U.S. patent application number 12/938645 was filed with the patent office on 2011-02-24 for disease-resistant plants and method of constructing the same.
Invention is credited to Yasuhiro Inoue, Yuji Ishida, Shigeru Kuwata, Yoshimitsu TAKAKURA, Fumiki Tsutsumi.
Application Number | 20110046352 12/938645 |
Document ID | / |
Family ID | 18757703 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110046352 |
Kind Code |
A1 |
TAKAKURA; Yoshimitsu ; et
al. |
February 24, 2011 |
DISEASE-RESISTANT PLANTS AND METHOD OF CONSTRUCTING THE SAME
Abstract
The present invention provides transgenic, disease-resistant
plants which have been transformed with an expression cassette
including a constitutive gene expression promoter; and a gene,
under the control of the promoter, encoding a harpin. The
transformed cells in the transgenic plant effect the constitutive
expression of the harpin in an amount effective for inducing a
defense reaction. The harpin is a protein consisting of an amino
acid sequence that is at least 90% homologous to the amino acid
sequence of SEQ ID NO: 2, and possesses a
hypersensitive-response-inducing activity. The present invention
also provides methods for producing the transgenic plants,
expression cassettes, recombinant vectors and genes encoding
harpins.
Inventors: |
TAKAKURA; Yoshimitsu;
(Shizuoka, JP) ; Inoue; Yasuhiro; (Ibaraki,
JP) ; Kuwata; Shigeru; (Kanagawa, JP) ;
Tsutsumi; Fumiki; (Kanagawa, JP) ; Ishida; Yuji;
(Shizuoka, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
18757703 |
Appl. No.: |
12/938645 |
Filed: |
November 3, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12406502 |
Mar 18, 2009 |
7851671 |
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12938645 |
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10363832 |
Sep 11, 2003 |
7525014 |
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PCT/JP01/07785 |
Sep 7, 2001 |
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12406502 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C12N 15/8282 20130101;
C07K 14/21 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/21 20060101
C07K014/21 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 7, 2000 |
JP |
271413-2000 |
Claims
1. A protein selected from: (a) a protein having the amino acid
sequence of SEQ ID NO: 2; (b) a protein having an amino acid
sequence derived from the amino acid sequence of SEQ ID NO: 2, by
deletion, substitution, addition or insertion of one or more amino
acids, and possessing a hypersensitive-response-inducing activity;
and (c) a protein having an amino acid sequence being at least 97%
homologous to the amino acid sequence of SEQ ID NO: 2, and
possessing a hypersensitive-response-inducing activity.
Description
[0001] This is a Division of co-pending U.S. application Ser. No.
12/406,502 filed on Mar. 18, 2009. U.S. application Ser. No.
12/406,502 is a Continuation of application Ser. No. 10/363,832,
now U.S. Pat. No. 7,525,014, which is the national stage
application of PCT International Application No. PCT/JP01/07785
filed on Sep. 7, 2001. PCT International Application No.
PCT/JP01/07785 claims the benefit of priority of Japanese
Application No. 271413/2000 filed on Sep. 7, 2000 under 35 U.S.C.
.sctn.119. The entire contents of all of the aforementioned
applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for producing
disease-resistant plants, gene expression cassettes for producing
disease-resistant plants, and transgenic, disease-resistant plants
produced by the method.
BACKGROUND OF THE INVENTION
[0003] Plant defense against pathogens differs in its mechanism
from that observed in animals. For example, there is known in
higher plants a hypersensitive response (HR) mechanism which
involves a dynamic resistance reaction to pathogen invasion. When a
pathogen invades a plant, plant cells at a site of invasion die in
response, whereby pathogens are trapped locally. This reaction is
known to be induced as a result of either an incompatible
host-pathogen interaction or a non-host-pathogen interaction. Such
cell suicide can be understood in terms of a localized, programmed
cell death (Dangl et al.: Plant Cell 8: 1973-1807 (1996)). In
addition to the mechanism involving HR, other defense reactions,
including generation of active oxygen species, reinforcement of a
cell wall, production of phytoalexin and biosynthesis of
defense-related proteins such as PR proteins, are also known
(Hammond-Kosack and Jones: Plant Cell 8: 1773-1791 (1996)).
Further, in addition to such localized defense responses, there is
known to take place in many cases a defense reaction spreads
whereby PR proteins accumulate also in non-infected parts of a
plant, whereby resistance is imparted to the entire plant. This
mechanism is referred to as systemic acquired resistance (SAR) and
continues for several weeks or longer. As a result, the entire
plant is made resistant to secondary infection (Sticker et al.:
Annu. Rev. Phytopathol. 35: 235-270 (1997)).
[0004] A first reaction of a plant of switching on a highly
organized defense reaction such as outlined above is the
recognition by the plant of a molecule called an "elicitor"
directly or indirectly produced by an invading pathogen.
Additionally, complex signal cascades including the subsequent
rapid generation of active oxygen species and reversible protein
phosphorylation are considered to be important as initial reactions
of the defense response (Yang et al.: Genes Dev. 11: 1621-1639
(1997)). There are a wide variety of elicitors, including so-called
non-specific elicitors e.g. oligosaccharides which are products by
degradation of cell wall components of many fungi including
chitin/chitosan and glucan, or oligogalacturonic acids derived from
a plant cell wall, variety-specific elicitors e.g. avirulence gene
products of pathogens such as AVR 9 (Avr gene products), and
elicitors with an intermediate specificity such as elicitin
(Boller: Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 189-214
(1995)).
[0005] Harpin is a bacterium-derived protein elicitor which induces
hypersensitive cell death in a non-host plant (Wei et al.: Science
257: 85-88 (1992), He et al.: Cell 73: 1255-1266 (1993)). Harpin
(harpin.sub.Ea) has been purified as a first bacterium-derived
HR-inducing protein from Erwinia amylovora Ea321, a pathogen of
pear and apple, and Escherichia coli transformed with a cosmid
containing the hrp gene cluster, and an hrpN gene encoding Harpin
has been cloned (Wei et al.: Science 257: 85-88 (1992)).
Thereafter, harpin.sub.pss encoded by hrpZ gene has been identified
and characterized from Pseudomonas syringae pv. syringae 61, a
pathogen of a bean, by screening an Escherichia coli expression
library with an activity of inducing HR to a tobacco leaf as an
index (He et al.: Cell 73: 1255-1266 (1993), and Japanese Patent
Application Domestic Announcement No. 1996-510127). The homology
between these two harpins is low, and a relatively high homology is
found only in 22 amino acids. Moreover, the role of a harpin in
pathogenicity has not been made clear. In addition to these, as a
third protein, PopA protein (which PopA encodes) is identified from
Pseudomonas solanacearum GMI1000, a pathogen of a tomato, as a
protein inducing HR to a non-host tobacco (Arlat et al.: EMBO. J.
13: 543-553 (1994)). Though PopA gene is located on the outside of
hrp cluster, differing from hrpN and hrpZ, they are identical in
that they are under the control of an hrp regulon. The above three
proteins are glycine-rich, heat stable proteins, induce HR to a
non-host tobacco and are secreted extracellularly at least in vitro
in a manner of depending upon hrp protein. In addition to these are
reported HrpW protein from Pseudomonas syringae pv. tomato DC3000
as a protein having the same function (Charkowski et al.: J.
Bacteriol. 180: 5211-5217 (1998)), hrpZ.sub.pst and hrpZ.sub.psg
proteins as harpin.sub.pss homologues (Preston et al.: Mol.
Plant-Microbe. Interact. 8: 717-732 (1995)), and harpin.sub.Ech
(Bauer et al.: Mol. Plant-Microbe. Interact. 8: 484-491 (1995)) and
hrpN.sub.Ecc protein (Cui el al.: Mol. Plant-Microbe. Interact. 9:
565-573 (1996)) as harpin.sub.Ea homologues.
[0006] It has been made apparent from studies upon various
metabolic inhibitors that the formation of localized necrosis spots
with harpin is not so-called necrosis due to the cytotoxicity of
harpin but a cell death resulting from a positive response on the
plant side (He et al.: Mol. Plant-Microbe. Interact. 7: 289-292
(1994), and He et al.: Cell 73: 1255-1266 (1993)), and this
hypersensitive cell death is thought to be a type of programmed
cell death (Desikan et al.: Biochem. J. 330: 115-120 (1998)). The
addition of harpin.sub.pss into a cell culture of Arabidopsis
induces a homologue of gp91-phox, a constituent of NADPH oxidase,
which is thought to have an important role in the oxidative burst
as an initial reaction of a disease-resistant reaction, (J. Exp.
Bot. 49: 1767-1771 (1998)), and mitogen-activated protein (MAP)
kinase (Desikan et al.: Planta. 210: 97-103 (1999)). Moreover, a
harpin can impart systemic acquired resistance (SAR) to a plant.
