Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PLANT PATHOGEN RESISTANCE GENES AND USES THEREOF
The present invention relates to pathogen resistance
in plants and more particularly the identification and
use of pathogen resistance genes. It is based on cloning
of the tomato Cf-2 gene.
Plants are constantly challenged by potentially
pathogenic microorganisms. Crop plants are particularly
vulnerable, because they are usually grown as genetically
uniform monocultures; when disease strikes, losses can
be severe. However, most plants are resistant to most
plant pathogens. To defend themselves, plants have
evolved an array of both preexisting and inducible
defences. Pathogens must specialize to circumvent the
defence mechanisms of the host, especially those
biotrophic pathogens that derive their nutrition from an
intimate association with living plant cells. If the
pathogen can cause disease, the interaction is said to be
compatible, but if the plant is resistant, the
interaction is said to be incompatible. Race specific
resistance is strongly correlated with the hypersensitive
response (HR), an induced response by which (it is
hypothesized) the plant deprives the pathogen of living
host cells by localized cell death at sites of attempted
pathogen ingress.
It has long been known that HR-associated disease
resistance is often (though not exclusively) specified by
dom~n~nt genes (R genes). Flor showed that when
pathogens mutate to overcome such R genes, these
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mutations are recessive. Flor concluded that for ~ genes
to function, there must also be corresponding genes in
the pathogen, denoted avirulence genes (Avr genes). To
become virulent, pathogens must thus stop making a
product that activates R gene-dependent defence
mechanisms (Flor, 1971). A broadly accepted working
hypothesis, often termed the elicitor/receptor model, is
that R genes encode products that enable plants to detect
the presence of pathogens, provided said pathogens carry
the corresponding Avr gene (Gabriel and Rolfe, 1990).
This recognition is then transduced into the activation
of a defence response.
Some interactions exhibit different genetic
properties. Helminthosporium carbonum races that express
a toxin (Hc toxin) infect maize lines that lack the ~ml
resistance gene. Mutations to loss of Hc toxin
expression are recessive, and correlated with loss of
virulence, in contrast to gene-for-gene interactions in
which mutations to virulence are recessive. A major
accomplishment was reported in 1992, with the isolation
by tagging of the ~ml gene (Johal and Briggs, 1992).
Plausible arguments have been made for how gene-for-gene
interactions could evolve from toxin-dependent virulence.
For example, plant genes whose products were the target
of the toxin might mutate to confer even greater
sensitivity to the toxin, leading to HR, and the
conversion of a sensitivity gene to a resistance gene.
However, this does not seem to be the mode of action of
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~ml, whose gene product inactivates Hc toxin.
Pathogen avirulence genes are still poorly
understood. Several bacterial Avr genes encode
hydrophilic proteins with no homology to other classes of
protein, while others carry repeating units whose number
can be modified to change the range of plants on which
they exhibit avirulence (Keen, 1992; Long and Staskawicz,
1993). Additional bacterial genes (hrp genes) are
required for bacterial Avr genes to induce HR, and also
for pathogenicity (Keen, 1992; Long and Staskawicz,
1993). It is not clear why pathogens make products that
enable the plant to detect them. It is widely believed
that certain easily discarded Avr genes contribute to but
are not required for pathogenicity, whereas other Avr
genes are less dispensable (Keen, 1992; Long and
Staskawicz, 1993). The characterization of one fungal
avirulence gene has also been reported; the Avr9 gene of
Cladosporium fulvum, which confers avirulence on C.
fulvum races that attempt to attack tomato varieties that
carry the Cf-9 gene, encodes a secreted cysteine-rich
peptide with a final processed size of 28 amino acids but
its role in compatible interactions is not clear (De Wit,
1992).
The technology for gene isolation based primarily on
genetic criteria has improved dramatically in recent
years, and many workers are currently attempting to clone
a variety of R genes. Targets include (amongst others)
rust resistance genes in maize, Antirrhinum and flax (by
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transposon tagging); down.y mildew resistance genes in
lettuce and Arabidopsis (by map based cloning and T-DNA
tagging); Cladosporium fulvum (Cf) resistance genes in
tomato (by tagging, map based cloning and affinity
labelling with avirulence gene products); virus
resistance genes in tomato and tobacco (by map based
cloning and tagging); nematode resistance genes in tomato
(by map based cloning); and genes for resistance to
bacterial pathogens in Arabidopsis and tomato (by map
based cloning).
The map based cloning of the tomato Pto gene that
confers "gene-for-gene" resistance to the bacterial speck
pathogen Psen~om~n~-~ syringae pv tomato (Pst) has been
~ reported (Martin et al, 1993). A YAC (yeast artificial
chromosome) clone was identified that carried restriction
~ fragment length polymorphism (RFLP) markers that~were
very tightly linked to the gene. This YAC was used to
isolate homologous cDNA clones. Two of these cDNAs were
fused to a strong promoter, and after transformation of a
disease sensitive tomato variety, one of these gene
fusions was shown to confer resistance to Pst strains
that carry the corresponding avirulence gene, AvrPto.
These two cDNAs show homology to each other. Indeed, the
Pto cDNA probe reveals a small gene family of at least
six members, 5 of which can be found on the YAC from
which Pto was isolated, and which thus comprise exactly
the kind of local multigene family inferred from genetic
analysis of other R gene loci.
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The Pto gene cDNA sequence is puzzling for
proponents of the simple elicitor/receptor model. It
reveals unambiguous homology to serine/threonine kinases,
consistent with a role in signal transduction
Intriguingly, there is strong homology to the kinases
associated with self incompatibility in Brassicas, which
carry out an analogous role, in that they are required to
prevent the growth of genotypically defined incompatible
pollen tubes. However, in contrast to the Brassica SRK
kinase (Stein et al 1991), the Pto gene appears to code
for little more than the kinase catalytic domain and a
potential N-terminal myristoylation site that could
promote association with membranes. It would be
surprising if such a gene product could act alone to
accomplish the specific recognition required to initiate
the defence response only when the AvrPto gene is
detected in invading microrganisms. The race-specific
elicitor molecule made by Pst strains that carry AvrPto
is still unknown and needs to be characterized before
possible recognition of this molecule by the Pto gene
product can be investigated.
