline.gb.vertline.-
BI472102.1.vertline.BI472102 sah99e03.y1 Gm-c1050 Glycine max cDNA
clone GENOME SYSTEMS CLONE 97aa Glycine max
[0074] SEQ ID NO:6 translation,
gi.vertline.15258392.vertline.gb.vertline.-
BI433702.1.vertline.BI433702 EST536463 P. infestans-challenged leaf
Solanum tuberosum cDNA clo 88aa Solanum tuberosum
[0075] SEQ ID NO: translation,
gi.vertline.14492357.vertline.gb.vertline.B-
I071737.1.vertline.BI071737 C063P09U Populus strain T89 leaves
Populus tremula.times.Populus trem 71aa P o
[0076] SEQ ID NO:8 translation,
gi.vertline.7981380.vertline.emb.vertline.-
CAB91874.1.vertline.(AJ277944) myb-related protein [Lycopersicon
esculentum] 88aa Lycopersicon esculentum
[0077] SEQ ID NO:9
gi.vertline.5091605.vertline.gb.vertline.AAD39594.1.ver-
tline.AC007858.sub.--8 (AC007858) 10A19I.9 [Oryza sativa] 126aa
Oryza sativa
[0078] SEQ ID NO:10
gi.vertline.5091604.vertline.gb.vertline.AAD39593.1.ve-
rtline.AC007858.sub.--7 (AC007858) 10A19I.8 [Oryza sativa] 236aa
Oryza sativa
[0079] SEQ ID NO:11
gi.vertline.18394750.vertline.ref.vertline.NP.sub.--56-
4087.1.vertline.(NM.sub.--101808) myb-related protein, putative
[Arabidopsis thaliana] 92aa Arabidopsis thaliana
[0080] SEQ ID NO:12
gi.vertline.15226604.vertline.ref.vertline.NP.sub.--17-
9759.1.vertline.(NM.sub.--127736) unknown protein [Arabidopsis
thaliana].quadrature.gi.vertline.4567225.vertline.gb.vertline.AAD236
101aa Arabidopsis thaliana
[0081] SEQ ID NO:13
gi.vertline.15234999.vertline.ref.vertline.NP.sub.--19-
5636.1.vertline.(NM.sub.--120086) putative protein [Arabidopsis
thaliana].quadrature.gi.vertline.7487341.vertline.pir.vertline.T08
97aa Arabidopsis thaliana
[0082] SEQ ID NO: 14
gi.vertline.8778436.vertline.gb.vertline.AAF79444.1.v-
ertline.AC025808.sub.--26 F18O14.26 [Arabidopsis thaliana]
[0083] SEQ ID NO:15
gi.vertline.20161824.vertline.dbj.vertline.BAB90739.1.-
vertline.[20161824] P0663E10.22 [Oryza sativa (japonica
cultivar-group)]
[0084] SEQ ID NO:16
gi.vertline.19387259.vertline.gb.vertline.AAL87171.1.v-
ertline.AF480496.sub.--25[19387259] putative myb-related protein
[Oryza sativa (japonica cultivar-group)]
[0085] The predicted protein from an EST contig identified from
Lycopersicon esculentum was also identified as an ortholog having
58% sequence identity to SEQ ID NO:2, and is presented as SEQ ID
NO:17.
[0086] The translation of an EST contig from Solanum tuberosum,
presented as SEQ ID NO:18, was identified as having 53% sequence
identify to SEQ ID NO:1.
Examples5
[0087] Confirmation of Pathogen Resistance Phenotype/Genotype
Association
[0088] PCR analysis, using primers to sequences in pSKI015 or
flanking the insert, was used to detect lines containing or lacking
the insert. W000058335 individuals analyzed in the tertiary screen
were genotyped. Results indicated that plants that were homozygous
or hemizygous for the insert were more resistant to P. parasitica
infection than plants that were homozygous wild-type; 100% of the
plants homozygous for the insert and 97% of the plants hemizygous
for the insertion received resistance scores of 0 or 1 while only
31% of the wild-type segregants scored 0 or 1. These results
suggest that the P. parasitica resistance trait in W000058335 is
caused by the overexpression of PRDT1 and is inherited in a
dominant manner
[0089] RT-PCR analysis showed that the PRDT1 gene was overexpressed
in plants from the line displaying the P. parasitica resistance
phenotype. Specifically, RNA was extracted from tissues derived
from plants exhibiting the resistance phenotype and from wild type
COL-0 plants. RT-PCR was performed using primers specific to the
sequence presented as SEQ ID NO:1, to other predicted genes in the
vicinity of the T-DNA insertion (At1g75240, At1g75260, and
At1g75270), and to a constitutively expressed actin (positive
control). The results showed that plants displaying the PRDT1
phenotype over-expressed the mRNA for the PRDT1 gene, indicating
the enhanced expression of the PRDT1 gene is correlated with the
PRDT1 phenotype.