For example, SAR meditated by salicylic acid and an NIM gene can be
induced to an Arabidopsis plant by artificially injecting
harpin.sub.Ea into the plant cells (Dong et al.: The Plant J. 20:
207-215 (1999)), and Harpin.sub.pss can induce SAR to a cucumber
and impart a wide spectrum of resistance to fungi, viruses and
bacteria (Strobel et al.: Plant J. 9: 431-439 (1996)).
[0007] Thus, there are reports about artificially injecting or
spraying purified harpin into a plant and analyzing the induction
of a hypersensitive cell death and an acquired resistance reaction
(Japanese Patent Application Domestic Announcement No. 1999-506938,
Strobel et al.: Plant J. 9: 431-439 (1996), and Dong et al.: The
Plant J. 20: 207-215 (1999)). However, there is no report about
introducing a gene encoding an elicitor protein such as a harpin
into a plant to produce a transgenic plant and analyzing it.
SUMMARY OF THE INVENTION
[0008] It has been anticipated that, when a gene encoding an
elicitor protein such as harpin is introduced into a plant, the
plant will express an elicitor protein at a certain amount, even in
a normal state with no pathogen, or that it will also express an
elicitor protein in a certain amount in organs other than those
invaded with a disease, and as a result, various unintended
reactions occur to prevent the plant from growing normally. The
object of the present invention is therefore to provide a
disease-resistant transgenic plant which has been transformed to
induce a proper defense reaction, and to provide a method for
producing the same.
[0009] The present inventors have engaged in studies assiduously,
and as a result have found that a transgenic tobacco with hrpZ gene
of Psedomonas syringae pv. syringae LOB2-1 introduced thereinto
induces hypersensitive-response-like localized necrosis spots in
response to the inoculation of a powdery mildew fungi (Erysiphe
cichoracearum) to become resistant, which has led to the completion
of the present invention. Surprisingly, a plant grew normally when
cell-death-inducing harpin was expressed with a constitutive
promoter (cauliflower mosaic virus 35S RNA gene promoter) capable
of promoting expression in cells of the whole body. In addition, a
hypersensitive cell-death-like reaction was induced only after
inoculation with a pathogen. Further, the present inventors have
found that a transgenic rice with the same hrpZ gene introduced
thereinto becomes blast (Magnaporthe grisea)-resistant, thus
showing the general-applicability of the present invention.
[0010] The present invention provides a transgenic,
disease-resistant plant which has been transformed with an
expression cassette comprising a promoter capable of promoting a
constitutive, inducible, or organ- or phase-specific gene
expression and a gene encoding an elicitor protein under the
control of said promoter, wherein said plant is capable of
effecting the constitutive, inducible, or organ- or phase-specific
expression of the elicitor protein in an amount effective for
inducing a defense reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the constructs constructed and introduced into
plants in the present invention.
[0012] FIG. 2 is a photograph showing exemplary of the detection
results using Western analysis for harpin.sub.pss accumulation in
transgenic tobacco and rice of the T.sub.0 generation. PC
represents harpin.sub.pss expression in Escherichia coli as a
control.
[0013] FIG. 3 is a photograph showing the appearances of localized
necrosis spots occurring in a transgenic tobacco of the T.sub.1
generation. A: PALL-hrpZ-introduced individual (5th day after
inoculation, harpin expression level: ++), B: 35S-hrpZ-introduced
individual (7th day after inoculation, harpin expression level:
++)
[0014] FIG. 4 is a photograph showing the resistance of a
transgenic tobacco of the T.sub.1 generation against powdery
mildew. (Right: 35S-hrpZ-introduced individual, harpin expression
level: ++, Left: SRI as a control, 11th day after inoculation in
both)
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention also provides methods for producing
transgenic, disease-resistant plants capable of effecting the
constitutive, inducible, or organ- or phase-specific expression of
an elicitor protein in an amount effective for inducing a defense
reaction. Such methods comprise the steps of: (a) obtaining
transgenic plant cells with expression cassettes comprising a
promoter capable of promoting a constitutive, inducible, or organ-
or phase-specific gene expression and a gene encoding an elicitor
protein under the control of said promoter; and (b) regenerating a
complete plant from said transgenic plant cell.
[0016] The present invention also provides expression cassettes
capable of being employed for producing a transgenic,
disease-resistant plants. Such expression cassettes comprise at
least: (a) a promoter capable of promoting a constitutive,
inducible, or organ- or phase-specific gene expression; and (b) a
gene, under the control of said promoter, encoding an elicitor
protein.
[0017] "Elicitor" is a general term used for substances inducing
defense reactions in plants, and including heavy metal ions, and
cell wall components of pathogens or plants, in addition to
proteins. The term "elicitor" as used in the present specification
refers to a protein elicitor unless otherwise specified.
[0018] The term "elicitor protein" as used in the present invention
can be any protein capable of inducing a proper defense reaction in
a plant to be transformed, and preferably a protein possessing a
hypersensitive-response-inducing activity against pathogenic
microorganisms. It includes harpin and a harpin-like protein having
the same function as harpin. "Harpin" is a protein expected to be
introduced into a plant in a manner of depending upon hrp gene
though the Type III secretion mechanism, and includes, in addition
to harpin.sub.pss (He et al.: Cell 73: 1255-1266 (1993), and
Japanese Patent Application Domestic Announcement[kohyo] No.
510127/96), harpin.sub.Ea (Wei et al.: Science 257: 85-88 (1992),
and Japanese Patent Application Domestic Announcement[kohyo]No.
506938/99), PopA (Arlat et al.: EMBO. J. 13: 543-553 (1994)), and
hrpW protein (Charkowski et al.: J. Bacteriol. 180: 5211-5217
(1998). Additionally the protein possessing a
hypersensitive-response-inducing activity can be, for example, (a)
a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(b) a protein consisting of an amino acid sequence derived from the
amino acid sequence of SEQ ID NO: 2 by deletion, substitution,
addition or insertion of one or more amino acids, and possessing a
hypersensitive-response-inducing activity; or (c) a protein
consisting of an amino acid sequence being at least 50% (preferably
at least 80%, more preferably at least 90%, and still more
preferably at least 97%) homologous to the amino acid sequence of
SEQ ID NO: 2, and possessing a hypersensitive-response-inducing
activity. A protein consisting of the amino acid of SEQ ID NO: 2 is
novel. Hence, the present invention provides one of the following
proteins: (a) a protein consisting of the amino acid sequence of
SEQ ID NO: 2; (b) a protein consisting of an amino acid sequence
derived from the amino acid sequence of SEQ ID NO: 2 by deletion,
substitution, addition or insertion of one or more amino acids, and
possessing a hypersensitive-response-inducing activity; and (c) a
protein consisting of an amino acid sequence being at least 97%
homologous to the amino acid sequence of SEQ ID NO: 2, and
possessing a hypersensitive-response-inducing activity (but known
proteins themselves are excluded from the scope of the present
invention).
[0019] By "Homology" referred to in connection with amino acid
sequences in the present specification is meant a degree of
identification of amino acid residues constituting each sequence
between sequences to be compared. In homology, the existence of a
gap(s) and the nature of an amino acid(s) are taken into
consideration (Wilbur, Proc. Natl. Acad. Sci. USA 80: 726-730
(1983) and the like). To calculate homology, commercially available
software such as BLAST (Altschul: J. Mol. Biol. 215: 403-410
(1990), and FASTA (Peasron: Methods in Enzymology 183: 63-69
(1990)) can be employed.
[0020] The description "deletion, substitution, addition or
insertion of one or more amino acids" as used in the present
specification in connection with an amino acid sequence in the
means that a certain number of an amino acid(s) are substituted
etc. by any well known technical method such as site-specific
mutagenesis, or naturally. The number is, for example, up to ten,
and is preferably from 3 to up to 5.
[0021] A gene encoding an elicitor protein to be employed in the
expression cassette of the present invention can easily be isolated
by methods well-known to those skilled in the art.
[0022] The gene encoding an elicitor protein can be, for example,
(a) a DNA molecule consisting of the nucleotide sequence of SEQ ID
NO: 1; (b) a DNA molecule consisting of a nucleotide sequence
derived from the nucleotide sequence of SEQ ID NO: 1 by deletion,
substitution, addition or insertion of one or more nucleotides, and
encoding a protein possessing a hypersensitive-response-inducing
activity; (c) a DNA molecule consisting of a nucleotide sequence
being hybridizable with a DNA molecule consisting of the nucleotide
sequence complementary to the nucleotide sequence of SEQ ID NO: 1
under stringent conditions, and encoding a protein possessing a
hypersensitive-response-inducing activity; or (d) a DNA molecule
consisting of a nucleotide sequence being at least 50% (preferably
at least 80%, more preferably at least 90%, and still more
preferably at least 97%) homologous to the nucleotide sequence of
SEQ ID NO: 1, and encoding a protein possessing a
hypersensitive-response-inducing activity. A DNA molecule
consisting of the nucleotide sequence of SEQ ID NO: 1 is novel.