Since the isolation of the Pto gene a number of
other resistance genes have been isolated. The isolation
of the tobacco mosaic resistance gene N from tobacco was
reported by Whitham et al (1994). The isolation of the
Ara~idopsis thaliana gene for resistance to Pseudomonas
syringae RPS2 was reported by Bent et al (1994) and by
Mindrinos et al (1994). These genes probably encode
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cytoplasmic proteins that carry a P-loop and a leucine-
rich repeat. The ligands with which they interact are
uncharacterised and it is not known what other plant
proteins they interact with to accomplish the defence
response. Our own laboratory has reported the isolation
of the tomato Cf-9 which confers resistance against the
fungus Cladosporium fulvum. This is a subject of a
previous patent application (PCT/GB94/02812 published as
WO 95/18230) and has been reported in Jones et al (1994).
Cf-9 and Avr9 sequences, and sequences of the encoded
polypeptides are given in WO95/18320 and Jones et al
(1994).
We have now cloned Cf-2 genes.
WO93/11241 reports the sequence of a gene encoding a
polygalacturonase inhibitor protein (PGIP) that has some
homology with Cf-9 and, as we have now discovered, Cf-2
(the subject of the present invention). Cf-9, Cf-2 and
others (Cf-4, .5 etc.) are termed by those skilled in the
art "pathogen resistance genes" or "disease resistance
genes". PGIP-encoding genes are not pathogen resistance
genes. A pathogen resistance gene (R) enables a plant to
detect the presence of a pathogen expressing a
corresponding avirulence gene (Avr). When the pathogen
is detected, a defence response such as the
hypersensitive response (HR) is activated. By such means
a plant may deprive the pathogen of living cells by
localised cell death at sites of attempted pathogen
ingress. On the other hand, the PGIP gene of WO93/11241
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(for example) is a gene of the kind that is induced in
the plant defence response resulting from detection of a
pathogen by an R gene.
Thus, a pathogen resistance gene may be envisaged as
encoding a receptor to a pathogen-derived and Avr
dependent molecule. In this way it may be likened to the
RADAR of a plant for detection of a pathogen, whereas
PGIP is involved in the defence the plant mounts to the
pathogen once detected and is not a pathogen resistance
gene. Expression of a pathogen resistance gene in a
plant causes activation of a defence response in the
plant. This may be upon contact of the plant with a
pathogen or a corresponding elicitor molecule, though the
possibility of causing activation by over-expression of
the resistance gene in the absence of elicitor has been
reported. The defence response may be activated locally,
e.g. at a site of contact of the plant with pathogen or
elicitor molecule, or systemically. Activation of a
defence response in a plant expressing a pathogen
resistance gene may be caused upon contact of the plant
with an appropriate, corresponding elicitor molecule,
e.g. as produced by a Cl adosporium f ul vum avr gene as
discussed. The elicitor may be contained in an extract
of a pathogen such as Cl adosporium f ul vum, or may be
wholly or partially purified and may be wholly or
partially synthetic. An elicitor molecule may be said to
"correspond" i~ it is a suitable ligand for the R gene
product to elicit activation of a defence response
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The "Cf-x"/"Avrx" terminology is standard in the
art. The Cf resistance genes and corresponding fungal
avirulence genes (Avr) were originally defined
genetically as interacting pairs of genes whose
measurable activities fall into mutually exclusive
interacting pairs. Avr9 elicits a necrotic response on
Cf-9 containing tomatoes but no response on Cf-2
containing tomatoes, the moeity recognised by Cf-2 being
different from that recognised by Cf-9.
Expression of Cf-2 function in a plant may be
determined by investigating compatibility of various C.
fulvum races.
A race of C. fulvum that carries functional copies
of all known Avr genes (race 0) will grow (compatible)
only on a tomato which lacks all the Cf genes. It will
not grow (incompatible) on a plant carrying any
functional Cf gene. If the C. fulvum race lacks a
functional Avr2 gene (race 2) it will be able to grow not
only on a plant lacking any Cf genes but also a plant
carrying the Cf-2 gene. A race also lacking a functional
Avr4 gene (race 2,4) will also be able to grow on a plant
carrying the Cf-4 gene. A race only lacking a functional
Avr4 gene (race 4) will not be able to grow on a plant
carrying Cf-2. Similarly, a C. fulvum race 5 (lacking a
functional Avr5 gene) will not be able to grow on a plant
carrying a Cf-2 gene. ~either a race 4 nor a race 2,4
will be able to grow on a plant carrying any of the other
Cf genes. Various races are commonly available in the
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art, e.g. from the Research Institute for Plant
Protection (IPO-DLO), PO Box 9060, 6700 GW Wageningen,
The Netherlands. A race 4 is available under accession
number IPO10379 and a race 2,4 available under Accession
number IPOS0379.
We have now isolated two almost identical tomato
genes, Cf-2 .1 and Cf-2 . 2, which confer resistance against
the fungus Cladosporium fulvum and we have sequenced the
DNA and deduced the amino acid sequence from these genes.
(Both genes are almost identical and any statement made
herein about one should be considered as applying to
both, unless context de~n~.q otherwise.) The DNA
sequence of the tomato Cf-2. 1 genomic gene is shown in
Figure 2 (SEQ ID NO. 1) and the deduced amino acid
sequences (for both genes) are shown in Figure 3A and B
(SEQ ID NO's 2 and 3).
As described in more detail below, the tomato ef-2
genes were isolated by map-based cloning. In this
technique the locus that confers resistance is mapped at
high resolution relative to restriction fragment length
polymorphism (RFLP) markers that are linked to the
resistance gene. We identified a marker that appeared to
be absolutely linked to the resistance gene and used
probes corresponding to this marker to isolate binary
vector cosmid clones from a stock that carried the Cf-2
gene locus. Two independent overlapping clones conferred
disease resistance and the region of overlap contains a
reading-frame which shows remarkable structural
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W O96~0518 PCTI~b~ C/o~
resemblance to the Cf-9 gene. Since this sequence is the
primary constituent of the DNA that overlaps the two
clones that complement, we are confident that this
sequence must correspond to the Cf-2 gene. A second
almost identicaI region on one of the cosmids was also
able to confer disease resistance, indicating that there
are two functional Cf -2 genes).