Example 6
[0090] Recapitulation of Pathogen Resistance Phenotype
[0091] The coding sequences of the PRDT1 gene were cloned behind
the strong constitutive CsVMV promoter and transformed into
wild-type Col-0 Arabidopsis thaliana plants. Primary transformants
(T1 plants) were selected and allowed to go to seed. Seed from
these plants were planted and evaluated for resistance to
Peronospora.
[0092] Approximately 54 individual seedlings from twenty different
primary transformants were planted, stratified and grown for 1 week
in a growth chamber. The 1 week old seedlings were inoculated with
Peronospora as described previously. After 1 week the seedlings
were scored for Peronospora infection by counting the number of
conidophores on a cotyledon. Plants with no conidiophores on a
cotyledon were scored 0, plants with 1-5 were scored 1, plants with
6-10 were scored 2, plants with 10-20 were scored 3 and plants with
more than 20 conidophores on a cotyledon were scored 4. Wild-type
Col-0 seedlings were included as a control. Six lines
over-expressing PRDT1 were significantly more resistant to
Peronospora than wild-type Col-0 plants. In the three most
resistant lines, over 70% of the plants scored 0 or 1 while in
wild-type Col-0 plants only 4% of the plants scored 0 or 1. These
results indicate that over-expression of PRDT1 in wild-type Col-0
confers resistance to Peronospora parasitica.
Example 7
[0093] Confirmation of Drought Tolerance Phenotype/Genotype
Association
[0094] RT-PCR analysis showed that the PRDT1 gene was specifically
overexpressed in plants from the line displaying the improved
drought tolerance phenotype. Specifically, RNA was extracted from
tissues derived from plants exhibiting the PRDT1 phenotype and from
wild type COL-0 plants. RT-PCR was performed using primers specific
to the sequence presented as SEQ ID NO:1 and to other predicted
genes in the vicinity of the T-DNA insertion. The results showed
that plants displaying the PRDT1 phenotype over-expressed the mRNA
for the PRDT1 gene by about 500 fold compared to wild type plants,
indicating that the enhanced expression of the PRDT1 gene is
correlated with the PRDT1 phenotype.
Example 8
[0095] Recapitulation of the Drought Tolerant Phenotype
[0096] The coding sequences of the PRDT1 gene were cloned behind
the strong constitutive CsVMV promoter and transformed into
wild-type Col-0 Arabidopsis thaliana plants. Primary transformants
(T1 plants) were selected and allowed to go to seed. Seed from
these plants were planted and evaluated for drought tolerance.
[0097] Approximately 18 seed from 2 T2 populations (PRDT1-1 and
PRDT1-2) transformed with the CsVMV:PRDT1 transgene were planted in
soil in 4 inch pots. The seed were stratified at 4.degree. C. for 3
days and then grown in the growth room for 4 weeks. Drought stress
was imposed on the plants by withholding water 28 days after
transfer to the growth room, at this time the plants were beginning
to bolt.
[0098] Observations on every individual plant in each pot were made
after the last watering (day 0), seven days after the last watering
(day 7), fourteen days after the last watering (day 14) and
seventeen days after the last watering (day 17). Plants that were
fully turgid were scored 0, plants that showed slight wilting were
scored 1, plants that showed significant wilting were scored 2 and
plants that were fully desiccated were scored 3.
[0099] PRDT1-1 and PRDT1-2 plants were significantly more tolerant
to drought than wild-type Col-0 plants. After seventeen days
without water, Col-0 plants had an average score of 2.7 indicating
that most plants were either significantly wilted or fully
desiccated. However, the average score of PRDT1-1 and PRDT1-2
plants was 0.1 and 0.4 respectively. These scores indicate that
most of the plants were fully turgid or slightly wilted. These
results indicate that overexpression of PRDT1 confers a drought
tolerant phenotype.
