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WO1997006259A2 - Resistance aux champignons provoquant la fletrissure - Google Patents

Resistance aux champignons provoquant la fletrissure Download PDF

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Publication number
WO1997006259A2
WO1997006259A2 PCT/EP1996/003480 EP9603480W WO9706259A2 WO 1997006259 A2 WO1997006259 A2 WO 1997006259A2 EP 9603480 W EP9603480 W EP 9603480W WO 9706259 A2 WO9706259 A2 WO 9706259A2
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Prior art keywords
plant
dna
sequence
dna sequence
plants
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PCT/EP1996/003480
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WO1997006259A3 (fr
Inventor
Marc Zabeau
Pieter Vos
Guus Simons
John Groenendijk
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Keygene NV
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Keygene NV
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Priority to IL12297396A priority Critical patent/IL122973A0/xx
Priority to AU68710/96A priority patent/AU722863B2/en
Priority to EP96929215A priority patent/EP0843727A2/fr
Priority to JP9508127A priority patent/JP2000507082A/ja
Priority to CA002227524A priority patent/CA2227524A1/fr
Publication of WO1997006259A2 publication Critical patent/WO1997006259A2/fr
Publication of WO1997006259A3 publication Critical patent/WO1997006259A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to resistance genes, DNA constructs, micro ⁇ organisms, plant cells and plants comprising said resistance genes. Furthermore the invention relates to genetically transformed plants which are resistant against wilt inducing fungi. In addition, the invention relates to probes, and primers for the identification of the resistance genes and diagnostic kits comprising said probes and/or primers. Finally, the invention relates to polypeptides encoded by said resistance genes and the use of said polypeptides.
  • Plant pathogens such as fungi are responsible for substantially losses of plants and plant products due to infection of the plant. Plant diseases, as a result of infection by plant pathogens, cause damage to the plants and/or plant products, reduce production and yield, limit the kind of plants that can grow in certain geographic areas and as a result cause severe (financial) losses to the grower.
  • Plants have developed a complex defense mechanism against attack and infection by pathogens.
  • their defense system is twofold, at the one hand it comprises a general resistance which is effective against different pathogen species, and at the other hand, it consists of a strong resistance against specific pathogen species. This latter resistance is generally based on a hypersensitivity reaction (HR).
  • HR hypersensitivity reaction
  • the resistance gene of a plant encodes for a product, a receptor molecule, that can recognize a product of the pathogen, an elicitor, encoded by a avirulence gene. If the receptor interacts with the elicitor molecule a hypersensitive response is triggered, resulting into the destruction of the infected plant cells and surrounding cells, and so preventing the multiplication and spread of the pathogen within the plant. This postulated mechanism has recently been confirmed by the isolation of a few avirulence genes of the pathogen with the corresponding resistance genes of the host plant.
  • RPS2 from Arabidopsis (resistance to Pseudomonas syringae expressing avrRpt2)
  • N from tobacco
  • Cf-9 from tomato
  • L 6 from flax (resistance to the corresponding leaf rust fungal race)
  • resistance genes are restricted due to several factors: (i) the limited occurrence of (known) resistance genes in the available germpiasm, (ii) incompatibility of crossing between different species and (iii) the limited availability of reproducible and reliable disease assays for certain pathogens, said assays being a prerequisite in selection breeding.
  • the pathogens causing serious damages to plants, one finds the group of soilborne cortical rots and vascular wilt inducing fungi, such as Fusarium and Verticillium (Toussoun, 1981 , in: Fusarium Diseases, Biology and Taxonomy, Nelson, Toussoun & Cook edit., Penn. State Univ. Press, 457 pp.).
  • Said wilt inducing fungi infect the plants through the roots via direct penetration or via wounds after which the xylem vascular tissue of the plant is colonized and symptoms of infection with said fungi are wilting, browning and dying of leaves followed by plant death. Entire plants or plant parts above the point of vascular invasion of the pathogen may die within a period of some weeks after infection.
  • the fungi usually spread internally through the xylem vessels as mycelium or conidia until the entire plant is killed. Because of the fact that those fungi are able to survive in the soil saprophytically, they become established forever once they are introduced in the field. They are distributed more or less worldwide causing tremendous losses on most species of vegetables and flowers, field crops, fruit trees, etc. Because those fungi are so widespread and so persistent in soils the only effective way of controlling said wilt inducing fungi is using resistant plant genotypes.
  • Fusarium species belong to the family of imperfect fungi. This class of fungi is characterized by the fact that only a vegetative stage of the fungus is known. The generative stage of those fungi has not been discovered yet. Due to the overall classification or taxonomy of fungi upon their mo ⁇ hological characteristics of the generative phase, one should bear in mind that fungi belonging to the class of imperfect fungi can be classified into another class once their generative stage is discovered and, subsequently, a change of classification and name can follow (Gerlach, 1981 , in: Fusarium, Diseases, Biology and Taxonomy, Nelson, Toussoun & Cook edit., Penn. State Univ. Press, p. 413-426). Moreover, it has been observed that some Fusarium species can "mutate" into another species depending on the plant infected and/or the environment (Bolton &
  • Fusarium isolates can infect different plant species.