According to one aspect, the present invention
provides a nucleic acid isolate encoding a pathogen
resistance gene, the gene being characterized in that it
comprises nucleic acid encoding the amino acid sequence
shown in SEQ ID N0 2 or SEQ ID N0. 3 or a fragment
thereof. The nucleic acid isolate may comprise DNA, and
may comprise the sequence shown in SEQ ID NO 1 or a
sufficient part to encode the desired polypeptide (eg.
from the initiating methionine codon to the first in
frame downstream stop codon). In one embodiment the DNA
comprises a sequence of nucleotides which are the
nucleotides 1677 to 5012 of SEQ ID NO 1, or a mutant,
derivative or allele thereof. A further aspect of the
invention provides a nucleic acid isolate encoding a
pathogen resistance gene, or a fragment thereof,
obtainable by screening a nucleic acid library with a
probe comprising nucleotides 1677 to 5012 of SEQ ID NO 1,
or a fragment, derivative, mutant or allele thereof, and
isolating DNA which encodes a polypeptide able to confer
pathogen resistance to a plant, such as resistance to
Cladosporium fulvum (eg. expressing Avr2) . The plant may
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be tomato. Suitable techniques are well known in the
art.
Nucleic acid according to the present invention may
encode the amino acid sequence shown in SEQ ID NO 2 or a
mutant, derivative or allele of the sequence provided
e.g. SEQ ID NO 3. Preferred mutants, derivatives and
alleles are those which retain a functional
characteristic of the protein encoded by the wild-type
gene, especially the ability to confer pathogen
resistance and most especially the ability to confer
resistance against a pathogen expressing the Avr2
elicitor molecule. Changes to a sequence, to produce a
mutant or derivative, may be by one or more of addition,
insertion, deletion or substitution of one or more
nucleotides in the nucleic acid, leading to the addition,
insertion, deletion or subsitution of one or more amino
acids. Of course, changes to the nucleic acid which make
no difference to the encoded amino acid sequence are
included.
Preferred embodiments of nucleic acid encoding the
amino acid sequences shown in Figure 3 (SEQ ID NO.'s 2
and 3) include encoding sequences shown in Figures 2 and
4, respectively. To encode amino acid SEQ ID NO. 3
(Figure 3b), DNA may comprise a nucleotide sequence shown
in Figure 4 (SEQ ID NO. 5).
Also provided by an aspect of the present invention
is nucleic acid comprising a sequence of nucleotides
complementary to a nucleotide sequence hybridisable with
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any encoding sequence provided herein. Another way of
looking at this would be for nucleic acid according to
this aspect to be hybridisable with a nucleotide sequence
complementary to any encoding sequence provided herein.
Of course, DNA is generally double-stranded and blotting
techniques such as Southern hybridisation are often
performed following separation of the strands without a
distinction being drawn between which of the strands is
hybridising. Preferably the hybridisable nucleic acid or
its complement encode a polypeptide able to confer
pathogen resistance on a host, i.e., includes a pathogen
re~istance gene. Preferred conditions for hybridisation
are familiar to those skilled in the art, but are
generally stringent enough for there to be positive
hybridisation between the sequences of interest to the
exlucsion of other sequences.
Although the polypeptides encoded by the Cf-2 and
Cf-9 genes share a high degree of homology, the genes
themselves are not sufficiently homologous to identify
each other in genomic Southern blotting using a
stringency of 2 x SSC at 60~C. In a BLASTN search, the
highest level of identity between the DNA sequences of
Cf-2 and Cf-9 is 69~ over a 428 base region.
Nucleic acid according to the present invention, for
instance mutants, derivatives and alleles of the specific
sequences disclosed herein, may be distinguished from Cf-
9 by one or more of the following:
- not being sufficiently homologous with Cf-9 for the
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nucleic acid of the invention and Cf-9 to identify each
other in Southern blotting using a stringency of 2 x SSC
at 60~C;
- havlng greater than 70~, preferably greater than
about 75~, greater than about 80~, greater than about 90
or greater than about 95~ homology with the encoding-
sequence shown in Figure 2 as nucleotides 1677-5012;
- eliciting ~ defence response, in a plant expressing
the nucleic acid, upon contact of the plant with Avr2
elicitor molecule, e.g. as provided by a Cladosporium
fulvum race expressing Avr2;
- eliciting a defence response, in a plant expressing
the nucleic acid, upon contact of the plant with the C.
fulvum race 4 deposited at and available from the
Research Institute for Plant Protection (IP0-DL0), PO Box
9060, 6700 GW Wageningen, The Netherlands, under
accession number IPO10379, or an extract thereof, but not
eliciting a defence response in the plant upon its
contact with the C. fulvum race 2,4 deposited at and
available from the same institute under Accession number
IPO50379, or an extract thereof;
- not eliciting a defence response, in a plant
expressing the nucleic acid, upon contact of the plant
with Avr9 elicitor molecule, e.g. as provided by a
Cladosporium fulvum race or other organism expressing
Avr9 (de Wit, 1992), the amino acid and encoding nucleic
acid sequences of chimaeric forms of which are given for
example in WO95/18230 as SEQ ID NO 3 and in w095/31564 as
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14
SEQ ID NO 4.
- comprising 38 leucine rich repeats (LRR's).
The nucleic acid isolate, which may contain DNA
encoding the amino acid sequence of SEQ ID NO 2 or SEQ ID
NO 3 as genomic DNA or cDNA, may be in the form of a
recombinant vector, for example a phage or cosmid vector.
The DNA may be under the control of an appropriate
promoter and regulatory elements for expression in a host
cell, for example a plant cell. In the case of genomic
DNA, this may contain its own promoter and regulatory
elements and in the case of cDNA this may be under the
control of an appropriate promoter and regulatory
elements for expression in the host cell.
Those skilled in the art are well able to construct
vectors and design protocols for recombinant gene
expression. Suitable vectors can be chosen or
constructed, containing appropriate regulatory sequences,
including promoter sequences, terminator fragments,
polyadenylation seuqences, enhancer sequences, marker
genes and other sequences as appropriate. For further
details see, for example, Molecular Cloning: a Laboratory
M~nu~ 7: 2nd edition, Sambrook et al, 1989, Cold Spring
Harbor Laboratory Press.