Sequence CWU 1
1
18 1 381 DNA Arabidopsis thaliana 1 atggcgtcaa actcaagaag
ttcaatctca ccatggacgt ttagtcaaaa caagatgttc 60 gagagggcct
tggcagttta cgacaaggac acacccgacc gatggcacaa tgtggcaaaa 120
gctgtcggag ggaaaactgt agaagaagtg aagcgccact atgacattct cgtcgaggat
180 ctcatcaaca tcgagactgg tcgtgtccct ttgcccaatt acaagacctt
cgaatctaac 240 tcaagaagca tcaatgactt tgacacaagg tatataacta
aatatctata tatgatgctc 300 tcgatatatt ttgataatca ttctagtgat
tttgagaaat tctctcaaaa agttcttgta 360 agttatattt ctttggttta a 381 2
126 PRT Arabidopsis thaliana 2 Met Ala Ser Asn Ser Arg Ser Ser Ile
Ser Pro Trp Thr Phe Ser Gln 1 5 10 15 Asn Lys Met Phe Glu Arg Ala
Leu Ala Val Tyr Asp Lys Asp Thr Pro 20 25 30 Asp Arg Trp His Asn
Val Ala Lys Ala Val Gly Gly Lys Thr Val Glu 35 40 45 Glu Val Lys
Arg His Tyr Asp Ile Leu Val Glu Asp Leu Ile Asn Ile 50 55 60 Glu
Thr Gly Arg Val Pro Leu Pro Asn Tyr Lys Thr Phe Glu Ser Asn 65 70
75 80 Ser Arg Ser Ile Asn Asp Phe Asp Thr Arg Tyr Ile Thr Lys Tyr
Leu 85 90 95 Tyr Met Met Leu Ser Ile Tyr Phe Asp Asn His Ser Ser
Asp Phe Glu 100 105 110 Lys Phe Ser Gln Lys Val Leu Val Ser Tyr Ile
Ser Leu Val 115 120 125 3 98 PRT Brassica rapa 3 Met Ala Ser Ser
Ser Met Ser Ser Ser Trp Thr Ser Lys Gln Asn Lys 1 5 10 15 Ile Phe
Glu Arg Ala Leu Ala Val Tyr Asp Lys Asp Thr Pro Asp Arg 20 25 30
Trp Gln Asn Val Ala Lys Ala Val Gly Asn Lys Ser Ala Glu Glu Val 35
40 45 Lys Arg His Tyr Asp Ile Leu Val Glu Asp Leu Met Asn Ile Glu
Gln 50 55 60 Asp Leu Val Pro Leu Pro Lys Tyr Lys Thr Val Asp Val
Gly Asn Lys 65 70 75 80 Ser Arg Gly Ile Asn Gly Tyr Gly Leu Arg Leu
Met Lys Asn Ile Glu 85 90 95 Val Gln 4 75 PRT Vitis vinifera 4 Met
Ala Ser Thr Ser Leu Lys Ser Ser Gly Ser Trp Thr Pro Lys Gln 1 5 10
15 Asn Lys Leu Phe Glu Lys Ala Leu Ala Leu Tyr Asp Arg Asp Thr Pro
20 25 30 Asp Arg Trp Gln Asn Val Ala Asn Ala Val Gly Gly Lys Ser
Ala Glu 35 40 45 Glu Val Lys Gln His Tyr Glu Ile Leu Ile Arg Asp
Leu Lys His Ile 50 55 60 Glu Ser Gly Arg Val Pro Ile Pro Asn Tyr
Lys 65 70 75 5 97 PRT Glycine max 5 Met Glu Ser Cys Ser Ile Ser Ala
Ser Gly Ser Trp Ser Val Lys Asp 1 5 10 15 Asn Lys Ala Phe Glu Lys
Ala Leu Ala Val Tyr Asp Lys Asp Thr Pro 20 25 30 Asp Arg Trp Tyr
Asn Val Ala His Ala Val Gly Gly Lys Thr Pro Glu 35 40 45 Glu Val
Lys Arg His Tyr Glu Leu Leu Val Gln Asp Val Lys His Ile 50 55 60
Glu Ser Gly Arg Val Pro Phe Pro Asn Tyr Lys Lys Thr Thr Ser Glu 65
70 75 80 Ser Thr Asp Gln Glu Glu Lys Arg Leu Arg Asn Leu Asn Leu
Asn Leu 85 90 95 Gln 6 88 PRT Solanum tuberosum 6 Met Ala Ser Ser