  • tomato Fusarium oxysporum f.sp. lycopersici
  • F. oxysporum f.sp. melonis melon
  • the present invention relates to a nucleic acid comprising the 1-2 resistance gene which when present and expressed in a plant is capable of conferring said plant resistant against wilt inducing fungi. Furthermore, the invention relates to the 1-2 resistance gene of which the DNA sequence is disclosed herein. The invention also relates to a gene product encoded by the 1-2 resistance gene which is capable of triggering a hypersensitive response in the plant when it comes into contact with a gene product encoded by a corresponding avirulence gene of the plant pathogen. In addition the present invention relates to DNA constructs, cosmids, vectors, bacterial strains, yeast cells and plant cells comprising the 1-2 resistance gene.
  • the present invention relates to a genetically transformed plant, which is resistant to a wilt inducing fungus, said fungus being capable of infecting the untransformed plant. Furthermore, the invention relates to resistance genes which are homologous to the 1-2 resistance gene, and which, when present in a plant, are able of conferring said plant resistant to infection by pathogens.
  • the invention relates to oligonucleotides corresponding to the sequence of the 1-2 resistance gene or part thereof, and detection kits comprising said oligonucleotides.
  • Figure 1 shows a schematic representation of YAC 1/546, with a size of 750 kb, and the position of the BssHII, Rs ⁇ , Sf ⁇ and SgrA1 restriction sites (indicated by small lines).
  • the hatched bar represents the 255 kb SgrA1 fragment comprising the 1-2 resistance gene.
  • the most lower line represents the size bar (in kb).
  • the circle/arrowhead combination represents the left arm of pYAC4, direction of the centromer.
  • FIG. 2 shows a schematic drawing of the binary cosmid vector pJJ04541 which is used to construct a cosmid library of YAC 1/546.
  • Plasmid pRK290 (20 kb large) (Ditta et al, 1980, Proc. Natl. Acad. Sci. USA, 77, 7347-7351 ) was used as starting vector.
  • 'Tet refers to the gene conferring resistance to tetracyclin.
  • LB signifies T- DNA left border repeat sequence
  • RB signifies the right border repeat.
  • the cauliflower mosaic virus 35S promoter sequence is indicated by "p35S", and "ocs3"' indicates the octopine synthase 3' end.
  • NTT' indicates neomycin phosphotransferase
  • cos refers to the bacteriophage lambda cos site 5 enabling in vitro packaging.
  • pDBS indicates the polylinker of pBluescript
  • Figure 3 shows part of a 4,5% denaturing polyacrylamide gel with DNA fingerprints of 24 cosmids using Restriction Fragment Amplification with the enzyme l o combination EcoR ⁇ /Mse ⁇ .
  • the templates used are depicted in the right part of the figure.
  • Figure 4 shows a schematic representation of the 255 kb SgrA1 fragment with the position of the Mlu ⁇ and Sa/I restriction sites (indicated with small lines) and the
  • Figure 5 shows a schematic representation of the overlapping cosmids A52 and B22 and partly of A55 and CC16 (the open arrow head indicates that the cosmid is continued).
  • the position of the various restriction sites is indicated with small lines.
  • the position of the AFLP markers EM05, EM14 and EM06 is indicated with an 5 arrow.
  • the DNA segment of which the nucleotide sequence was determined is indicated with a line with a bidirectional arrow.
  • Figure 6 shows the nucleotide sequence of a DNA segment of almost the complete overlap between cosmids A52, B22 and A55, and the deduced amino acid 0 sequence of the 1-2 resistance gene.
  • the initiation codon (ATG position 1798-
  • the position of the AFLP marker EM06 is from nucleotide position 3470 (5'- AATTCAGA-3') to nucleotide position 3565 (5'-AGATTA-3').
  • the positions of three intron sequences are given in italics: one intron of 86 nucleotides located upstream of the ATG initiation codon from nucleotide position 1703 to 1788 and two introns of respectively 399 and 82 nucleotides located downstream of the TAA termination codon from respectively nucleotide position 5628 to 6026 and 6093 to 6174.
  • the transcriptional initiation site is predicted to be located at least 201 nucleotides upstream of the ATG initiation codon.
  • the transcriptional termination site is predicted to be located at least 893 nucleotides downstream of the TAA termination codon.
  • AAUAAA is located at nucleotide position 6406-6411 and is given in bold.
  • FIG. 7 shows a schematic drawing of plasmid pKG6016.
  • sm/sp adtr refers to the streptomycin/spectinomycin resistance gene
  • the origin of replication of pBR322 is indicated by “OriV”
  • bla refers to the ampicilin resistance gene.
  • LB signifies T-DNA left border repeat sequence
  • RB signifies the right border repeat.
  • the nopaline synthase promoter sequence is indicated by “nos pr”, and “nos 3'” indicates the nopaline synthase 3' end.
  • nptll indicates the kanamycin resistance gene.
  • 12-upstream refers to the 1.3 kb DNA segment upstream of the coding sequence of the 1-2 resistance gene
  • Fusl2 refers to the coding sequence of the 1-2 resistance gene from nucleotide position 1798 to nucleotide position 5598
  • 3' untrans refers to the 1.1 kb DNA segment downstream of the coding sequence of the 1-2 resistance gene. The relevant restriction sites are indicated.