Nucleic acid molecules and vectors according to the
present invention may be provided isolated and/or
purified from their natural environment, in substantially
pure or homogeneous form, or free or substantially free
of nucleic acid or genes of the species of interest or
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origin other than the sequence encoding a polypeptide
with the required function. Nucleic acid according to
the present invention may comprise cDNA, RNA, genomic DNA
and may be wholly or partially synthetic. The term
"isolate" encompasses all these possibilities.
When introducing a chosen gene construct into a
cell, certain considerations must be taken into account,
well known to those skilled in the art. The nucleic acid
to be inserted may be assembled within a construct which
contains effective regulatory elements which will drive
transcription. There must be available a method of
transporting the construct into the cell. Once the
construct is within the cell membrane, integration into
the endogenous chromosomal material may or may not occur
according to different embodiments of the invention.
Finally, as far as plants are concerned the target cell
type must be such that cells can be regenerated into
whole plants.
Plants transformed with the DNA segment containing
the sequence may be produced by standard techniques which
are already known for the genetic manipulation of plants.
DNA can be transformed into plant cells using any
suitable technology, such as a disarmed Ti-plasmid vector
carried by Agrobacterium exploiting its natural gene
trans~er ability (EP-A-270355, EP-A-0116718, NAR 12(22)
8711 - 87215 1984), particle or microprojectile
bombardment (US 5100792, EP-A-444882, EP-A-434616)
microinjection (WO 92/09696, WO 94/00583, EP 331083, EP
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16
175966), electroporation (EP 290395, WO 8706614) or other
forms of direct DNA uptake (DE 4005152, WO 9012096, US
4684611). Agrobacterium transformation is widely used by
those skilled in the art to transform dicotyledonous
species. Although Agrobacterium has been reported to be
able to transform foreign DNA into some monocotyledonous
species (WO 92/14828), microprojectile bombardment,
electroporation and direct DNA uptake are preferred where
Agrobacterium is inefficient or ineffective.
Alternatively, a combination of different techniques may
be employed to enhance the efficiency of the
transformation process, eg. bombardment with
Agrobacterium coated microparticles (EP-A-486234) or
mircoprojectile bombardment to induce wounding followed
by co-cultivation with Agrobacterium (EP-A-486233).
The particular choice of a transformation technology
will be determined by its efficiency to transform certain
plant species as well as the experience and preference of
the person practising the invention with a particular
methodology of choice. It will be apparent to the
skilled person that the particular choice of a
transformation system to introduce nucleic acid into
plant cells is not essential to or a limitation of the
invention.
A Cf-2 gene and modified versions thereof (alleles,
mutants and derivatives thereof), and other nucleic acid
provided herein may be used to confer resistance in
plants, in particular tomatoes, to a pathogen such as c.
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fulvum. This may include cloned DNA from Lycopersicon
pimpinellifolium which has the same chromosomal location
as the Cf-2 gene or any subcloned fragment thereof. For
this purpose a vector as described above may be used for
the production of a transgenic plant. Such a plant may
possess pathogen resistance conferred by the Cf-2 gene.
The invention thus further encompasses a host cell
transformed with such a vector, especially a plant or a
microbial cell. Thus, a host cell, such as a plant cell,
comprising nucleic acid according to the present
invention is provided. Within the cell, the nucleic acid
may be incorporated within the chromosome.
A vector comprising nucleic acid according to the
present invention need not include a promoter,
particularly if the vector is to be used to introduce the
nucleic acid into cells ~or recombination into the
genome.
Also according to the invention there is provided a
plant cell comprising, e.g. having incorporated into its
genome a sequence of nucleotides as provided by the
present invention, under operative control of a promoter
~or control of expression of the encoded polypeptide. A
~urther aspect of the present invention provides a method
of making such a plant cell involving introduction of a
vector comprising the sequence of nucleotides into a
plant cell. Such introduction may be followed by
recombination between the vector and the plant cell
genome to introduce the sequence o~ nucleotides into the
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genome. The polypeptide encoded by the introduced
nucleic acid may then be expressed.
A plant which comprises a plant cell according to
the invention is also provided, along with any part or
clone of such a plant, seed, selfed or hybrid progeny and
descendants, and any part of these, such as cuttings,
seed. The invetion provides any plant propagule, that is
any part which may be used in reproduction or
propagation, sexual or asexual, including cuttings, seed
and so on.
The invention further provides a method of
comprising expression from nucleic acid encoding the
amino acid sequence SEQ ID N0 2 or SEQ ID N0 3, or a
mutant, allele or derivative of either sequence, within
cells of a plant (thereby producing the encoded
polypeptide), following an earlier step of introduction
of the nucleic acid into a cell of the plant or an
ancestor thereof. Such a method may confer pathogen
resistance on the plant. This may be used in combination
with the Avr2 gene according to any of the methods
described in WO91/15585 (Mogen) or, more preferably,
PCT/GB95/01075 (published as WO 95/31564), or any other
gene involved in conferring pathogen resistance.
The Cf-2 and Cf-9 genes function in a similar manner
in that they both confer a resistance to tomato that
prevents the growth of tomato leaf mould C. f ul vum .
They, however, by recognition of different Avr products
and have subtle differences in the speed with which they
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stop growth of the pathogen and stimulate a resistance
response (Hammond-Kosack and Jones 1994; Ashfield et al
1994). These differences may be exploited to optimise
applications disclosed herein.
A gene stably incorporated into the genome of a
plant is passed from generation to generation to
descendants of the plant, cells of which decendants may
express the encoded polypeptide and so may have enhanced
pathogen resistance. Pathogen resistance may be
determined by assessing compatibility of a pathogen (eg.
Cladosporium fulvum) or using recombinant expression of a
pathogen avirulence gene, such as Avr-2 or delivery of
the Avr-2 gene product.
Sequencing of the Cf-2 gene has shown that like the
Cf-9 gene it includes DNA sequence encoding leucine-rich
repeat (LRR) regions and homology searching has revealed
strong homologies to other genes containing hRRs. The
Cf-2 and Cf-9 genes contain all the same general features
and as such form a new class of disease resistance genes
separate from other disease resistance genes
characterised to date. As discussed in WO 95/18230, and
validated herein, the presence of LRRs may be
characteristic of many pathogen resistance genes and the
; presence of LRRs may be used in identifying further
pathogen resistance genes.