Ser Leu Gln Ser Ser Ser Trp Thr Pro Gln Gln Asn 1 5 10 15 Lys Leu
Phe Glu Arg Ala Leu Ala Gln Phe Asp Lys Asp Thr Pro Asp 20 25 30
Arg Trp Gln Asn Val Ala Arg Ala Val Gly Gly Gly Lys Ser Ala Asp 35
40 45 Glu Val Lys Arg His Tyr Glu Ile Leu Ile Glu Asp Leu Lys Arg
Ile 50 55 60 Glu Ser Gly Arg Val Pro Leu Pro Thr Tyr Thr His Glu
Gln Gln Arg 65 70 75 80 Leu Leu Arg Tyr Met Asn Leu His 85 7 71 PRT
Populus tremula 7 Met Ser Ser Ser His Gln Thr Pro Arg Asn Ser Ser
Ser Ser Trp Thr 1 5 10 15 Pro Arg Glu Asn Lys Leu Phe Glu Lys Ala
Leu Ala Leu Phe Asp Lys 20 25 30 Asp Thr Pro Asp Arg Trp Lys Asn
Val Ala Lys Ala Val Gly Gly Val 35 40 45 Lys Ser Ala Glu Glu Val
Lys Arg His Tyr Glu Ile Leu Ile Glu Asp 50 55 60 Leu Lys His Ile
Glu Pro Ala 65 70 8 88 PRT Lycopersicon esculentum 8 Met Ser Ser
Met Ser Ser Gln His Gly Ser Ser Gly Ser Trp Thr Ala 1 5 10 15 Lys
Gln Asn Lys Ala Phe Glu Lys Ala Leu Ala Val Tyr Asp Lys Glu 20 25
30 Thr Arg Asp Arg Trp Ser Asn Val Ala Lys Ala Val Gly Gly Lys Thr
35 40 45 Ala Glu Glu Val Lys Arg His Tyr Glu Ile Leu Leu Arg Asp
Val Phe 50 55 60 Phe Ile Asp Asn Gly Met Val Pro Phe Pro Lys Tyr
Lys Thr Thr Gly 65 70 75 80 Gly Ser His Asn Ser Thr Ser Asp 85 9
126 PRT Oryza sativa 9 Met Ala Ser Ala Ala Gly Ser Lys Gln Gln Gln
Ala Met Met Ser Leu 1 5 10 15 Pro Ser Ser Arg Gly Gly Gly Gly Gly
Gly Trp Thr Gln Arg Gln Asn 20 25 30 Lys Gln Phe Glu Cys Ala Leu
Ala Val Tyr Asp Lys Glu Thr Pro Asp 35 40 45 Arg Trp His Asn Ile
Ala Arg Tyr Met Gly Gly Ala Lys Ser Ala Asp 50 55 60 Glu Val Arg
Arg His Phe Asp His Leu Val Glu Asp Val Ser Arg Ile 65 70 75 80 Glu
Ser Gly Arg Val Pro Phe Pro Arg Tyr Ser Ser Ser Ser Ser Ser 85 90
95 Arg Gly Ala Asp Asp Gly Asn Arg Leu Leu Thr Val Phe His Leu Ser
100 105 110 Ser Val Pro Arg Thr Arg Asn Ala Asn His Lys Phe Asn Thr
115 120 125 10 236 PRT Oryza sativa 10 Met Ala Gln Gln Ala Arg Ala
Gln Trp Pro Gln Lys Gln Asn Lys Leu 1 5 10 15 Phe Glu Gln Ala Leu
Ala Val Tyr Asp Lys Glu Thr Pro Asp Arg Trp 20 25 30 His Asn Ile
Ala Arg Ala Val Gly Gly Gly Lys Ser Ala Glu Asp Val 35 40 45 Lys
Arg Tyr Tyr Glu Met Leu Glu Glu Asp Ile Lys His Ile Glu Ser 50 55
60 Gly Lys Val Pro Phe Pro Ala Tyr Arg Cys Pro Ala Ala Ala Gly Tyr
65 70 75 80 Gln Ala Glu Ser Arg Pro Ser Thr Ala Ala Glu Pro Ser Arg
Leu Pro 85 90 95 Leu Ser Asp Ser Gly Leu Ser Gly Ile Arg Pro Thr
Gln Tyr Pro Pro 100 105 110 Asp Gly Glu Leu Ser Pro Pro Arg His Arg
Leu Arg Arg Arg Gly Asn 115 120 125 Gln Pro Ile Pro Ser Tyr Lys Pro