Hence, the present invention also provides a gene consisting of one
of the following DNA molecules: (a) a DNA molecule consisting of
the nucleotide sequence of SEQ ID NO: 1; (b) a DNA molecule
consisting of a nucleotide sequence derived from the nucleotide
sequence of SEQ ID NO: 1 by deletion, substitution, addition or
insertion of one or more nucleotides, and encoding a protein
possessing a hypersensitive-response-inducing activity; (c) a DNA
molecule consisting of a nucleotide sequence being hybridizable
with a DNA molecule consisting of the complementary nucleotide
sequence to the nucleotide sequence of SEQ ID NO: 1 under stringent
conditions, and encoding a protein possessing a
hypersensitive-response-inducing activity; or (d) a DNA molecule
consisting of a nucleotide sequence being at least 50% homologous
to the nucleotide sequence of SEQ ID NO: 1, and encoding a protein
possessing a hypersensitive-response-inducing activity (but known
genes themselves such as hrpZ gene of Pseudomonas syringae pv.
syringae 61 are excluded from the scope of the present invention).
To calculate homology in connection with nucleotide sequences,
commercially available software can be employed.
[0023] By "deletion, substitution, addition or insertion of one or
more nucleotides" in connection with a nucleotide sequence in the
present specification is meant that a certain number of a
nucleotide(s) are substituted etc. by a well-known technical method
such as a site-specific mutagenesis or naturally. The number is,
for example, up to ten, preferably from 3 to up to 5. By "stringent
conditions" referred to in the present specification is meant
hybridization conditions wherein the temperature is at about
40.degree. C. or above and that the salt concentration is of about
6.times.SSC (1.times.SSC=15 mM sodium citrate buffer; pH: 7.0; 0.15
M sodium chloride; 0.1% SDS), preferably at about 50.degree. C. or
above, more preferably at about 65.degree. C. or above.
[0024] The promoter to be employed in the present invention can be
any promoter capable of functioning as a promoter for a gene
encoding an elicitor protein in a plant to be transformed. In the
present invention, a promoter capable of promoting a constitutive,
inducible, or organ- or phase-specific gene expression can be
employed.
[0025] By "promoter promoting a constitutive gene expression (often
referred to as a "constitutive promoter")" is meant a promoter
whose organ specificity and/or phase specificity are (is) not high
in connection with the transcription of the gene. Examples of the
constitutive promoter include cauliflower mosaic virus 35S
promoter, ubiquitin promoter (Cornejo et al.: Plant Mol. Biol. 23:
567-581 (1993)), actin promoter (McElroy et al.: Plant Cell 2:
163-171 (1990)), alpha tubulin promoter (Carpenter et al.: Plant
Mol. Biol. 21: 937-942 (1993)) and Sc promoter (Schenk et al.:
Plant Mol. Biol. 39: 1221-1230 (1999)). In a transgenic plant, the
expression cassette promoting the constitutive expression of an
elicitor protein includes, for example, a known promoter that is
known as a constitutive promoter.
[0026] By "promoter promoting an inducible gene expression (often
referred to as an "inducible promoter")" is meant a promoter which
induces transcription by physical or chemical stimulation, such as
light, disease, injury or contact with an elicitor. Examples of the
inducible promoter include pea PAL promoter, Prp1 promoter
(Japanese Patent Application No. 1998-500312), hsr203J promoter
(Pontier et al.: Plant J. 5: 507-521 (1994)), EAS4 promoter (Yin et
al.: Plant Physiol. 115: 437-451 (1997)), PR1b1 promoter (Tornero
et al.: Mol. Plant Microbe. Interact. 10: 624-634 (1997)), tap1
promoter (Mohan et al.: Plant Mol. Biol. 22: 475-490 (1993)) and
AoPR1 promoter (Warner et al.: Plant J. 3: 191-201 (1993)). In a
transgenic plant, the expression cassette promoting an inducible
elicitor protein expression includes, for example, a known promoter
known as an inducible promoter.
[0027] By "promoter promoting an organ-specific gene expression
(often referred to as an "organ-specific promoter")" is meant a
promoter giving, to the transcription of the gene, a specificity to
an organ, such as a leaf, a root, a stem, a flower, a stamen and a
pistil. Examples of the organ-specific promoter include a promoter
promoting a high gene expression in green tissues of a
photosynthesis-related gene, such as PPDK (Matsuoka et al.: Proc.
Natl. Acad. Sci. USA 90: 9586-9590 (1993)), PEPC (Yanagisawa and
Izui: J. Biochem. 106: 982-987 (1989) and Matsuoka et al.: Plant J.
6: 311-319 (1994)) and Rubisco (Matsuoka et al.: Plant J. 6:
311-319 (1994)). In a transgenic plant, the expression cassette
promoting an organ-specific elicitor protein expression includes,
for example, a known promoter that is known as an organ-specific
promoter.
[0028] By "promoter promoting a phase-specific gene expression
(often referred to as a "phase-specific promoter")" is meant a
promoter giving, to the transcription of the gene, a phase
specificity to a phase, such as a initial, middle and later growth
phase. Examples of the phase-specific promoter include a promoter
functioning specifically in aged leaves such as SAG12 promoter (Gan
and Amashino: Science 270: 1986-1988 (1985)).
[0029] Vectors for sub-cloning each DNA fragment as a component of
the expression cassette of the present invention can be simply
prepared by connecting an intended gene into a vector for
recombination (plasmid DNA) available in the art by any common
technique. Specific examples of suitable vectors include plasmids
derived from Escherichia coli, such as pBluescript, pUC18, pUC19
and pBR322, but are not limited only to these plasmids.
[0030] As a vector for introducing the expression cassette of the
present invention into a plant to be transformed, a vector for
transforming plants can be used. The vectors for plants are not
particularly limited, so far as they are capable of expressing the
concerned gene and producing the concerned protein in a plant cell,
and examples thereof include pBI221, pBI121 (both being
manufactured by Clontech) and vectors derived therefrom. In
addition, for the transformation of a monocotyledonous plant in
particular, there can be exemplified pIG121Hm, pTOK233 (both by
Hiei et al.: Plant J. 6: 271-282 (1994)), pSB424 (Komari et al.:
Plant J. 10: 165-174 (1996)), superbinary vector pSB21 and vectors
derived therefrom. A recombination vector having the expression
cassette of the present invention can be constructed by introducing
a gene encoding an elicitor protein into any of these known vectors
(if required, a promoter region being recombined) by a procedure
known well to those skilled in the art. For example, a recombinant
vector having an expression cassette comprising a constitutive
promoter and hrpZ gene can be constructed by integrating hrpZ gene
into superbinary vector pSB21. A recombinant vector having an
expression cassette comprising an inducible promoter and hrpZ gene
can be constructed by removing the existing promoter from the above
recombinant vector and integrating an inducible promoter in
place.
[0031] A plant-transforming vector preferably comprises at least a
promoter, a translation initiator codon, a desired gene (a DNA
sequence of the invention of the present application or a part
thereof), a translation termination codon and a terminator.
Moreover, it may comprise a DNA molecule encoding a signal peptide,
an enhancer sequence, a non-translation region on the 5' side and
the 3' side of the desired gene and a selection marker region as
appropriate. Examples of marker genes include antibiotic-resistant
genes such as tetracyclin, ampicillin, kanamycin or neomycin,
hygromycin or spectinomycin; and genes such as luciferase,
.beta.-galactosidase, .beta.-glucuronidase(GUS), green fluorescence
protein (GFP), .beta.-lactamase and chloramphenicol acetyl
transferase (CAT).
[0032] As methods for introducing a gene into a plant can be
mentioned a method employing an agrobacterium (Horsch et al.:
Science 227: 129 (1985), Hiei et al.: Plant J. 6: 271-282 (1994)),
a leaf disc method (Horsch et al.: Science 227: 1229-1231 (1985),
an electroporation method (Fromm et al.: Nature 319: 791 (1986)), a
PEG method (Paszkowski et al.: EMBO. J.3: 2717 (1984)), a
micro-injection method (Crossway et al.: Mol. Gen. Genet. 202: 179
(1986)) and a minute substance collision method (McCabe et al.:
Bio/Technology 6: 923 (1988)), but any method for introducing a
gene into a desired plant may be employed without any particular
limitation. Of these methods for transfection, a method comprising
transferring a vector into an agrobacterium by mating and then
infecting a plant with the agrobacterium is preferred. Methods for
infection is also well-known to those skilled in the art. Examples
include a method comprising damaging a plant tissue and infecting
it with a bacterium; a method comprising infecting an embryo tissue
(including an immature embryo) of a plant with the bacterium; a
method comprising infecting with a callus; a method comprising
co-culturing protoplasts and the bacterium; and a method comprising
culturing a fragment of a leaf tissue together with the bacterium
(leaf disc method).