  • nucleic acid a double-stranded DNA molecule
  • oligonucleotide a short single-stranded DNA molecule
  • primers in general, the term primer refers to a single-stranded DNA molecule which can prime the synthesis of DNA
  • nucleic acid hybridization a method for detecting related DNA sequences by hybridization of single-stranded DNA on supports such as nylon membrane or nitrocellulose filter papers. Nucleic acid molecules that have complementary base sequences will reform the double-stranded structure if mixed in solution under the proper conditions.
  • the double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a support.
  • hybridization probe to detect a particular DNA sequence in the Southern hybridization procedure, a labelled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to a support such as nylon membrane or nitrocellulose filter paper.
  • the areas on the filter that carry DNA sequences complementary to the labelled DNA probe become labelled themselves as a consequence of the reannealing reaction. The areas of the filter that exhibit such labelling can then be detected according to the type of label used.
  • the hybridization probe is generally produced by molecular cloning of a specific DNA sequence or by synthesizing a synthetic oligonucleotide; homologous sequence: a sequence which can hybridize under stringent conditions to a particular sequence, and/or a DNA sequence coding for a polypeptide which has the same properties as the polypeptide encoded by the particular DNA sequence, and/or a DNA sequence coding for a polypeptide having the same amino acid sequence as the polypeptide encoded by the particular DNA sequence and/or an amino acid sequence in which some amino acid residues have been changed with respect to the amino acid sequence of the particular polypeptide without substantial effect on the major properties of said polypeptide and/or a sequence which has at least 50 %, preferably 60 %, more preferably 70 %, most preferably 80 % or even 90 % sequence identity with the particular sequence, whereby the length of sequences to be compared for nucleic acids is generally at least 120 nucleotides, preferably 200 nucleotides and more preferably 300
  • high stringent conditions refer to the hybridization conditions which allow a nucleic acid sequence of at least 50 nucleotides and preferably about 200 or more nucleotides to hybridize to a particular sequence at about 65 °C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65 °C in a solution comprising' about 0,1 M salt, or less, preferably 0,2 x SSC or any other solution having a comparable ionic strength.
  • These conditions allow the detection of sequences having about 90 % or more sequence identity.
  • lower stringent conditions refer to the hybridization conditions which allow a nucleic acid sequence of at least 50 nucleotides and preferably about 200 or more nucleotides to hybridize to a particular sequence at about 45 °C in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength.
  • These conditions allow the detection of sequences having up to 50 % sequence identity.
  • the person skilled in the art will be able to modify these hybridization conditions in order to identify sequences varying in identity between 50 % and 90 %.
  • stringent conditions refer to hybridization conditions which allow a nucleic acid sequence to hybridize selectively to the 1-2 resistance gene in its genomic environment, substantially to the exclusion of hybridization with other DNA sequences of said genomic environment.
  • Fusarium 2 Fusarium oxysporum f.sp. lycopersici race 2 or any other genotype which is not able to infect a host having a resistance gene according to the invention; other genotypes are such as but not limited to, wilt inducing fungi, soil born fungi, or any other plant pathogens, resistance gene product: a polypeptide having an amino acid sequence as depicted in Figure 6, or part thereof, or any homologous amino acid sequence;
  • Ro plant primary regenerant from a transformation experiment, also denoted as transformed plant or transgenic plant; Ri line: the progeny of a selfed R 0 plant. R 2 line: the progeny of a selfed Ri plant. - RiBC line: the progeny of a backcross between a R ⁇ plant and a plant of the genotype which was originally used for the transformation experiment.
  • Ri line the progeny of a selfed R 0 plant.
  • R 2 line the progeny of a selfed Ri plant.
  • - RiBC line the progeny of a backcross between a R ⁇ plant and a plant of the genotype which was originally used for the transformation experiment.
  • 1-2 lmmunity-2 resistance gene. The gene was cloned from a tomato genotype which is resistant to Fusarium oxysporum f.sp. lycopersici race 2.
  • the isolated 1-2 resistance gene according to the invention can be transferred to a susceptible host plant using Agrobacterium mediated transformation or any other known transformation method, and is able to confer the host plant resistant against Fusarium 2.
  • the host plant can be tomato or any other genotype that is infected by Fusarium 2.
  • the present invention provides also the nucleic acid sequence of the 1-2 resistance gene which is depicted in Figure 6. With the 1-2 resistance gene according to the invention, one has an effective means for control against wilt inducing fungi, since the gene can be used for transforming susceptible plant genotypes thereby producing genetically transformed plants having a reduced susceptibility or being preferably resistant to infection by wilt inducing fungi.
  • the 1-2 resistance gene comprises the coding sequence preceded by a promoter region and followed by a terminator region.
  • the promoter region should be functional in plant cells and, preferably, corresponds to the native promoter region of the 1-2 resistance gene.
  • any heterologous promoter region can be used in conjunction with the coding sequences, as long as it is functional in plant cells.
  • a constitutive promoter is used, such as the CaMV 35 S promoter or T-DNA promoters, all well known to those skilled in the art.
  • a suitable terminator region should be functional in plant cells all well known to those skilled in the art.
  • the invention relates to the 1-2 resistance gene product which is encoded by the 1-2 resistance gene according to the invention and which has an amino acid sequence provided in Figure 6, or which is homologous to the deduced amino acid sequence or part thereof as listed in Figure 6.