Ser Pro Ser Arg Glu Gly Ile Phe 130 135 140 Tyr Trp Glu Val Val Val
Ala Ala Leu Lys Ser Arg Gly Thr Gly Ala 145 150 155 160 Thr Ser Thr
Pro Trp Ile Arg Leu Leu Leu Pro Gly Leu Thr Val Cys 165 170 175 Arg
Leu Leu Gly Ser Ser Gly Cys Phe Asp Ala Trp Met Leu Ser Thr 180 185
190 Ala Arg Leu Met Val Val Asn Thr Tyr Trp Met Ser Tyr Leu Thr Arg
195 200 205 Ser Pro Glu Phe His Leu Asn Phe Pro His Ile Asn Leu Arg
Lys Tyr 210 215 220 Glu Val Val Cys Val Gln Pro Gly Phe Met Gln Glu
225 230 235 11 92 PRT Arabidopsis thaliana 11 Met Ala Ser Ser Ser
Met Ser Ser Ser Ser Ser Trp Thr Ser Lys Gln 1 5 10 15 Asn Lys Met
Phe Glu Arg Ala Leu Ala Val Tyr Asp Lys Asp Thr Pro 20 25 30 Asp
Arg Trp Gln Asn Val Ala Lys Ala Val Gly Ser Lys Ser Ala Glu 35 40
45 Glu Val Lys Arg His Tyr Asp Ile Leu Val Glu Asp Leu Met Asn Ile
50 55 60 Glu Gln Asp Leu Val Pro Leu Pro Lys Tyr Lys Thr Val Asp
Val Gly 65 70 75 80 Ser Lys Ser Arg Gly Ile Asp Asp Phe Asp Leu Arg
85 90 12 101 PRT Arabidopsis thaliana 12 Met Ala Ser Gly Ser Met
Ser Ser Tyr Gly Ser Gly Ser Trp Thr Val 1 5 10 15 Lys Gln Asn Lys
Ala Phe Glu Arg Ala Leu Ala Val Tyr Asp Gln Asp 20 25 30 Thr Pro
Asp Arg Trp His Asn Val Ala Arg Ala Val Gly Gly Lys Thr 35 40 45
Pro Glu Glu Ala Lys Arg Gln Tyr Asp Leu Leu Val Arg Asp Ile Glu 50
55 60 Ser Ile Glu Asn Gly His Val Pro Phe Pro Asp Tyr Lys Thr Thr
Thr 65 70 75 80 Gly Asn Ser Asn Arg Gly Arg Leu Arg Asp Glu Glu Lys
Arg Met Arg 85 90 95 Ser Met Lys Leu Gln 100 13 97 PRT Arabidopsis
thaliana 13 Met Ala Ser Ser Ser Met Ser Ser Gln Ser Ser Gly Ser Trp
Thr Ala 1 5 10 15 Lys Gln Asn Lys Ala Phe Glu Gln Ala Leu Ala Thr
Tyr Asp Gln Asp 20 25 30 Thr Pro Asn Arg Trp Gln Asn Val Ala Lys
Val Val Gly Gly Lys Thr 35 40 45 Thr Glu Glu Val Lys Arg His Tyr
Glu Leu Leu Val Gln Asp Ile Asn 50 55 60 Ser Ile Glu Asn Gly His
Val Pro Phe Pro Asn Tyr Arg Thr Ser Gly 65 70 75 80 Gly Cys Thr Asn
Gly Arg Leu Ser Gln Glu Glu Lys Arg Tyr Val Leu 85 90 95 Ser 14 639
PRT Arabidopsis thaliana 14 Met Ala Ser Ser Ser Met Ser Ser Ser Ser
Ser Trp Thr Ser Lys Gln 1 5 10 15 Asn Lys Met Phe Glu Arg Ala Leu
Ala Val Tyr Asp Lys Asp Thr Pro 20 25 30 Asp Arg Trp Gln Asn Val
Ala Lys Ala Val Gly Ser Lys Ser Ala Glu 35 40 45 Glu Val Lys Arg
His Tyr Asp Ile Leu Val Glu Asp Leu Met Asn Ile 50 55 60 Glu Gln
Asp Leu Val Asn Glu Glu Tyr Glu Asn Pro Val Lys Leu Leu 65 70 75 80
His Asp Val Lys Ile Ala Ile Cys Leu Arg Ile Gln Arg Asp Met Met 85
90 95 Ala Lys Ile Ser Val Ala Val Leu Leu Ser Val Met Leu Leu Val