[0033] Successfully transformed cells can be selected from other
cells by employing an appropriate marker as an index or examining
the expression of a desired trait. The transformed cell can further
be differentiated employing a conventional technique to obtain a
desired transgenic plant.
[0034] Analysis of the resultant transformant can be performed by
employing various methods that are well-known to those skilled in
the art. For example, oligonucleotide primers can be synthesized
according to the DNA sequence of the introduced gene, and the
chromosome DNA of the transgenic plant can be analyzed by PCR
employing the primers. In addition, the analysis can be performed
on the basis of the existence of mRNA corresponding to the
introduced gene and the existence of the protein expression.
Moreover, the analysis can be performed on the basis of the
appearance of the plant (for example, in the case of transformation
with a gene encoding a protein capable of inducing localized
necrosis spots, the presence of localized necrosis spots, or the
size, number and the like of the localized necrosis spots), disease
resistance (for example, the existence of resistance or its degree
upon contacting the plant with a pathogen) and the like.
[0035] In the transgenic plant of the present invention, a
constitutive, inducible, or organ- or phase-specific expression of
an elicitor protein in an amount effective for inducing a defense
reaction can be achieved. The amount effective for inducing a
defense reaction is such an amount that the expressed elicitor
protein can induce at least a localized defense-related reaction
(for example, induction of a hypersensitive cell death (localized
necrosis)) to the plant. Preferably, the amount is such that the
defense reaction extends to the whole body of the plant, and as a
result, the whole plant becomes resistant (systemic acquired
disease-resistant). Moreover, preferably, the amount is not so
large that causes death of the localized tissue having the necrosis
spots as a result of the localized necrosis spots becoming too
large.
[0036] Moreover, in the transgenic plant of the present invention,
an elicitor protein is preferably expressed in an amount which,
while being effective for inducing a defense reaction in response
to stimulation such as the invasion of a pathogen, does not, under
normal conditions, remarkably prevent the growth of the plant due
to the negligible or low expression, if any. For example, in the
case of employing harpin.sub.pss as an elicitor protein, usually no
harpin.sub.pss is expressed, or is expressed only in an amount that
does not allow localized necrosis spots to cause the death of the
organ, and preferably it is expressed in an amount that induces a
hypersensitive response at the time of the invasion of a pathogen.
Further, it is preferably expressed in such an amount that, even if
a pathogen invades to cause harpin.sub.pss to accumulate, localized
necrosis spots are hardly observable by the naked eye, but the
whole body acquires a systemic disease-resistannce.
[0037] In order to induce such a proper defense reaction, for
example, a promoter capable of promoting an inducible gene
expression is employed. Hence, in one embodiment of the present
invention, an inducible promoter and a harpin gene are
combined.
[0038] In addition, a proper defense reaction can be accomplished
not only in the case of employing an inducible promoter but also in
the case of employing a constitutive promoter. Hence, in another
embodiment of the present invention, a constitutive promoter and a
harpin gene are used in combination. In this embodiment, as a
mechanism of the occurrence of a proper defense reaction, it is
considered that an elicitor protein, for example, harpin.sub.pss,
is recognized at the outside of cell membranes or on the cell wall
of plant cells, and hence, harpin.sub.pss accumulating in cytoplasm
is not recognized by plant cells until degradation of cells occurs
due to invasion of fungus, and as a result, the hypersensitive
response appears after the inoculation of the pathogen or it is
deduced that there exists a further factor which is related to the
inoculation of a pathogen in the mechanism of the occurrence of the
elicitor activity of harpin.sub.pss.
[0039] The transgenic plants of the present invention include a
transgenic, powdery mildew-resistant tobacco which has been
transformed with an expression cassette comprising a constitutive
or inducible promoter and a gene, under the control of said
promoter, encoding an elicitor protein such as harpin.sub.pss, or a
transgenic, blast-resistant rice which has been transformed with an
expression cassette comprising a constitutive promoter and a gene,
under the control of the promoter, encoding an elicitor protein
such as harpin.sub.pss.
[0040] It is thought that the present invention can be applied to
plants other than rice and tobacco described in the examples to be
described later. Examples of such plants include, as crops, wheat,
barley, rye, corn, sugar cane, sorghum, cotton, sunflower, peanut,
tomato, potato, sweet potato, pea, soybean, azuki bean, lettuce,
cabbage, cauliflower, broccoli, turnip, radish, spinach, onion,
carrot, eggplant, pumpkin, cucumber, apple, pear, melon, strawberry
and burdock; and, as ornamental plants, arabidopsis thaliana,
petunia, chrysanthemum, carnation, saintpaulia and zinnia. The
"transgenic plants" referred to in the present invention include
not only transgenic plants (T.sub.0 generation) obtained by
obtaining a transgenic plant cell according to the method of the
present invention and regenerating, from said plant cell, a
complete plant, but also later-generation (T.sub.1 generation and
the like) plants obtained from said transgenic plants so far as the
disease-resistant trait is contained. In addition, the "plants"
referred to in the present invention include, unless otherwise
specified, in addition to plants (individuals), seeds (including
germinated seeds and immature seeds), organs or parts thereof
(including a leaf, a root, a stem, a flower, a stamen, a pistil and
pieces thereof), a plant culture cell, a callus and a
protoplast.
[0041] The diseases analyzed in the following examples are tobacco
powdery mildew and rice blast, but as other diseases of tobacco
there can be mentioned wildfire, bacterial wilt and TMV; and as
other diseases of rice there can be mentioned sheath blight disease
and bacterial leaf blight disease. According to the method for
producing a disease-resistant plant of the present invention, it is
possible to impart resistance in plants to these diseases.
Examples
Example 1
Cloning of HrpZ Gene
[0042] A pair of primers for amplifying the open leading frame of
hrpZ gene were synthesized in reference to the nucleotide sequence
of the reported hrpZ gene of Pseudomonas syringae pv. syringae 61
(He et al.: Cell 73: 1255-1266 (1993)), and Japanese Patent
Application Domestic Announcement[Kohyo] No. 1996-510127):
TABLE-US-00001 (SEQ ID NO: 3) Hrp1: AAA ATC TAG AAT GCA GAG TCT CAG
TCT TAA (SEQ ID NO: 4) Hrp2: AAA AGT CGA CTC AGG CTG CAG CCT GAT
TGC
[0043] Employing these primers, PCR was performed with a DNA
molecule of a cosmid clone containing an hrp cluster derived from
Pseudomonas syringae pv. syringae LOB2-1 (a casual agent for
bacterial blight of lilac) (Inoue and Takikawa: J. Gen. Plant
Pathol. 66: 238-241 (2000)) as a template. PCR was performed under
the following conditions: the amount of a reaction solution: 20
.mu.l; each primer: 0.5 .mu.l M; dNTP: 0.2 mM; 1.times. ExTaq
buffer; ExTaq DNA polymerase (from Takara Shuzo): 1 U; once at
95.degree. C. for 5 minutes, then 30 cycles at 94.degree. C. for 30
seconds, at 60.degree. C. for 30 seconds and at 72.degree. C. for 2
minutes, and once at 72.degree. C. for 10 minutes. The PCR product
was ligated to a vector pCR2.1 (from Invitrogen) using Takara
ligation kit (from Takara Shuzo) and transformed into an
Escherichia coli TB1 strain. As a result of determining the entire
nucleotide sequence of the PCR product, it consisted of 1029 bp in
the length, longer than the reported hrpZ gene (He et al.: Cell
73:1255-1266(1993)) by three bases (one amino acid), and showed a
homogoly of 96.7% in nucleotides and a homology of 96.5% in amino
acids. The reason that the nucleotide sequences are not completely
the same is thought to be due to a variation among the pathover.
The nucleotide sequence of the cloned hrpZ gene is shown in SEQ ID
NO: 1 and the deduced amino acid sequence obtained therefrom is
shown in SEQ ID NO: 2, respectively.