  • the 1-2 resistance gene product can be used for the identification and/or isolation of the corresponding gene product encoded by an avirulence gene of the pathogen.
  • the relationship between the 1-2 resistance gene product, which is assumed to be acting like a receptor molecule, and the gene product of the pathogen, which is assumed to be acting like an elicitor molecule, is characterized by the occurrence of a defense mechanism reaction in the plant.
  • the 1-2 resistance gene product can be used for raising antibodies against it, which antibodies can be used for the detection of the presence of the 1-2 resistance gene product.
  • the 1-2 resistance gene can be used for the design of oligonucleotides which are complementary to one strand of the DNA sequence as described in Figure 6, or part thereof, which can be used as hybridization probes, being accordingly labelled to allow detection, for the screening of genomic DNA or cDNA libraries for homologous genes.
  • Homologous sequences which can hybridize to the probe, and which encode for a gene product that is able to confer resistance to a plant against a fungus which normally infects said plant, or both are comprised within the scope of the present invention.
  • oligonucleotides are designed based on the 1-2 resistance gene sequence, such that they can be used as hybridization probes in Southern analysis. These probes can be used as molecular markers to distinguish plant genotypes having the resistance gene and plant genotypes lacking the resistance gene. Such a probe can be used as an additional tool in selection breeding.
  • oligonucleotides are designed based on the 1-2 resistance gene sequence, such that they can be used as primers in an amplification reaction, such as polymerase chain reaction
  • PCR telomere sequence corresponding to the coding sequence of the 1-2 resistance gene and regulatory sequences functional in plant cells.
  • Said regulatory sequences are either homologous or heterologous to the coding sequences of the 1-2 resistance gene.
  • the invention relates also to DNA constructs (B) comprising the regulatory sequences, and more preferably the promoter region of the 1-2 resistance gene in conjunction with a structural gene sequence heterologous to said regulatory sequences.
  • the invention relates also to a DNA vector comprising a DNA construct (A) and/or a DNA construct (B).
  • Suitable vectors can be cloning vectors, transformation vectors, expression vectors, etc...., which are well known to the person skilled in the art.
  • cells harbouring a vector comprising a DNA sequence corresponding to the sequence as described in Figure 6 or part thereof, DNA constructs (A) or DNA constructs (B), are within the scope of the invention.
  • a genetically transformed plant is obtained by introducing the 1-2 resistance gene within the genome of said plant, having a susceptible genotype to Fusarium 2, using standard transformation techniques, wherein said genetically transformed plant is resistant to Fusarium 2.
  • the 1-2 resistance gene is inherited in following generations of the said genetically transformed plant and is able to confer the next generations of said plant resistant to Fusarium 2.
  • part of the DNA sequence comprising the 1-2 resistance gene is used for transforming a plant which is susceptible to Fusarium 2.
  • Such part can be obtained by digesting the DNA sequence comprising the 1-2 resistance gene, in one or more steps, with one or more appropriate restriction enzymes, chosen on the basis of the presence of their recognition site in the 1-2 resistance gene according to the invention, or in the sequences flanking the 1-2 resistance gene.
  • the obtained DNA segment can be transferred to a susceptible host plant and genetically transformed plants having a resistant phenotype can be identified when inoculated with Fusarium 2.
  • the 1-2 resistance gene according to the present invention is functional in homologous systems and/or heterologous systems, such as but not limited to tomato, melon, tobacco, Arabidopsis, egg plant, potato species, and is involved in reducing the susceptibility and/or is capable of conferring these plant species resistance against Fusarium 2 as defined above, and especially against one or more wilt inducing fungi.
  • a homologous system refers to a plant species which is the same plants species from which the resistance gene was isolated and a heterologous system refers to a plant species which is different from the plant species from which the resistance gene was isolated.
  • the DNA sequence comprising the 1-2 resistance gene as provided in the present invention has numerous applications of which some are described herein but which are not limiting the scope of the invention.
  • the present invention will be further described in detail in view of the isolation of the 1-2 resistance gene present in tomato lines which are resistant against Fusarium oxysporum f.sp. lycopersici race 2.
  • For the isolation of the 1-2 resistance gene we have used a map-based cloning (positional cloning) strategy,
  • AFLPTM selective restriction fragment amplification technology
  • AFLP markers In general, total DNA of different genotypes of the same plant species are subjected to the AFLP technology and the different AFLP fingerprints obtained from the different genotypes are compared. Fragments that are present in one genotype and absent in another genotype are polymorphic fragments and are 5 denoted as AFLP markers.
  • the selectivity in AFLP reactions is obtained by using randomly chosen selective nucleotides at the 3' end of the PCR primers immediately adjacent to the nucleotides of the restriction enzyme site.
  • the total amount of different 0 primers that can be used is determined by the number of selective nucleotides that are added to the 3' end (4 primers with 1 selective nucleotides, 16 primers with 2 selective nucleotides, 64 primers with 3 selective nucleotides). If two different restriction enzymes are used than there are twice the amount of primers. Those primers can be used in different combination.
  • Msel-primers 5'-GATGAGTCCTGAGTAANNN-3'
  • the N's indicate the variable selective nucleotides.
  • the objective of the screening was to identify AFLP markers linked to the 1-2 resistance gene, i.e. present in the finge ⁇ rints of the resistant pools, and absent in the fingerprints of susceptible pools.