Furthermore, there are some striking homologies
between Cf-9 and Cf-2. These homologies may also be used
to identify further resistance genes of this class, for
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example using oligonucleotides (e.g. a degenerate pool)
designed on the basis of sequences conserved (preferably
at the amino acid level) between the Cf -9 and the
Cf-2 genes.
According to a further aspect, the present invention
provides a method of identi~ying a plant pathogen
resistance gene comprising use of an oligonucleotide
which comprises a sequence or sequences that are
conserved between pathogen resistance genes such as Cf-9
and Cf-2 to search for new resistance genes. Thus, a
method o~ obtaining nucleic acid comprising a pathogen
resistance.gene (encoding a polypeptide able to confer
pathogen resistance) is provided, Somprising
hybridisation of an oligonucleotide (details of which are
discussed herein) or a nucleic acid molecular comprising
such an oligonucleotide to target/candidate nucleic acid.
Target or candidate nucleic acid may, for example,
comprise a genomic or cDNA library obtainable from an
organism known to encode a pathogen resistance gene.
Successful hybridisation may be identified and
target/candidate nucleic acid isolated for further
investigation and/or use.
Hybridisation may involve probing nucleic acid and
identifying positive hybridisation under suitably
stringent conditions (in accordance with known
techniques) and/or use of oligonucleotides as primers in
a method of nucleic acid amplification, such as PCR. For
probing, preferred conditions are those which are
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stringent enough for there to be a simple pattern with a
small number of hybridisations identified as positive
which can be investigated further. It is well known in
the art to increase stringency of hybridisation gradually
until only a few positive clones remain.
As an alternative to probing, though still employing
nucleic acid hybridisation, oligonucleotides designed to
amplify DNA sequences may be used in PCR reactions or
other methods involving amplification of nucleic acid,
using routine procedures. See for instance "PCR
protocols; A Guide to Methods and Applications", Eds.
Ihnis et al. 1990, Academic Press, New York.
Preferred amino acid sequences suitable for use in
the design of probes or PCR primers are sequences
conserved (completely, substantially or partly) between
polypeptides able to confer pathogen resistance such as
those encoded by Cf-2 and Cf-9.
On the basis of amino acid sequence information,
oligonucleotide probes or primers may be designed, taking
into account the degeneracy of the genetic code, and,
where appropriate, codon usage of the organism from the
candidate nucleic acid is derived. Preferred nucleotide
sequences may include those comprising or having a
sequence encoding amino acids (i) SGEIPOO; (ii)
YE/OGNDG; (iii) FEGHIPS; or (iv) SGEIPOOLASLTSLE, or a
sequence complementary to these encoding sequences.
Suitable fragments of these may be employed.
Preferred oligonucleotide sequences include:-
CA 0221~496 1997-09-16
W O96/30518 PCT/GB96/00785
(i) TCX-GGX-GAA/G-AAT.C.A-CCX-CAA/G-CA;
(ii) TAT!C-G/CAA/G-GGX-AAT/C-GAT/C-GGX-CTX-CG; and
(iii) CG-XAG-XCC-A/GTC-A/GTT-XCC-T/CTC/G-A/GTA.
(All sequences given 5' to 3'; see Figure 6).
Sequences (ii) and (iii) are complementary: (iii) is
useful as a back (reverse) primer in PCR.
Preferably in oligonucleotide in accordance with the
invention, e.g. for use in nucleic acid amplification,
has about 10 or fewer codons (e.g. 6, 7 or 8), i.e. is
about 30 or fewer nucleotides in length (e.g. 18, 21 or
24).
Assessment of whether or not a PCR product
corresponds to a resistance genes may be conducted in
various ways. A PCR band may contain a complex mix of
products. Individual products may be cloned and each
sreened for linkage to known disease resistance genes
that are segregating in progeny that showed a
polymorphism for this probe. Alternatively, the PCR
product may be treated in a way that enables one to
display the polymorphism on a denaturing polyacrylamide
DNA sequencing gel with specific bands that are linked to
the resistance gene being preselected prior to cloning.
Once a candidate PCR band has been cloned and shown to be
linked to a known resistance gene, it may be used to
isolate clones which may be inspected for other features
and homologies to Cf-9, Cf-2 or other related gene It
may subsequently be analysed by transformation to assess
its function on introduction into a disease sensitive
CA 0221~496 1997-09-16
W O96/30518 PCT/~ ..'~
variety of the plant of interest. Alternatively, the
PCR band or sequences derived by analysing it may be used
to assist plant breeders in monitoring the segregation of
a useful resistance gene.
These techniques are of general applicability to the
identification of pathogen resistance genes in plants.
Examples of the type of genes that can be identified in
this way include Phytophthora resistance in potatoes,
mildew resistance and rust resistance in cereals such as
barley and maize, rust resistance in Antirrhinnm and
flax, downy mildew resistance in lettuce and Arabidopsis,
virus resistance in potato, tomato and tobacco, nematode
resistance in tomato, resistance to bacterial pathogens
in Arabidopsis and tomato and Xanthomonas resistance in
peppers.
Once a pathogen resistance gene has been identified,
it may be reintroduced into plant cells using techniques
well known to those skilled in the art to produce
transgenic plants. According to a further aspect, the
present invention provides a DNA isolate encoding the
protein product of a plant pathogen resistance gene which
has been identified by use of the presence therein of
LRRs or, in particular, by the technique defined above.
According to a yet further aspect, the invention provides
transgenic plants, in particular crop plants, which have
been engineered to carry pathogen resistance genes which
have been identified by the presence of LRRs or by
nucleic acid hybridisation as disclosed. Examples of
CA 0221~496 1997-09-16
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24
suitable plants include tobacco, cucurbits, carrot,
vegetable brassica, lettuce, strawberry, oilseed
brassica, sugar beet, wheat, barley, maize, rice,
soyabeans, peas, sorghum, sunflower, tomato, potato,
pepper, chrysanth~mllm, carnation, poplar, eucalyptus and
pine.