Example 2
Expression in an Escherichia coli and Production of an Antibody
[0044] The above plasmid with an hrpZ gene integrated into pCR2.1
was digested with restriction enzymes BamHI and SaII, and was
subjected to electrophoresis on 0.7% agarose to separate a fragment
of about 1.1 kb. This fragment was ligated to an expression vector
pQE31 (from QIAGEN) digested with the same enzymes and transformed
into Eschrichia coli M15 strain. The thus obtained Eschrichia coli
was cultured in an LB medium in the presence of 1 mM of IPTG at
37.degree. C., harpin.sub.pss was accumulated as insoluble
fraction. Since this protein showed poor adsorption to a nickel
resin adsorbent, the purification of harpin.sub.pss was conducted
in the following procedure. The Eschrichia coli M15 strain having
the pQE31 vector with the hrpZ gene integrated thereinto was
cultured in 2 ml of an LB medium containing 100 mg/l of ampicillin
and 25 mg/l of kanamycin at 37.degree. C. overnight, and
transferred into 250 ml of the LB medium and cultured for about
three hours; then 1 mM of IPTG was added thereto and the culture
was further conducted at 37.degree. C. for 4 hours. Cells were
collected by centrifugation, the insoluble fraction was dissolved
in 4 ml of an eluation buffer (8 M urea, 0.1 M sodium dihydrogen
phosphate, 0.01 M Tris, pH 8.0), and a supernatant liquid was
obtained by centrifugation and subjected to electrophoresis on a
12.5% acrylamide gel containing 0.1% SDS, and then stained with
Coomassie Brilliant Blue to cut a band appearing at around 40 kDa.
The gel was cut into small pieces, and an elution buffer (1% SDS,
0.02 M Tris-HCl, pH of 8.0) was added thereto in an amount ten
times the volume of the gel, and shaken for three days. The
supernatant was transfered to a dialysis membrane with a cut off
molecular weight of 6,000 to 8,000, and the dialysis was conducted
with 80% acetone as an external liquid once for 4 hours and once
overnight. The whole content in the dialysis tube was moved into an
Eppendorf tube, subjected to centrifugation to discard the
supernatant, and the pellet was dried to obtain a purified
harpin.sub.pss preparation. 3 mg of the purified harpin.sub.pss was
sent to Sawady Technology for the production of an antibody
(anti-rabbit harpin.sub.pss serum).
Example 3
Construction of a Gene and Transformation of a Plant
[0045] The hrpZ gene integrated into pCR2.1 was excised from the
vector by digestion with restriction enzymes XbaI and SacI (from
Takara Shuzo). On the other hand, superbinary vector pSB21
(35S-GUS-NOS, Komari et al.: Plant J. 10: 165-174 (1996)) was
digested with the same enzymes to remove the GUS gene, and the hrpZ
gene was integrated thereinto. According to the above procedure, a
construct named 35S-hrpZ (35S promoter-hrpZ gene-NOS terminator)
was constructed. The cauliflower mosaic virus 35S promoter is a
promoter capable of constitutively promoting a high expression, and
it is anticipated that rice and tobacco transformed with this
construct will accumulate harpin.sub.pss, the hrpZ gene product, in
the whole body.
[0046] pSB21 was digested with restriction enzymes HindIII and XbaI
to remove the 35S promoter, and a 0.9 kb fragment of corn PPDK
promoter (Taniguchi et al.: Plant Cell Physiol. 41: 42-48 (2000))
was integrated thereinto. The resulting plasmid was digested with
XbaI and SacI to remove the GUS gene, and then the above-described
hrpZ XbaI-SacI fragment was inserted thereinto. Thus, PPDK-hrpZ
(PPDK promoter-hrpZ gene-NOS terminator) was constructed. The corn
PPDK promoter is a promoter capable of promoting a strong
expression in photosynthesis organs such as mesophyl cells
(Taniguchi et al.: Plant Cell Physiol. 41: 42-48 (2000)), and it is
anticipated that rice plants transformed with this construct will
accumulate harpin.sub.pss, the hrpZ gene product, in green organs
(leaves).
[0047] PAL promoter was cloned as below. Plasmid DNA was extracted
from agrobacterium LBA4404 strain (gifted from Prof. Shiraishi of
Okayama University) having a construct containing PSPAL1 (PSPAL1
promoter-GUS gene-NOS terminator) (Yamada et al.: Plant Cell
Physiol. 35: 917-926 (1994), and Kawamata et al.: Plant Cell
Physiol. 38: 792-803 (1997)). On the other hand, a reverse primer
and two forward primers were designed on the basis of the
nucleotide sequence of the reported PSPAL1 promoter (Patent: JP
1993153978-A 1 22 Jun. 1993; TAKASAGO INTERNATL. CORP.):
TABLE-US-00002 PALRVXba: (SEQ ID NO: 5) GGG GTC TAG AAT TGA TAC TAA
AGT AAC TAA TG PALFFHin: (SEQ ID NO: 6) TTG GAA GCT TAG AGA TCA TTA
CGA AAT TAA GG PALFSHin: (SEQ ID NO: 7) CTA AAA GCT TGG TCA TGC ATG
GTT GCT TC
[0048] A promoter region (PAL-S) of about 0.45 kb in the upstream
of the starting point of translation (about 0.35 kb at the upstream
of the initiation point of transcription) was amplified by the
combination of PALRVXba and PALFSHin, and a promoter region (PAL-L)
of about 1.5 kb by the combination of PALRVXba and PALFFHin. The
above-mentioned agrobacteruium plasmid DNA was used as a template
and PCR was conducted with these primers. The reaction conditions
of PCR were as below: reaction solution: 50 .mu.l; each primer: 0.5
.mu.M, dNTP: 0.2 mM; 1.times. ExTAq buffer, ExTAq DNA polymerase
(from Takara Shuzo): 1 U; and the reaction was conducted once at
94.degree. C. for three minutes, then 30 cycles at 94.degree. C.
for one minute, at 50.degree. C. for one minute and at 72.degree.
C. for two minutes, and once at 72.degree. C. for 6 minutes. A PCR
product was cloned to vector pCRII (from Invitrogen).
[0049] Since the PsPAL1 promoter had a HinIII site at the upstream
142 bp from the starting point of translation, PAL-S was digested
completely with restriction enzyme XbaI and then partially with
HindIII to obtain a 0.45 kb of fragment from pCRII. The above
mentioned pSB21 was digested with HindIII and XbaI to remove the
35S promoter, and PAL-S was integrated thereinto. In the pSB21
vector employed here the unique Pvull site existing in the basic
structure had been removed, and, instead, a PvuII linker had been
placed at the unique ECoRI site (just after the Nos terminator).
The plasmid with PAL-S integrated thereinto was further digested
with XbaI and SacI to remove the GUS gene, and then the above
mentioned 1.1 kb hrpZ XbaI-SacIl fragment was inserted therein.
PALS-hrpZ was constructed according to the above procedure. Next,
PAL-L integrated into pCRII was digested with restriction enzymes
XhoI and XbaI to take out a 1.45 kb PAL promoter, which was
integrated into vector pSB11 (Komari et al.: Plant J. 10: 165-174
(1996)) co-digested with the same enzymes. The formed plasmid was
digested with XbaI and SmaI, and an XbaI-PvuII fragment of
PALS-hrpZ (hrpZ-NOS terminator) was inserted therein. In this
manner, PALL-hrpZ was produced. The PAL promoter promotes a
low-level expression constitutively, but it is a promoter strongly
induced with a pathogen and an injury (Yamada et al.: Plant Cell
Physiol. 35: 917-926 (1994), and Kawamata et al.: Plant Cell
Physiol. 38: 792-803 (1997)), and it is anticipated that a tobacco
plant transformed with PALS-hrpZ or PALL-hrpZ accumulates more
harpin.sub.pss at the place of stress when these stresses occur. In
this case, it is anticipated that more harpin.sub.pss will
accumulate in the case of PALL relative to the case of PALS.
[0050] According to the tri-parental mating system, of Escherichia
coli LB392 strain containing the thus produced four constructs
35S-hrpZ, PALS-hrpZ, PALS-hrpZ and PALL-hrpZ (summarized in FIG.
1), agrobacterium LBA4404 strain containing a vector pSB4U with a
selection marker gene integrated thereinto (corn ubiquitin
promoter-hygromycin-resistant gene (hptII)-NOS terminator) and
Escherichia coli HB101 containing a helper plasmid pRK2013, the
hrpZ gene containing construct was introduced into an agrobacterium
utilizing homologous recombination.
[0051] The transformation of a tobacco was performed by the leaf
disc method (Horsch et al.: Science 227: 1229-1231 (1985)). A leaf
of tobacco variety SR1 grown in a greenhouse was sterilized by
treatment with ethanol for 30 seconds and with antiformin diluted 5
times for 5 minutes, and after it was cleaned with sterilized water
twice, it was cut into one-centimeter squares, and an agrobacterium
suspension was inoculated thereto. The concentrations of hygromycin
at the time of induction and selection of a transfected shoot and
at the time of rooting were 50 or 100 mg/ml and 0 or 50 mg/ml,
respectively. For the transformation of rice,
immature-embryo-derived cali of varieties of paddy rice,
Tsukinohikari, and Koshihikari were transformed employing
agrobacterium according to the method of Hiei et al.: Plant J. 6:
271-282 (1994).