  • AFLP finge ⁇ rints a total of 18 AFLP markers were identified which were present in the resistant pools and absent in the sensitive pools: these markers are referred to as candidate 1-2 linked AFLP markers.
  • the position of the AFLP markers with respect to the 1-2 resistance gene and the distance between the 1-2 resistance gene and the respective markers has still to be determined.
  • pu ⁇ ose a genetic map of the 1-2 locus was made by making genetic crosses between Fusarium 2 resistant tomato lines and susceptible tomato lines having mo ⁇ hological markers, followed by a screening for recombinants in the segregating F2 populations and screening of the F2 recombinants with the AFLP markers.
  • the results indicated that the 1-2 locus is flanked by marker EM 18 at one end of the DNA segment comprising the /- 2 resistance gene and markers EM03, EM12 and EM16 at the other end. All the other AFLP markers co-segregated with the 1-2 resistance gene.
  • the AFLP markers were screened on a high molecular weight genomic library.
  • the cloning of very large segments of DNA as large artificial chromosomes in yeast has become an essential step in isolating genes via positional cloning.
  • the cloning capacity of the YAC vector allows the isolation of DNA fragments up to one million base pairs in length.
  • the tomato line Lycopersicon esculentum E22, homozygous for the 1-2 locus, was used as source DNA to construct a YAC library.
  • YAC 1/546 One positive clone was obtained after an AFLP screening with the 1-2 linked AFLP markers, and all markers were present on this individual YAC clone, designated as YAC 1/546. The size of this YAC clone is determined to be 750 kb.
  • a cosmid library was constructed of the yeast strain containing YAC 1/546 using cosmid vectors which are suitable for Agrobacterium mediated transformation.
  • the size of this binary cosmid vector amounts 29 kb and is shown schematically in Figure 2.
  • the cloning capacity of this binary cosmid vector, using phage lambda packaging extract is within the range of 9 to 24 kb.
  • a bank of approximately 250,000 cosmid clones was obtained from size fractionated yeast DNA. The cosmid bank was screened by colony hybridization using the labelled
  • SgrAI fragment as probe. Of about 10,000 colonies approximately 150 positive cosmid clones were identified. In the following step, the position of the AFLP markers on the 255 kb SgrAI fragment was determined on the basis of a cosmid contig. The positive cosmid clones were screened with the 18 AFLP markers and their position was determined. A schematic outline of the cosmid contig and the physical fine mapping of the 18 AFLP markers is depicted in Figure 4.
  • the final step in the identification of the 1-2 resistance gene via positional cloning is the complementation of the corresponding susceptible phenotype.
  • the cosmid clones were introduced in Agrobacterium tumefaciens through conjugative transfer in a tri-parental mating. The presence of the cosmid in the A. tumefaciens strains was determined comparing various restriction enzyme patterns as well as
  • DNA finge ⁇ rints from the A. tumefaciens strains with the Escherichia coli strain containing the cosmid Only those A. tumefaciens cultures harbouring a cosmid with the same DNA pattern as the corresponding E. coli culture were used to transform a susceptible tomato line.
  • a susceptible tomato line was transformed with several cosmids forming the cosmid contig using standard transformation methods.
  • the primary regenerants (Ro plants) of the transformation experiments were grown in the greenhouse for seed set to obtain R ⁇ lines. These were tested for disease symptoms in order to identify cosmids with the resistance gene.
  • the disease assay is performed on seedlings. Their roots are immersed in a conidial suspension of Fusarium oxysporum f.sp. lycopersici race 2 and disease symptoms are scored three to four weeks after inoculation. Plants are scored resistant when they are healthy without wilting symptoms and/or without browning of the stem tissue. Plants being dead or having yellow wilting leaves and having severe browning of the stem tissue are scored susceptible.
  • the observations of the disease assay revealed that 3 cosmids were able to complement the susceptible phenotype, thereby providing definitive evidence that a functional 1-2 resistance gene is located on each of these 3 cosmids.
  • resistant plants of the Ri lines were selfed and grown in the greenhouse for seed set to obtain R 2 lines. Seedlings of the R 2 lines were subjected to a disease assay as described above and were scored for disease symptoms: Wilting plants were considered to be susceptible, whereas plants showing no wilting were considered to be resistant. The results obtained 5 indicated the stable Mendelian inheritance of the 1-2 resistance gene. Additionally, resistant plants of the R T lines were backcrossed with the susceptible tomato genotype used for the transformation experiments to obtain RiBC lines. The results of the disease assay performed on seedlings of the RiBC lines confirmed both the inheritance as well as the dominance of the 1-2 resistance gene.
  • a DNA segment corresponding to part of the DNA sequence, as provided in Figure 6, starting at nucleotide position 464 and ending at nucleotide position 6658 was used for transforming a susceptible tomato genotype.
  • the DNA segment was obtained by digesting cosmid B22 with restriction enzymes BamHI and Sa 1, providing a 3.8 kb fragment, and with Seal and BamHI, providing a 2.4 kb fragment, resulting in a 6.2 kb fragment comprising the coding sequence of the 1-2 resistance gene flanked upstream by a 1.3 kb DNA sequence and downstream by a 1.1 kb DNA sequence.