Ser 100 105 110 Ile Asn Ser Val Asp Ile Leu Ala Glu Glu Glu Pro Thr
Val Gly Gln 115 120 125 Arg Val Asp Ser Ala Met Thr Ser Val Thr Asp
Ala Phe Asn Glu His 130 135 140 Gly Gly Pro Gln Ala Val Asp Thr Val
Ser Ser Thr Phe Lys Ser Val 145 150 155 160 Tyr Gly Trp Phe Gly Asp
Lys Ala Lys Tyr Leu Glu Pro Ile Ser Ser 165 170 175 Ser Cys Cys Ser
Ser Ser Ser Ser Ser Ser Gly Glu Glu Asn Thr Ala 180 185 190 Ala Ala
Asn Met Thr Glu Met Glu Ala Ala Glu Ala Leu Ala Asp Leu 195 200 205
Ala Gln Leu Ala Ile Met Arg Glu Gln Val Phe Glu Ser Ala Ala Ser 210
215 220 Trp Gly Ser Lys Gly Lys Arg Val Arg Lys Arg Val Lys Thr Glu
Ser 225 230 235 240 Pro Pro Ser Asp Ser Leu Leu Lys Pro Pro Asp Ser
Asp Thr Leu Pro 245 250 255 Thr Pro Asp Leu Ala Glu Glu Arg Leu Val
Lys Glu Glu Glu Glu Glu 260 265 270 Glu Glu Val Glu Pro Ile Thr Lys
Glu Leu Thr Lys Ala Pro Val Lys 275 280 285 Ser Glu Ile Asn Gly Glu
Thr Pro Lys Pro Ile Leu Ala Ser Thr Leu 290 295 300 Ile Arg Cys Ser
Arg Ser Asn Gly Cys Gly Arg Ser Arg Gln Asn Leu 305 310 315 320 Ser
Glu Ala Glu Arg Glu Glu Arg Arg Ile Arg Arg Ile Leu Ala Asn 325 330
335 Arg Glu Ser Ala Arg Gln Thr Ile Arg Arg Arg Gln Ala Met Cys Glu
340 345 350 Glu Leu Ser Lys Lys Ala Ala Asp Leu Thr Tyr Glu Asn Glu
Asn Leu 355 360 365 Arg Arg Glu Lys Asp Trp Ala Leu Lys Glu Phe Gln
Ser Leu Glu Thr 370 375 380 Ile Asn Lys His Leu Lys Glu Gln Val Leu
Lys Ser Val Lys Pro Asp 385 390 395 400 Thr Lys Glu Pro Glu Glu Ser
Pro Lys Pro Ser Gln Val Glu Met Ser 405 410 415 Thr Ser Ser Thr Pro
Phe Tyr Phe Tyr Asn Gln Asn Pro Tyr Gln Leu 420 425 430 Phe Cys Trp
Pro His Val Thr Gln Ser Ser Asn Pro Met Ile Ser Pro 435 440 445 Leu
Glu Phe Pro Thr Ser Gly Gly Ala Ser Ala Lys Thr Ile Thr Thr 450 455
460 Gln Glu His Glu Asn Ala Ala Asp Asp Asn Gly Gln Lys Thr His Phe
465 470 475 480 Tyr Val Val Pro Cys Pro Trp Phe Leu Pro Pro Pro Asp
His Ser Asn 485 490 495 Gly Val Pro Phe Gly Leu Gln Asp Thr Gln Arg
Gly Thr Phe Ser Asn 500 505 510 Gly His His Ile Asp Asp Ser Ser Ala
Arg Pro Met Asp Val Thr Glu 515 520 525 Thr Pro Arg Ser His Leu Pro
Thr Arg Ile Lys Glu Glu Asp Ser Gly 530 535 540 Ser Pro Glu Thr Arg
Pro Leu Tyr Asp Leu Asn Glu Ser Ala Thr Glu 545 550 555 560 Val Leu
Ser Glu Gly Gly Asp Gly Phe Pro Val Thr Gln Gln Ala Tyr 565 570 575
Ser Leu Lys His Glu Asp Val Ser Glu Thr Thr Asn Gly Val Thr Leu 580
585 590 Met Pro Pro Gly His His Val Leu Ile Ser Leu Pro Glu Lys Lys
His 595 600 605 Gly Ser Leu Ala Ala Ala Glu Ala Arg Lys Arg Arg Lys