Modifications to these and further aspects and
embodiments of the present invention will be apparent to
those skilled in the art. All documents mentioned herein
are incorporated by reference.
As already indicated, the present invention is based
on the cloning and sequencing of the tomato Cf-2 genes
and this experimental work is described in more detail
below with reference to the fol~owing figures.
Figure 1 shows a physical map of the tomato Cf-2
locus generated from overlapping cosmids (38, 82, 89, 90,
92, 94, 96 and 141) isolated from the Cf-2/Cf-9 cosmid
library. Also included are the modified cosmids (112Bl
and 112B2) which contain sequences derived from cosmid 94
(also known as 2.2). The extent of each cosmid and
location of the Cf-2 genes are shown schematically. Also
indicated is the predicted direction of transcription
(arrow). The boxed regions represent expanded views of
areas encoding Cf-2 genes. The open boxes show regions
not sequenced, the hatched boxes show the sequenced
reglons .
Figure 2 shows the genomic DNA se~uence o~ the
Cf-2.1 gene (SEQ ID NO 1). Features: Nucleic acid
CA 0221~496 1997-09-16
W O 96130~18 ~1/~9'/~C
sequence - Translation start at nucleotide 1677;
translation stop at nucleotide 5012; a consensus
polyadenylation signal (AATA~A) exists in the
characterised sequence downstream of the translation stop
starting at nucleotide 3586. Predicted Protein Sequence
- primary translation product 1112 amino acids; signal
peptide sequence amino acids 1-26; mature peptide amino
acids 27-1112.
Figure 3A shows Cf-2 protein amino acid sequence,
designated Cf-2, 1 (SEQ ID NO 2~. Figure 3B shows the
amino acid sequence encoded by the Cf-2.2 gene (SEQ ID
NO. 3). Amino acids which differ between the two Cf-2
genes are underlined.
Figure 4 shows shows the sequence of an almost full
length cDNA clone (SEQ ID NO. 4) which corresponds to the
Cf2-2 gene.
Figure 5 shows a comparison of the carboxy-terminal
regions of the Cf-2 and Cf-9 genes (SEQ ID No's 5 and 6,
respectively). The protein sequences are aligned
according to predicted protein domains. Identical amino
acid residues are indicated by bold type. Figure 6
shows an alignment of part of the Cf-2 and Cf-9 proteins
(SEQ ID NO's 7 and 8, respectively). Two identical
regions are shown in bold type and are also shown as PEP
SEQ 1 (SEQ ID NO. 9) and PEP SEQ 2 (SEQ ID NO. 10)
respectively. OLIGO 1 (SEQ ID NO. 11) and OLIGO 2 (SEQ
ID NO. 12) show the sequence of degenerate
oliggonucleotides which encode these regions of protein
CA 0221~496 1997-09-16
W 096/30518 P~ 96loo785
similarity.
Figure 7 shows the primary amino acid saequence of
Cf-2 ( SEQ ID NO. 2) divided into domains of predicted
differing functions.
Cloning of the tomato Cf-2 gene
The Cf-2 gene was cloned using a map-based cloning
strategy similar in principle to that used for the
isolation of the tomato Pto gene, described briefly
earlier.
(i) Assignment of Cf- gene map locations
We have mapped several Cf genes, including Cf-2, to
their chromosomal locations (Dickinson et al 1993; Jones
et al 1993; Balint-Kurti et al 1994). We showed that
Cf-4 and Cf-9 map to approximately the same location on
the-short arm ~ chromosome 1, and Cf-2 and Cf-5 map to
approximately the same location on chromosome 6.
20-
(ii) High resolution mapping of the physical location ofthe Cf-2 gene
We have ordered a number of restriction fragment
length polymorphism (RFLP) markers by ex~m;ning the DNA
isolated from recombinant tomato plants. In this way, we
have assembled a detailed linkage map of the location of
the C~-2 gene on tomato chromosome 6 (Dixon et al 1995]).
We determined that the Cf-2 gene maps between the RFLP
CA 0221~496 1997-09-16
W 096130518 P~I~D.-~e7~
markers MG112A and CT119. These RFLP markers were made
available from the laboratory of S. Tanksley (Cornell).
Using available YACs, also made available by the Tanksley
laboratory, we have also shown that in tomato (L.
esculentum) the physical distance between the markers
MG112A and CTll9 is only 40 kb. We isolated two further
RFLP markers, MG112B (a weak homologue of MG112A) and
SC3-8 that were shown to map to this region and as such
represented candidate Cf-2 genes.
To determine more precisely the position of the Cf-2
gene, tomato crosses were set up to look for
recombination between the Cf-2 and Cf-5 resistance genes,
A plant that was heterozygous for both Cf-2 and Cf-5 was
crossed to a C. fulvum-sensitive tomato line.
Approximately 12,000 resulting F1 progeny were screened
for resistance to C. fulvum, and a single sensitive plant
was identified. DNA from this plant was analysed with
the molecular markers which map closely to the Cf-2 gene
and this plant was found to carry a chromosome that was
recombinant between MG112A and CT119. This analysis
strongly indicated that the RFLP marker MG112B identified
DNA which mapped very closely linked to the Cf-2 gene or
was the Cf-2 gene itself (Dixon et al 1996).
(iii) Isolation of binary cosmid vector clones that
carry a genomic Cf-2 gene
To determine whether DNA identified by the molecular
marker MG112B carried the Cf-2 gene, DNA sequences were
CA 0221~496 1997-09-16
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28
isolated from a plant that carried the
Cf-2 gene and transformed into Cf-O tomato plants.
A genomic DNA library was constructed from a stock
that carried both the Cf - 9 gene on chromosome 1, and the
Cf-2 gene on chromosome 6, so that the library could be
used for isolating both genes. The library was
constructed in a binary cosmid cloning vector pCLD04541,
obtained from Dr C. Dean, John Innes Centre, Colney Lane,
Norwich (see also Bent et al 1994). This vector is
essentially similar to pOCA18 (Olszewski et al 1988). It
contains a bacteriophage lambda cos site to render the
vector packageable by lambda packaging extracts and is
thus a cosmid (Hohn and Collins, 1980). It is also a
binary vector (van den Elzen et al 1985), so any cosmid
clones that are isolated can be introduced directly into
plants to test for the function of the cloned gene.