Example 4
Analysis of Transformants
(1) Transgenic Tobacco
[0052] 15 individuals of the re-generated plant were obtained from
35S-hrpZ, 10 individuals were from PALS-hrpZ and 16 individuals
were from PALL-hrpZ. There was observed no remarkable difference
between the constructs in transformation efficiency. Western
analysis was performed on the primary generation (T.sub.0) of the
transformant, and Western analysis and disease assays were
performed on the self-pollinated next generation (T.sub.1).
1) Western Analysis of T.sub.0 Generation
[0053] 2.times.2 cm of a leaf of a transgenic tobacco of the 4 or 5
leaf stage and 2.times.2 cm of a leaf of a non-transgenic tobaco
(SR1) were pulverized in 0.1 M HEPES-KOH pH 7.6 buffer in a mortar.
The supernatant liquid after centrifugation with 15000 g for 10
minutes was made a protein sample. The amount of the protein was
determined with a Bio-Rad Protein Assay kit (from BIO-RAD). About
20 .mu.g of the protein was fractioned by the SDS-PAGE method
according to the method of Laemmni et al. (Nature 227: 680-685
(1970)), on 12.5% PAGEL (from ATTO). After electrophoresis, the
protein bands on the gel were transferred to a PVDF membrane (from
Millipore). The PVDF membrane was placed in a 1.times. TBS buffer
containing 0.5% skim milk for 30 minutes, and shaken in the same
buffer containing 1/1000 (v/v) of anti-harpin.sub.pss serum at room
temperature overnight. As a secondary antibody was employed an
anti-goat rabbit IgG peroxidase labeled conjugate (from MBL) or an
anti-goat rabbit IgG alkaline phosphatase conjugate (from BIO-RAD)
at the concentration of 1/1000 (v/v). As color development systems
were employed HRP Color Development Reagent (from BIO-RAD),
alkaline phosphatase substrate kit II (from Vector Laboratories).
The amounts of the protein expressed were calculated by comparison
with the color development of the harpin.sub.pss sample of a known
concentration, by using a densitometer (model GS-670, from
BIO-RAD). Some of the results of the Western analysis of the
T.sub.0 generation is shown in FIG. 2, and the whole results are
summarized in Table 1.
[0054] The expression level is shown in four stages (+++, ++, +,
-), which show 0.1% or more of the total soluble proteins (+++),
0.05 to 0.1% (++), 0.05% or less (+) and below the detection
limitation (-) in the amount of expression, respectively. This is
true also in Tables 2, 3 and 4 to be described later.
TABLE-US-00003 TABLE 1 Results of the Western Analysis of the
Tobacco T.sub.0 Generation Number of Expression level re-generated
of Harpin.sub.pss.sup.a Construct individuals - + ++ +++.sup.b
PALS-hrpZ 10 1 8 1 0 PALL-hrpZ 16 2 10 4 0 35S-hrpZ 15 6 2 1 6 SR1
3 0 0 0 .sup.aEach numerical value shows the number of individuals
showing each expression level. .sup.bThe expression level of
harpin.sub.pss is shown in four stages (+++: particularly high
expression, ++: high expression, +: moderate to poor expression, -:
below the detection limitation).
[0055] In the case of the constructs having a PAL promoter, the
accumulation of harpin.sub.pss was detected in 80% or more of
individuals. As anticipated, PALL had a larger proportion of
high-expression individuals (++) than PALS. On the other hand, in
the case of the construct having a 35S promoter, though no
accumulation of harpin.sub.pss was detected in 6 individuals of the
15 individuals, high-expression individuals were obtained in 7
individuals, near half of the total individuals. Besides, a very
high expression (+++) was shown in 6 individuals. Interestingly, no
morphological change was observed in the organ of any of a leaf, a
stem, a root or a flower of these high-expression individuals, and
seed fertility was normal in almost all of them.
2) Western Analysis of the T.sub.1 Generation and Disease
Resistance Assay
[0056] Reaction to powdery mildew fungus (Erysiphe cichoracearum)
was analized in about 8 lines of KH1-2 (PALS-hrpZ), KC6-7
(PALL-hrpZ), KC8-1 (PALL-hrpZ), KK1-1 (35S-hrpZ), KK3-8 (35S-hrpZ),
KK4-2 (35S-hrpZ), KK4-3 (35S-hrpZ), KK7-6 (35S-hrpZ), in which the
amount of harpin.sub.pss accumulated was high in the T.sub.0
generation.
[0057] Tobacco individuals in which harpin.sub.pss was accumulated
at a high level in the T.sub.0 generation were selected, and seeds
of self-pollinated next generation (T.sub.1) thereof were obtained.
The seeds were sowed and observed for about two months, but no
visual morphological change was observed for this period; they grew
normally in the same manner as the T.sub.0 generation, and no
hypersensitive response was observed on the surface of a leaf.
Then, powdery mildew fungi were sprayed to inoculate upon the
T.sub.1 generation of the transgenic tobacco of the 4 or 5 leaf
stage and a disease resistance assay was performed. About 2 L of a
suspension of powdery mildew fungi spores (1.4.times.10.sup.6
spores/ml) was spray-inoculated to 244 recombinants and 41 original
individuals. As a result, hypersensitive-response-like localized
necrosis spots were induced onto a lower leaf of the recombinant 4
or 5 days after inoculation (FIG. 3A, B). Surprisingly, not only in
the case of the PAL-hrpZ constructs but also in the case of the
35S-hrpZ constructs employing a constitutive promoter, specific
localized necrosis spots were induced after the pathogen infection
(FIG. 3B). The expression frequency of localized necrosis spots on
the 5th day after the inoculation was about 5% in the
non-transformants, but the frequency was from 6 to 14 times grater
in the 35S-hrpZ construct (30 to 71%), from 4 to 5 times greater in
the PAL-hrpZ constructs (20 to 27%) (Table 2), and thereafter, in
the case of the PAL-hrpZ constructs, the number of local necrosis
spots gradually increased. This was assumed to be due to the
response of the PsPAL1 promoter to Erysiphe cichoracearum. Though
the amount of harpin.sub.pss accumulated and the degree of the
formation of localized necrosis spots tended to be positively
correlative (Table 3), there were some exceptional transformants in
which no accumulation of harpin.sub.pss was detected at least in
our Western analysis but localized necrosis spots occurred.
[0058] Next, in order to examine whether the localized necrosis
spots having occurred after the powdery mildew infection were
related to disease resistance, the symptom of powdery mildew on the
11th day after the inoculation thereof was examined. As a result,
while there existed no individual in which the spread of powdery
mildew hyphae was prevented in the non-transformants, from 15 to
57% individuals in the case of 35S-hrpZ constructs and from 13 to
18% individuals in the case of PAL-hrpZ constructs showed
apparently less significant symptom as compared to the
non-transformants (FIG. 4, Table 2). The prevenstion of that the
spread of powdery mildew was observed not only in leaves with
localized necrosis spots but also in middle or upper leaves with no
localized necrosis spots, and this is thought to be due to systemic
acquired resistance (SAR). As a result of observing the hyphae of
powdery mildew by cotton blue dyeing, the hyphae of powdery mildew
extended sharply and spread around the surface in infested leaves
of the SR1 of the original line as a control, whereas, though
haustorium is formed on the surface of a leaf in the transformants,
the spreading of hyphae was prevented and stopped halfway. The
promoters employed in the present studies are 35S promoter
(constitutive) and PAL promoter (inducible); and it was found that
when 35S promoter was employed instead of PAL promoter, the
frequency of localized necrosis spots was higher, and it was
further found that at least according to examination on the 11th
day after inoculation, more individuals with a strong disease
resistance were obtained (Table 2). However, it was observed that,
in the case of employing the 35S promoter, the localized necrosis
spots formed in response to the pathogen became larger (occupying
10% or more of the leaf area) in some individuals, and as a result,
lower leaves died out. In addition, inversely, in some individuals
with harpin.sub.pss accumulated therein, localized necrosis spots
were not observable by the naked eye (Table 2), but some of such
individuals had resistance to powdery mildew (of individuals with -
of localized necrosis spots in Table 2, individuals of the number
in parentheses; the amount of harpin.sub.pss expressed is ++ in
all). This is thought to be probably due to the occurrence of a
hypersensitive response in very small range, and it is possible
that a disease-resistant plant with a high practicability can be
obtained by the selection of such individuals. According to the
fact that no localized necrosis spot occurred without the invasion
of the pathogen even in the case where the transription of hrpZ
gene was controlled with a constitutive promoter, it is possible to
deduce that, since harpin.sub.pss was recognized on the outside of
a transmembrane or cell wall of plant cells, probably
harpin.sub.pss accumulated in cytoplasm was not recognized for
plant cells till the degradation of cells due to the invasion of
the fungi, and as a result, it caused a hypersensitive response
after the inoculation of the pathogen. Another possibility may be
that the elicitor activity of harpin.sub.pss requires the existence
of some other factors derived from the pathogen or the plant,
induced by the inoculation of the pathogen.