  • the DNA segment was cloned into a suitable cointegrate type vector and subsequently introduced through Agrobacterium tumefaciens mediated transformation into a tomato plant which is susceptible to Fusarium 2.
  • the Ro plants were grown in the greenhouse for seed set to obtain Ri lines and these Ri lines were subjected to the disease assay as described above. The observations indicated that the DNA segment is involved in conferring to the transformed plants a reduced susceptibility to Fusarium 2.
  • Cosmid B22 was used for the transformation of susceptible genotypes of melon, tobacco as well as Arabidopsis according to general known transformation methods. The Ro plants were grown in the greenhouse for seed set to obtain R lines. These were tested for disease symptoms in order to identify the functionality of the 1-2 resistance gene. The disease assay was performed on seedlings as described herein. Cosmid B22 was also used for the transformation of a susceptible genotype of potato. Vegetatively propagated transformed plants were
  • genomic and cDNA libraries were screened o with the coding sequence of the 1-2 resistance gene as a probe under stringent hybridization conditions. Positive clones were isolated and were used for complementation analysis.
  • Cosmid B22 has been deposited on July 14, 1995 as plasmid pKGI2-B22 at Centraalbureau voor Schimmelcultures at Baarn, The Netherlands, under deposit 5 number CBS 546.95.
  • Cosmid A55 has been deposited on August 5, 1996 as plasmid pKGI2-A55 at Centraalbureau voor Schimmelcultures at Baarn, The Netherlands, under deposit number CBS 820.96.
  • Seeds of tomato were germinated in soil in the greenhouse at 25° Celsius. Ten to 14 days-old seedlings were used for inoculation with the fungus. The seedlings were carefully pulled out of the soil and the roots were dipped in water for removing most of the adhering soil. Subsequently, the roots were immersed in the conidial suspension for two minutes and the plants were repotted in soil. The plantlets were grown in the greenhouse at a temperature of 25° C at daytime (16 hours) and 22° C at night (8 hours). After three to four weeks the plants were scored for disease symptoms.
  • Resistant plants resemble non-inoculated control plants; they are large non-wilting and/or without browning of stem tissue. Susceptible plants are dead or show typical symptoms: small plants with yellow, wilting leaves and severe browning of stem tissue.
  • Cuttings were prepared from greenhouse-grown Ro plants. Small sideshoots were cut from the plants and put in soil under 100% humidity at 20° Celsius. After one to two weeks the cuttings started rooting. Two to three weeks old cuttings were used for inoculation with the fungus. The plantlets were carefully pulled out of the soil.
  • the roots were dipped in water for removing most of the adhering soil. Subsequently, the roots were immersed in the conidial suspension for five minutes and the plants were repotted in soil.
  • the plantlets were grown in the greenhouse at a temperature of 25 °C at daytime (16 hours) and 22 °C at night (8 hours). After three to four weeks the plants were scored for disease symptoms.
  • EXAMPLE 2 IDENTIFICATION OF AFLP MARKERS LINKED TO A DNA SEGMENT COMPRISING THE 1-2 RESISTANCE GENE
  • Tomato lines (Lycopersicon esculentum) A total of 10 F. oxysporum f.sp. lycopersici race 2 resistant and 10 F. oxysporum f.sp. lycopersici race 2 susceptible tomato lines were used, and are depicted below:
  • Enza Zaden E22 resistant Enia Zaden, de Enkhuiier Zaadhandel B.V. Enkhuize ⁇ , The Netherlands
  • the structure of the EcoRI-adapter was:
  • the structure of the Msel-adapter was:
  • Adapters were prepared by adding equimolar amounts of both strands; adapters were not phosphorylated. After ligation, the reaction mixture was diluted to 500 ⁇ l with 10 mM Tris. HCI, 0.1 mM EDTA pH 8.0, and stored at -20°C. The diluted reaction mixture is further referred to as template DNA.
  • the primers used for the AFLP screening are depicted below:
  • AFLP reactions employed a radio-actively labelled EcoRI-primer and a non- labelled Msel-primer.
  • the EcoRI-primers were end-labelled using ( ⁇ - ⁇ PJATP and T4 polynucleotide kinase.
  • the labelling reactions were performed in 50 ⁇ l 25 mM
  • oligonucleotide primer 100 ⁇ Ci ( ⁇ - 33 P)ATP
  • T4 polynucleotide kinase 10 units T4 polynucleotide kinase.
  • 20 ⁇ l reaction mixture were prepared containing 5 ng labelled EcoRI-primer (0.5 ⁇ l from the labelling reaction mixture), 30 ng Msel-primer, 5 ⁇ l template-DNA, 0.4 units Taq-polymerase, 10 mM Tris.HCI pH 8.3, 1.5 mM MgCI 2 ,
  • amplification reaction was performed using the following cycle profile: a 30 seconds DNA denaturation step at 94 °C, a 30 seconds annealing step (see below), and a 1 minute extension step at 72 °C.
  • the annealing temperature in the first cycle was 65 °C, was subsequently reduced each cycle by 0.7 °C for the next 12 cycles, and was continued at 56 °C for the remaining 23 cycles. All amplification reactions were performed in a PE-9600 thermocycler (Perkin Elmer Corp., Norwalk, CT, USA).