Glu Leu Thr 610 615 620 Arg Leu Lys Asn Leu His Gly Arg Gln Cys Arg
Met Gln Val Gly 625 630 635 15 90 PRT Oryza sativa 15 Met Ala Ser
Met Ser Val Ser Ser Ser Arg Ala Pro Gln Trp Thr Ala 1 5 10 15 Arg
Gln Asn Glu Gln Phe Glu Arg Ala Leu Ala Val Tyr Asp Arg Asp 20 25
30 Thr Pro Glu Arg Trp His Asn Ile Ala Arg Ala Val Ala Gly Lys Ser
35 40 45 Ala Asp Glu Val Lys Leu Tyr Tyr Asp Leu Leu Val Glu Asp
Val Lys 50 55 60 Arg Ile Glu Thr Gly Lys Val Pro Phe Pro Ala Tyr
Arg Cys Pro Gln 65 70 75 80 Pro Ala Ile Ala Glu Asn Ser Gly Ile Trp
85 90 16 101 PRT Oryza sativa 16 Met Ser Ser Ser Trp Thr Thr Lys
Gln Asn Lys Val Phe Glu Arg Ala 1 5 10 15 Leu Ala Ile Tyr Asp Arg
Asp Thr Pro Asp Arg Trp Gln Asn Val Ala 20 25 30 Arg Ala Val Gly
Gly Gly Lys Ser Val Asp Asp Val Lys Arg His Tyr 35 40 45 Glu Lys
Leu Ile Lys Asp Val Asp Arg Ile Asp Ser Thr Gly Gly His 50 55 60
Gln Gly Ser His Tyr Asn Ser Ser Asn Ala Ser Ser Ser Ser Ser Ser 65
70 75 80 Ser Ser Ser Asn Ser Arg Gly Ser Ala Asn Glu Asp Gln Arg
Arg Arg 85 90 95 Tyr His Asn Phe Gln 100 17 97 PRT Lycopersicon
esculentum misc_feature (93)..(93) Xaa can be any naturally
occurring amino acid 17 Gln Lys Ile Ile Met Ser Ser Met Ser Ser Gln
His Gly Ser Ser Gly 1 5 10 15 Ser Trp Thr Ala Lys Gln Asn Lys Ala
Phe Glu Lys Ala Leu Ala Val 20 25 30 Tyr Asp Lys Glu Thr Arg Asp
Arg Trp Ser Asn Val Ala Lys Ala Val 35 40 45 Gly Gly Lys Thr Ala
Glu Glu Val Lys Arg His Tyr Glu Ile Leu Leu 50 55 60 Arg Asp Val
Phe Phe Ile Asp Asn Gly Met Val Pro Phe Pro Lys Tyr 65 70 75 80 Lys
Thr Thr Gly Gly Ser His Asn Ser Thr Ser Asp Xaa His Tyr Phe 85 90
95 Tyr 18 732 DNA Solanum tuberosum 18 agtaatggca tcaagctcac
ttcaatcttc atcatggaca ccgcagcaaa acaagttatt 60 tgaaagggca
ctagctcaat tcgataagga cacacctgac cggtggcaga atgtggcgcg 120
ggcggttgga ggtggaaaat ccgccgatga agtaaagaga cactatgaaa tacttattga
180 ggatctcagg cgcattgaat ctggacgtgt tcctcttcct acttacaccc
atgaacaaca 240 aaggtattct taatcattct ctttaagtct tttgtccgtt
attatttaaa attacaacat 300 tcaaaagttc tttcaaattc aattggatgg
agtgaataaa tatgatattt tttgtttcaa 360 aggaatagca aagtatatat
actttgatct tgaacatttt gaaatgtgaa atgagacggt 420 tccatactta
aaccctactt tactagtcta tacttttgaa tgagacagtt acatatttct 480
aacttttgtc tatttgtaaa
acataagaaa tattcttatc ttttttagaa ttcagttaat 540 attttctttt
caaccttttg ttgtatttta gtcgattcga gtcatgcaaa cagttcggat 600
atgaatgaaa tttagaaatc ttaaatttca taaattaaca aaacagacat ggtgcggtgt
660 ttgaaagtta ttgcatgtaa ccctataccc tattctaatt aagagtcctt
aaatctttat 720 tagtatcttt tt 732
* * * * *