High molecular weight DNA was isolated from leaves
of 6 week old greenhouse-grown plants by techniques well
known to those skilled in that art (Thomas et al 1994)
and partially digested with MboI restriction enzyme. The
partial digestion products were size fractionated using a
sucrose gradient and DNA in the size range 20-25
kilobases (kb) was ligated to BamHI digested pCLD04541
DNA, using techniques well known to those skilled in the
art. After in vitro packaging using Stratagene packaging
extracts, the cosmids were introduced into a tetracycline
sensitive version (obtained from Stratagene) o~ the
Stratagene Escherichia coli strain SURETM . Recombinants
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29
were selected using the tetracycline resistance gene on
pCLD04541.
The library was randomly distributed into 144 pools
containing about 1500 clones per pool, cells were grown
from each pool and from 10 ml of cells, 9 ml were used
for bulk plasmid DNA extractions, and 1 ml was used after
addition of 0.2 ml of glycerol, to prepare a frozen
stock. Plasmid DNA from the pools was isolated by
alkaline lysis (Birnboim and Doly, 1979), and DNA samples
were analyzed by hybridisation in "slot blots" with the
molecular marker MG112B. Pools 38, 82, 89, 90, 92, 94,
96 and 141 proved positive by this assay. "94n is also
known as " 2.2".
For each pool, approximately 10,000 colonies were
plated out and inspected for MG112B homology by colony
hybridisation with a radioactive MG112B probe, and from
each pool, single clones were isolated that carried such
homology. These techniques are all well known to those
skilled in the art.
These clones have been further characterized by
Southern blot hybridisation using a MG112B probe, and by
restriction enzyme mapping. Our current assessment of the
extent of contiguous DNA around MG112B, as defined by
these overlapping cosmids is shown in Figure 1. These
cosmids revealed two regions with very similar
restriction maps that hybridised to MG112B (now labelled
MG112B1 and B2 respectively). Four of these cosmids ( 82,
89, 94, 141) were subsequently used in plant
CA 0221~496 1997-09-16
W O96/30518 PCTI~,.'V0785
transformation experiments, selecting for plant cells
transformed to kanamycin resistance, using techniques
well known to those skilled in the art. Transgenic tomato
and tobacco plants were produced (Fillatti et al 1987;
Horsch et al 1985) with at least one of each of cosmids
82, 89, 94 and 141.
(iv) Assessment of cosmid function in transgenic tomato
The function of a putative cloned Cf-2 gene was
assessed in transformed tomato by testing transformants
for resistance to Avr2-carrying C. fulvum.
Most transgenic plants containing cosmid 94 (11 of
16) or cosmid 82 (all of 4) were resistant to C. fulvum
carrying Avr2. All transgenic plants containing cosmid
89 or 141 were sensitive to C. fulvum. These data
indicate that the genomic DNA which carries the Cf-2 gene
is that piece which corresponds to the overlap between
cosmids 82 and 94. Thus the Cf-2 gene lies in one of the
regions identified by marker MG112Bl.
The region on cosmid 94 identified by MGl12B2 has
many similarities with the cosmid 82/94 overlap. This
region was subcloned to generate cosmid 112B2 (Figure 1)
which was also transformed into a sensitive tomato line.
Of the transgenic tomato plants carrying cosmid 112B2, 16
of 18 were resistant to C. fulvum carrying Avr2. The
overlap between cosmids 82 and 112B2 is very small and
unlikely to carry the Cf-2 gene. Additionally, the
region identified as MG112Bl was subcloned to generate
W 096/30518 P ~/~,.-~C7X~
31
cosmid 112B1 (Figure 1) which was transformed into a
sensitive tomato line. This cosmid ll2B1 only contains
the sequences characterised in figure 2 (SEQ ID NO. 1).
Of the transgenic tomato carrying cosmid 112B1,all (5 out
of 5) were resistant to C. f ul vum carrying Avr-2.
Therefore, these data indicate the presence of 2
functional Cf -2 genes ( Cf -2 . 1 and Cf -2 . 2 respectively)
characterised by the molecular probe MG112B (Dixon et al
1996). The results of all transformation experiments are
summarized in Table 1.
Progeny from all resistant nonpolyploid transformed
plants were screened with matched races of C. f ul vum
either carrying or lacking Avr-2 (race 5,9 compared with
race 2,5,9). Races of C. fulvum are named after the
resistance genes they can overcome. All progeny were
susceptible to C. fulvum lacking Avr-2 (race 2,5,9)
whereas approximately 75~ of progeny from each
transformant were resistant to C. fulvum carrying Avr-2
(race 5,9). These data confirm the race-specific nature
of the resistance genes cloned as Cf-2.
(v) DNA sequence analysis of the regions characterised by
MG112B.
The DNA sequence of the 6.5 kb region representing
the central core of the cosmid 82/94 overlap has been
determined. Two small regions of 2 3 and 1.1 kb
corresponding to the extremities of the cosmid overlap
have not been sequenced (Fig. 1).
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32
The central core sequence carries a single major
open reading frame which upon conceptual translation has
revealed an interesting motif (the leucine rich repeat,
or LRR) that may be diagnostic of other resistance genes,
as previously noted for the Cf-9 gene (WO 95/18320). The
open reading frame initiates with the translation start
codon (ATG) at position 1677 and finishes with the
translation termination codon TAG at position 5012 with
an intervening 3336 bp sequence that encodes a 1112 amino
acid protein. This is the Cf-2.1 gene.
The sequence of the region labelled MGl12B2 carried
on cosmid 112B2 has also been determined. This sequence
also carries a single open reading frame which differs by
only 3 nucleotides from the Cf-Z.l gene sequence. Upon
conceptional translation this also encodes a 1112 amino
acid protein which differs by only 3 amino acids from the
Cf-2.1 protein. These amino acid differences are all
clustered in the carboxy-terminal region of the protein
and are indicated as underlined residues in Figure 3B
~SEQ ID NO. 3). We therefore designate this to be Cf-
2.2.