TABLE-US-00004 TABLE 2 Relationship among the Amount of
harpin.sub.pss Accumulated, the Formation of Localized Necrosis
Spots and Disease Resistance of the Tobacco T.sub.1 Generation
Expression level Number of Line Name Construct (T.sub.0)
individuals analyzed (T.sub.1) KH1-2 PALS-hrpZ ++ 18 KC6-7
PALL-hrpZ ++ 43 KC8-1 PALL-hrpZ ++ 44 KK1-1 35S-hrpZ +++ 23 KK3-8
35S-hrpZ +++ 33 KK4-2 35S-hrpZ ++ 35 KK4-3 35S-hrpZ +++ 7 KK7-6
35S-hrpZ +++ 41 SR1 (control) - 41 Number of individuals with
localized necrosis spots Rate of individuals Rate of individuals
(Number of individuals with with localized with less progress less
progress of disease necrosis spots of disease spots spots) (5th day
after (11th day after Line Name +++ ++ + -.sup.a inoculation)
inoculation) KH1-2(PALS) 0 0 5 (3) 13 (0) 27% 16% KC6-7(PALL) 0 1
(1) 8 (6) 34 (1) 20% 18% KC8-1(PALL) 0 1 (0) 11 (5) 32 (1) 27% 13%
KK1-1(35S) 0 0 7 (3) 16 (1) 30% 17% KK3-8(35S) 0 2 (0) 11 (5) 20
(0) 39% 15% KK4-2(35S) 1 (1) 4 (3) 15 (6) 15 (0) 57% 28% KK4-3(35S)
0 3 (3) 2 (1) 2 (0) 71% 57% KK7-6(35S) 1 (1) 4 (4) 18 (4) 18 (1)
56% 24% SR1(control) 0 0 2 (0) 39 (0) 5% 0% .sup.aThe degree of
localized necrosis spots is shown in four stages (+++: very high,
++: high, +: low, -: nil).
TABLE-US-00005 TABLE 3 Relationship between the Expression level of
Harpin.sub.pss and the Number of Localized Necrosis Spots in the
Tobacco T.sub.1 Generation Incidence of Expression level localized
of harpin.sub.pss.sup.a Degree of localized necrosis spots.sup.b
necrosis (Western analysis) +++ ++ + - spots +++ 1 4 19 19 56% ++ 0
5 32 77 32% + 1 6 18 38 40% - 0 1 5 18 25% SR1 0 0 2 39 5%
.sup.aThe expression level of harpin.sub.pss is shown in four
stages (+++: particularly high expression, ++: high expression, +:
moderate to poor expression, -: below the detection limit) (SR1,
--). .sup.bThe degree of localized necrosis spots is shown in four
stages (+++: great many, ++: many, +: few, -: nil).
(2) Transgenic Rice
1) Western Analysis of the T.sub.0 Generation
[0059] Harpin.sub.pss was introduced into a rice variety,
Tsukinohikari. 35 individuals of the regenerated plant were
obtained from the 35S-hrpZ construct, and 26 individuals of the
regenerated plant were obtained from the PPDK-hrpZ construct. There
was observed no remarkable difference between the constructs in
transformation efficiency. Western analysis was performed on the
primary generation (T.sub.0) of the transformation and individuals
with a high expression were selected.
[0060] Protein was extracted from the regenerated transgenic rice
(Tsukinohikari) in the same manner as in the example of the tobacco
and subjected to Western analysis. The results of Western analysis
of the T.sub.0 generation are shown in Table 4.
TABLE-US-00006 TABLE 4 Results of the Western Analysis of the
T.sub.0 Generation of Rice (Tsukinohikari) Number of Expression
level regenerated of harpin.sub.pss.sup.a Construct individuals - +
++ +++.sup.b 35S-hrpZ 35 17 5 13 0 PPDK-hrpZ 26 9 13 4 0 .sup.aEach
numerical value shows the number of individuals showing each
expression level. .sup.bThe Expression level of harpin.sub.pss is
shown in four stages (+++: particularly high expression, ++: high
expression, +: moderate to poor expression, -: below the detection
limit).
[0061] In the case of the rice (Tsukinohikari), similar to the case
of the tobacco, individuals with a high-expression of
harpin.sub.pss were obtained (see also FIG. 2). In the case of a
construct having a 35S promoter, the accumulation of harpin.sub.pss
was detected in about half of the individuals, and the rate of
high-expression individuals (++) was about one-third or more of the
whole. Also, in the case of a PPDK promoter the accumulation of
harpin.sub.pss was detected in about two-thirds of the individuals,
and of them, 4 individuals showed a high expression. Interestingly,
no morphological change was observed in the organ of any of a leaf,
a root or a flower of these high-expression individuals. And seed
fertility was normal in almost all of them, and T.sub.1 seeds of
high-expression individuals could be obtained.
2) Western Analysis of the T.sub.0 Generation and the Disease
Resistance Assay of the T.sub.1 Generation
[0062] Next, harpin.sub.pss was introduced into Koshihikari, one of
the most important varieties of rice of Japan. The results of the
Western analysis of the T.sub.0 generation are shown in Table
5.
TABLE-US-00007 TABLE 5 Results of the Western Analysis of the
T.sub.0 Generation of Rice (Koshihikari) Number of Expression level
regenerated of harpin.sub.pss.sup.a Construct individuals - + ++
+++.sup.b 35S-hrpZ 78 18 33 21 6 PPDK-hrpZ 27 7 13 7 0 .sup.aEach
numerical value shows the number of individuals showing each
expression level. .sup.bThe expression level of harpin.sub.pss is
shown in four stages (+++: amount of accumulation of 0.5% or more
to the total soluble leaf proteins, ++: amount of accumulation of
from 0.1 to 0.5%, +: amount of accumulation of from 0.01 to 0.1%,
-: below the detection limit).
[0063] Of the individuals of the T.sub.0 generation with the
35S-hrpZ construct introduced thereinto, four individuals showning
a large amount (+++ in Table 5) of the accumulation of
harpin.sub.pss (hrp5-8, hrp23-5, hrp24-1, hrp42-9) were selected,
and their vulnerability to rice blast in the T.sub.1 generation was
examined. The seed fertility of the selected four high-expression
individuals was normal, and many self-fertilized seeds could be
obtained. T.sub.1 seeds were sowed in a seedling case with culture
soil in a manner of 8 seeds.times.2 rows, cultivated in a
greenhouse, and subjected to a disease assay at the 4.8 to 5.2 leaf
stage. As a rice blast fungus (Magneporthe grisea) was employed
race 007. For inoculation, a conidium formed by culturing the blast
fungi on an oatmeal sucrose agar medium at 28.degree. C. under dark
condition and then, after the spread of the fungi, at 25.degree.
C., irradiating near ultraviolet light for three days was employed.
The inoculation of the blast fungi was performed by
spray-inoculating 30 ml of a suspension adjusted to
1.5.times.10.sup.5 condia/ml in 0.02% Tween 20 per three seedling
cases. The spray-inoculated rice was held in a moistening incubator
(SLPH-550-RDS, manufactured by Nippon Medical & Chemical
Instruments Co. Ltd.) for 24 hours after the inoculation at
25.degree. C. at a humidity of 100%, and then transferred into the
greenhouse. The conditions of the greenhouse were set at 25.degree.
C. under light conditions for 16 hours, and at 22.degree. C. under
dark conditions for 8 hours. The evaluation of disease resistance
was performed by visually counting the number of progressive
disease spots on the 5th leaf at 6th day after the inoculation,
said leaf being the topmost development leaf at the time of
inoculation. Significant differences among the results were
evaluated according to the Mann-Whitney U test.
[0064] As a result, though no localized necrosis spot due to the
inoculation of the blast fungi was observed, the average number of
progressive disease spots was reduced by to 38% relative to the
control Koshihikari in three lines (hrp5-8, hrp42-9, hrp23-5) out
of the four lines of the harpin.sub.pss-introduced rice. Moreover,
this reduction was statistically significant (Table 6). The above
results show that the disease resistance of rice could be increased
by the introduction of harpin.sub.pss.