  • reaction products were mixed with an equal volume (20 ⁇ l) of formamide dye (98% formamide, 10 mM EDTA pH 8.0, and bromo phenol blue and xylene cyanol as tracking dyes). The resulting mixtures were heated for 3 minutes at 90°C, and then quickly cooled on ice. 2 ⁇ l of each sample was loaded on a 5% denaturing (sequencing) polyacrylamide gel (Maxam and Gilbert, 1980, Methods in Enzymology 65, 499-560). The gel matrix was prepared using 5% acrylamide,
  • the template DNAs of the 20 tomato lines were pooled in the following way:
  • resistant pool 1 tomato lines 1 - 5 resistant pool 2: tomato lines 6 - 10 susceptible pool 3: tomato lines 11 - 15 susceptible pool 4: tomato lines 16 - 20
  • AFLP screening was performed using all possible 4096 EcoRI-Msel primer combinations on the 4 pools. The aim was to identify AFLP markers present in both resistant pools, and absent in both sensitive pools. AFLP gels contained the AFLP finge ⁇ rints of 24 primer combinations of the 4 pools, giving a total of 171 gels. Additional gels were run to reanalyse unsuccessful AFLP reactions and to confirm candidate markers. A total of 18 AFLP markers were identified present in both resistant pools and absent in both susceptible pools: these markers were referred to as candidate 1-2 linked markers.
  • Marker- refers to the sequence 5'-GATGAGTCCTGAGTAA-3'.
  • marker EM06 can be identified using the EcoRI-primer having the following sequence: 5'-GACTGCGTACCAATTCAGA-3', and the Msel-primer having the following sequence: 5'-GATGAGTCCTGAGTAATCT-3'.
  • the susceptible tomato line GCR210 is homozygous for the recessive mo ⁇ hological marker gene a (anthocyaninless).
  • the susceptible line GCR508 is homozygous for the recessive mo ⁇ hological marker gene sub (subtilis) (Stevens and Rick, 1986, in: The Tomato Crop, Atherton & Rudich edit., Chapman and Hall, p. 35-109). Both susceptible lines were obtained from the Institute of Horticultural Research (Littlehampton, United Kingdom).
  • the dominant 1-2 gene (conferring resistance to F. oxysporum f.sp. lycopersici 2), and recessive genes a and sub have all been mapped to the long arm of chromosome 11 of tomato (Stevens and Rick, 1986, in: The Tomato Crop,
  • Fi plants from all crosses were selfed for generating F 2 seeds.
  • the resulting F 2 populations will segregate for resistance to F. oxysporum f.sp. lycopersici 2 and for the mo ⁇ hological markers.
  • the recombinants are susceptible wild type plants and resistant anthocyaninless plants.
  • the recombinants are susceptible wild type plants and resistant 'subtilis' plants.
  • F 2 plants were grown in the greenhouse for seed set (by induced or spontaneous selfing).
  • F 3 seeds were obtained from most of the resistant recombinant F 2 plants and from a few of the susceptible recombinants. These F 3 lines were tested for resistance/ susceptibility in order to check the phenotype of the F 2 plants. Twenty to 30 seedlings of each F 3 line were inoculated with F. oxysporum f.sp. lycopersici
  • EM10, EM11, EM13, EM14, EM15 and EM17 were present in all 199 resistant F 2 plants and absent in all 37 susceptible plants; these markers are closely linked to the 1-2 gene.
  • the markers EM03, EM12 and EM16 were present in all resistant plants and absent in the susceptible plants with the exception of one anthocyaninless plant.
  • the marker EM18 is present in all resistant plants except for seven anthocyaninless plants, and absent in all susceptible plants.
  • the 1-2 locus is flanked by marker EM18 at one end of the DNA segment comprising the 1-2 resistance gene and on the other end by the markers EM03, EM12 and EM16. All of the remaining markers completely co-segregate with the 1-2 resistance gene based on the analysis of recombinants of crosses as described above.
  • 1-2 locus was used as source DNA to construct a YAC-library.
  • Plasmid pYAC4 containing an unique EcoRI cloning site was used as cloning vector and the yeast strain AB1380 was used as a host (Burke etal., 1987, Science 236, 806-812).
  • 3840 clones with a average insert size of 520 kb, which corresponds to 2.2 genome equivalents were finally obtained and the individual clones were stored in 40 96-wells microtiter plates containing 75 ⁇ l YPD solution (1% yeast extract, 2% peptone and 2% dextrose).
  • the cells of one 96-well microtiter plate was pooled (a plate pool) and used for DNA isolation as described by Ross et al. (1991 , Nucleic Acids Res., 19, 6053).
  • the 2.2 genome equivalent tomato YAC library consists of 40 96-wells microtiter wells and as a result DNA of the 40 plate pools were screened with the AFLP-markers EM01, EM12 and EM18 (see
  • Example 2 using the AFLP-protocol as described in Example 2.
  • One positive plate pool out of the 40 was identified with all three AFLP-markers.
  • a secondary screening of the 96 individual YAC clones was employed to find the correct address of the YAC or YACs.
  • One individual YAC clone was identified, designated 1/546, and subsequently analyzed with the remaining AFLP markers.
  • YAC-clone As expected, all of identified markers EM01 to EM18 were present on this YAC- clone since the flanking markers and one co-segregating marker were used in the screening.