An almost full length cDNA clone (SEQ ID NO. 4) has
been isolated corresponding to the Cf-2 gene (Fig. 4).
this confirms the predicted amino acid sequence of the
Cf-2 genes as it is colinear with the genomic sequence
through the entire open reading frame. The cDNA clone
lacks the most 5' sequences including any untranslated
leader sequence and the codon encoding the initiator
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W O 96/30518 PCT/C~9~ 5
methionine. The first ~ull codon of this cDNA encodes
for Valine which is amino acid number 4. A single intron
of 182 bases exists in the 3' untranslated sequences
(Dixon et al 1996).
G~nh~nk accession numbers for the sequences reported
within are U42444 ~or Cf-2.1 and U42445 for ~f-2.2.
(Vi) I-l~nt; fication of a leucine-rich repeat region in
Cf-2.
A genomic DNA sequence of the Cf-2.1 gene is shown
in Figure 2 (SEQ ID NO. 1). The deduced amino acid
sequence of the C~-2 protein is shown in Figure 3A (SEQ
ID NO.,2).
Homology searching of the resulting sequence against
sequences in the databases at the US National Centre of
Biological Information (NCBI) reveals strong homologies
to other genes that contain leucine rich repeat regions
(LRRs). The Cf-2 gene identifies Cf-9 with a blast score
of 483. Other homologies include the Arabidopsis genes
TMKl (Chang et al 1992), TMKLl (Valon et al 1993), RLK5
(Walker, 1993), as well as expressed sequences with
incomplete sequence and unknown ~unction (e.g.
Arabidopsis thaliana transcribed sequence [ATTS] 1447).
The presence of LRRs has been observed in other genes,
. many of which probably function as receptors (see Chang
et al [1992] for further references).
The TMKl and RLK5 genes have structures which
suggest they encode transmembrane serine/threonine
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W O 96/30518 PCT/GB96/00785
34
kinases and carry extensive LRR regions. As yet no known
function has been assigned to them. Disease resistance
genes are known to encode gene products which recognize
pathogen products and subsequently initiate a signal
transduction chain leading to a defence response. It is
known that another characterized disease resistance gene
(Pto) i~ a protein kinase (Martin et al 1993). However,
in Cf-2 there is no apparent protein kinase domain based
on genomic DNA and cDNA sequence analysis.
The predicted Cf-2 amino acid sequence can be
divided into 7 domains (Fig. 7).
Domain A is a 26 amino acid probable signal peptide.
Domain B is a 37 amino acid region with some
homology to polygalacturonase inhibitor proteins.
Domain C is a 930 amino acid comprising 33 perfect
copies and 5 imperfect copies of a 24 amino acid leucine
rich repeat (LRR).
Domain D is a 30 amino acid domain with some
homology to polygalacturonase inhibitor proteins.
Domain E is a 28 amino acid domain rich in
negatively charged residues.
Domain F is a 24 amino acid hydrophobic domain
encoding a putative transmembrane domain.
Domain G is a 3 7 amino acid domain rich in
positively charged residues.
Domains E, F and G together comprise a likely
membrane anchor.
The Cf- 2 and Cf-9 proteins are predicted to have the
CA 0221~496 1997-09-16
WO 96/30518 PCr/~;D' .
same general features in that they can both be sub-
divided into the above 7 domains. They are, however,
very different in length, 1112 verses 863 amino acids
respectively. The majority of this size difference
resides in the number of LRRs in ~om~; n~ C. Although the
LLRs are characterised by specific conserved amino acids
(mainly leucine), they are generally spaced apart such
that no block of conserved amino acids exists.
Additionally, leucine can be encoded by 6 different
codons and as a result it would be difficult to exploit
the similarities of the conserved amino acids in the LLR
domain at the level of DNA hybridisation to identify new
related genes. Indeed, at the level of genomic Southern
hybridisation the Cf-2 and Cf-9 genes did not identify
each other under the conditions we used.
(vii) Co~r~ison of the amino acid sequence of Cf-2 and
Cf-9
Comparison of the amino acid sequence of Cf-2 and
Cf-9 shows a remarkable degree of homology at the end of
Domain C and in Domain D. These are shown in bold in
Figure 6. Regions of Cf-2 such as the sequence F E G H I
P S (SEQ ID NO. 13) starting at position 915 and the
sequence S G E I P Q Q L A S L T S L E (SEQ ID NO. 14)
starting at position 965 are absolutely identical to Cf-
9. Other regions o~ identity or strong conservation also
exist. Since Cf-2 and Cf-9 are on different chromosomes,
it seems likely that they did not diverge recently This
CA 0221~496 1997-09-16
W 096130518 PCT/~r'~/o5
36
conservation then suggests functional conservation due to
selection of the maintenance of an association between
the amino acids in Cf-9 and Cf-2 and some other protein
required ~or Cf-9 or Cf-2 to provide disease resistance.
Accordingly, these homologies may be used in the
isolation of further disease resistance genes. Using
techniques well known to those skilled in the art these
sequences and fragments thereof may be used to design
oligonucleotide probes/primers for the purpose of
isolating further disease resistance genes that carry
these amino acid sequence motifs.
For example, based upon the identities between the
Cf-2 and Cf-9 genes (Figures 5 and 6), synthetic
degenerate oligonucleotide primers like those indicated
in Figure 6 might be produced. These primers correspond
to the different DNA sequences which potentially encode
amino acids conserved between the Cf-2 and Cf-9
polypeptides. These synthetic oligonucleotide primers
may be used in a PCR to identify related sequences from
any species which contains them.
,
CA 02215496 1997-09-16
W O 96130518 PCT/~ C/~S
37
T~B ~ 1
Cosmid Cosmid Cosmid Cosmid Cosmid Cosmid
hine 94 82 89 141 112B1 112B2
A S R S S R R
B S R S S R R
C R R S S R R
D R R S S R R
E R S S R R
F R S S R
G R S R
H R S R
I R S R
J R R
K R R
L R R
M S R
N R S
O S R
P S R
Q S
R R
The reponse of transgenic tomato plants (primary
transformants) carrying different cosmids. Tomato
transformants were tested for resistance (R) or
susceptibility (S) to a race of C. fulvum carrying Avr-2
(Race 4 GUS).
- - =
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38
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