TABLE-US-00008 TABLE 6 Results of the Disease Test against Rice
Blast of the Four Lines of Harpin.sub.pss-Intorduced Rice (T.sub.1
Generation) Number of average Number of progressive tested disease
spots.sup.a Strain individuals (standard error) Significant
Test.sup.b hrp5-8 16 9.3 (.+-.1.0) significant (significance level
1%,) hrp23-5 21 11.4 (.+-.1.3) significant (significance level 5%)
hrp24-1 20 14.4 (.+-.1.4) No significant difference hrp42-9 14 9.4
(.+-.1.4) significant (significance level 1%) Koshihikari 64 15.0
(.+-.0.7) -- .sup.aResults of the 5th leaf on the 6th day after
inoculation .sup.bSignificant difference to Koshihikari in the
Mann-Whitney U test
[0065] As a result of the present invention, it has become apparent
for the first time that disease resistance can be imparted to a
plant by connecting a gene enconding harpin to a constitutive
promoter or an inducible promoter and introducing the gene into the
plant. This harpin-introduced plant is thought to be useful for
explicating the function of harpin as a protein elicitor, and also
for explicating the mechanism of localized or systemic acquired
resistance. In addition, it is revealed that the production of a
harpin-introduced resistant plant, which has been thought to be
difficult without the use of an inducible promoter, can
sufficiently be realized by employing a constitutive promoter, and
the extension of the application range of the present approach can
be shown. The present invention shows that a method for producing a
disease-resistant plant by integrating a DNA sequence encoding a
harpin into an expression cassette comprising a sequence of an
appropriate constitutive, or organ- or phase-specific promoter
capable of functioning in a plant cell, or a promoter induced with
stress or pests, and a sequence of a terminator capable of
functioning in a plant cell, and introducing it into the plant cell
to obtain a regenerated individual is a useful and effective
approach in view of genetic engineering.
Sequence CWU 1
1
711029DNAPseudomonas syringae pv. syringae LOB2-1 1atg cag agt ctc
agt ctt aac agc agc tcg ctg caa acc ccg gca atg 48Met Gln Ser Leu
Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met1 5 10 15gcc ctt gtc
ctg gta cgt cct gaa acc gag acg act ggc gcc agt acg 96Ala Leu Val
Leu Val Arg Pro Glu Thr Glu Thr Thr Gly Ala Ser Thr 20 25 30tcg agc
aag gcg ctt cag gaa gtt gtc gtg aag ctg gcc gag gaa ctg 144Ser Ser
Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu 35 40 45atg
cgc aat ggt caa ctc gac gac agc tcg cca ttg ggc aaa ctg ctg 192Met
Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu 50 55
60gcc aag tcg atg gcc gcg gat ggc aag gca ggc ggc ggt atc gag gat
240Ala Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu
Asp65 70 75 80gtc atc gct gcg ctg gac aag ctg att cat gaa aag ctg
ggt gac aac 288Val Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu
Gly Asp Asn 85 90 95ttc ggc gcg tct gcg gac aac gcc tcg ggt acc gga
cag cag gac ctg 336Phe Gly Ala Ser Ala Asp Asn Ala Ser Gly Thr Gly
Gln Gln Asp Leu 100 105 110atg act cag gtg ctc agt ggc ctg gcc aag
tct atg ctc gat gat ctt 384Met Thr Gln Val Leu Ser Gly Leu Ala Lys
Ser Met Leu Asp Asp Leu 115 120 125ctg acc aag cag gat ggc ggg gca
agc ttc tcc gaa gac gat atg ccg 432Leu Thr Lys Gln Asp Gly Gly Ala
Ser Phe Ser Glu Asp Asp Met Pro 130 135 140atg ctg aac aag atc gcg
cag ttc atg gat gac aat ccc gca cag ttt 480Met Leu Asn Lys Ile Ala
Gln Phe Met Asp Asp Asn Pro Ala Gln Phe145 150 155 160ccc aag ccg
gac tcg ggt tcc tgg gtg aac gaa ctc aag gaa gac aac 528Pro Lys Pro
Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn 165 170 175ttc
ctt gat ggc gac gaa acg gct gcg ttc cgc tcg gca ctc gac atc 576Phe
Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile 180 185
190att ggc cag caa ctg ggt aat cag cag agt ggc gct ggc ggt ctg gcg
624Ile Gly Gln Gln Leu Gly Asn Gln Gln Ser Gly Ala Gly Gly Leu Ala
195 200 205ggg acg ggt gga ggt ctg ggc act ccg agc agt ttt tct aac
aac tcg 672Gly Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn
Asn Ser 210 215 220tcc gtg acg ggt gat ccg ctg atc gac gcc aat acc
ggt ccc ggt gac 720Ser Val Thr Gly Asp Pro Leu Ile Asp Ala Asn Thr
Gly Pro Gly Asp225 230 235 240agc ggc aat agc agt ggt gag gcg ggg
caa ctg atc ggc gag ctt atc 768Ser Gly Asn Ser Ser Gly Glu Ala Gly
Gln Leu Ile Gly Glu Leu Ile 245 250 255gac cgt ggc ctg caa tcg gta
ttg gcc ggt ggt gga ctg ggc aca ccc 816Asp Arg Gly Leu Gln Ser Val
Leu Ala Gly Gly Gly Leu Gly Thr Pro 260 265 270gta aac acc ccg cag
acc ggt acg gcg gcg aat ggc gga cag tcc gct 864Val Asn Thr Pro Gln
Thr Gly Thr Ala Ala Asn Gly Gly Gln Ser Ala 275 280 285cag gat ctt
gac cag ttg ctg ggc ggc ttg ctg ctc aag ggc ctt gaa 912Gln Asp Leu
Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu 290 295 300gcg
acg ctc aag gat gcc ggt caa acc gct acc gac gtg cag tcg agc 960Ala
Thr Leu Lys Asp Ala Gly Gln Thr Ala Thr Asp Val Gln Ser Ser305 310
315 320gct gcg caa atc gcc acc ttg ctg gtc agt acg ctg ctg caa ggc
acc 1008Ala Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly
Thr 325 330 335cgc aat cag gct gca gcc tga 1029Arg Asn Gln Ala Ala
Ala 3402342prtPseudomonas syringae pv. syringae LOB2-1 2Met Gln Ser
Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met1 5 10 15Ala Leu
Val Leu Val Arg Pro Glu Thr Glu Thr Thr Gly Ala Ser Thr 20 25 30Ser
Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu 35 40
45Met Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu
50 55 60Ala Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu
Asp65 70 75 80Val Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu
Gly Asp Asn 85 90 95Phe Gly Ala Ser Ala Asp Asn Ala Ser Gly Thr Gly
Gln Gln Asp Leu 100 105 110Met Thr Gln Val Leu Ser Gly Leu Ala Lys
Ser Met Leu Asp Asp Leu 115 120 125Leu Thr Lys Gln Asp Gly Gly Ala
Ser Phe Ser Glu Asp Asp Met Pro 130 135 140Met Leu Asn Lys Ile Ala
Gln Phe Met Asp Asp Asn Pro Ala Gln Phe145 150 155 160Pro Lys Pro
Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn 165 170 175Phe
Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile 180 185
190Ile Gly Gln Gln Leu Gly Asn Gln Gln Ser Gly Ala Gly Gly Leu Ala
195 200 205Gly Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn
Asn Ser 210 215 220Ser Val Thr Gly Asp Pro Leu Ile Asp Ala Asn Thr
Gly Pro Gly Asp225 230 235 240Ser Gly Asn Ser Ser Gly Glu Ala Gly
Gln Leu Ile Gly Glu Leu Ile 245 250 255Asp Arg Gly Leu Gln Ser Val
Leu Ala Gly Gly Gly Leu Gly Thr Pro 260 265 270Val Asn Thr Pro Gln
Thr Gly Thr Ala Ala Asn Gly Gly Gln Ser Ala 275 280 285Gln Asp Leu
Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu 290 295 300Ala
Thr Leu Lys Asp Ala Gly Gln Thr Ala Thr Asp Val Gln Ser Ser305 310
315 320Ala Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly
Thr 325 330 335Arg Asn Gln Ala Ala Ala 340330DNAArtificial
SequenceSynthetic primer derived from Pseudomonas syringae pv.
Syringae 61 3aaaatctaga atgcagagtc tcagtcttaa 30430DNAArtificial
SequenceSynthetic primer derived from Pseudomonas syringae pv.
Syringae 61 4aaaagtcgac tcaggctgca gcctgattgc 30532DNAArtificial
SequenceSynthetic primer derived from the reported PSPAL1 promoter
5ggggtctaga attgatacta aagtaactaa tg 32632DNAArtificial
SequenceSynthetic primer derived from the reported PSPAL1 promoter
6ttggaagctt agagatcatt acgaaattaa gg 32729DNAArtificial
SequenceSynthetic primer derived from the reported PSPAL1 promoter
7ctaaaagctt ggtcatgcat ggttgcttc 29
* * * * *