  • the size of the YAC-clone was determined by Pulse-field gel electrophoretic (PFGE) analysis using contour-clamped homogeneous electric field (CHEF; Chu et al. 1986, Science, 235, 1582-1585) and appeared to be 750 kb.
  • PFGE Pulse-field gel electrophoretic
  • EXAMPLE 5 CONSTRUCTION OF A PHYSICAL MAP OF YAC 1/546 AND LOCATION OF THE AFLP MARKERS
  • YAC 1/546 was subjected to partial digestion with increasing concentration of the restriction enzymes SgrAI, Rs/11, S//I and BssHII.
  • the samples were fractionated by PFGE, transferred to a Gene Screen Plus membrane (DuPont NEN, Boston, MA, USA) and assayed by hybridization using end-adjacent sequence probes according to the protocol for indirect end-label mapping as described by Burke ef al. (1987, Science 236, 806-812).
  • FIG. 1 a schematic representation is given of the physical map of YAC 1/546.
  • the binary cosmid vector pJJ04541 is a derivative of pJJ1881 (Jones ef al., 1992, Transgenic Research 1 , 285-297) and is based on plasmid pRK290 containing the tetracyclin resistance gene for selection in Escherichia coli and Agrobacterium tumefaciens. Into the unique EcoRI site of pRK290, T-DNA carrying sequences
  • LB left border repeat
  • RB signifies the right border repeat
  • pJJ04541 The size of pJJ04541 amounts 29 kb and is shown schematically in Figure 2.
  • the cloning capacity of this binary cosmid vector, using phage lambda packaging extracts is within the range of 9 to 24 kb.
  • the binary cosmid vector pJJ04541 was digested completely with Xho ⁇ and the linear fragment was partially filled-in with dTTP and dCTP as described by Korch (1987, Nucleic Acids Res. 15, 3199-3220).
  • the 20-kb fragments were ligated to the cosmid vector and transduced to E coli strain XL1-Blue MR (Stratagene, La Jolla, CA, USA) using phage lambda Gigapack II XL packaging extracts (Stratagene, La Jolla, CA, USA) as recommended by the manufacturers.
  • transformants were stored into the wells of microtiter plates (96-wells, 100 ⁇ l of LB medium containing 10 mg/l of tetracyclin).
  • Replicas of the 96-well grid of cosmid clones in microtiter plates were stamped onto Gene Screen Plus membrane filters (DuPont NEN, Boston, MA, USA) and allowed to grow into colonies on media.
  • Colony hybridization using 32 P-labelled SgrAI fragment revealed positive cosmids. Of about 10.000 colonies approximately 150 positive cosmid clones were identified.
  • Cosmid DNA was isolated by alkaline lysis using the method as described by Ish-Horowicz ef al. (1981 , Nucl. Acids Res. 9, 2989-
  • GACTGCGTACCAATTC-3' having no selective nucleotide and the Msel-primer 5'- GATGAGTCCTGAGTAA-3' having no selective nucleotide as described in
  • Example 2 The EcoRI-primer was labelled at the 5' end and each of the 150 DNAs was amplified using the described primer set.
  • the DNA finge ⁇ rints contained about 8 to 20 amplified fragments.
  • Sets of DNA samples containing amplified fragments of identical size were selected and were rerun on polyacrylamide gels as described in Example 2 until a contiguous array of all the amplified fragments throughout the SgrAI fragment was obtained. The final fingerprint of the cosmid contig is shown in Figure 3.
  • AFLP markers were used as probes on the Mlu ⁇ and Sa/1 partial digests and secondly the AFLP markers were used as probes on DNAs of the cosmid contig using standard hybridization techniques as described by Sambrook ef al. (in
  • the cosmid clones were introduced in Agrobacterium tumefaciens through conjugative transfer in a tri-parental mating with helper strain HB101 (pRK2013) essentially according to Deblaere et al. (1987, Methods in Enzymology 153, 277- 292).
  • E.coli were grown in LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract and 1% NaCl, pH 7.5) supplemented with 5 mg/l tetracyclin at 37°C.
  • the helper strain HB101 (pRK2013) was grown under identical conditions in LB medium supplemented with 100 mg/l kanamycin sulphate.
  • Agrobacterium tumefaciens strain C ⁇ CIRif (pGV3101 ) (Van Larebeke et al., 1974, Nature, 252, 169-170) was grown in LB medium supplemented with 100 mg/l rifampicin at 28°C.
  • A. tumefaciens transconjugants Small-scale cultures were grown from selected colonies and grown in LB medium containing 10 mg/l tetracyclin. Plasmid DNA was isolated by alkaline lysis using the method as described by Ish-Horowicz et al. (1981 , Nucl. Acids Res. 9, 2989- 2997). and digested with Sg/ll using standard techniques. In addition, restriction fragment amplification on miniprep DNA of A. tumefaciens was performed using the enzyme combination EcoRI/Msel and primers having no selective nucleotide as described in Example 7. Subsequently, the Sg/ll restriction enzyme pattern as well as the DNA finge ⁇ rint of the A.
  • tumefaciens transconjugant were compared with those of miniprep DNA of the E. coli strain containing the cosmid. Only those A. tumefaciens transconjugants harbouring a cosmid with the same DNA pattern as the corresponding E coli culture were used to transform a susceptible tomato line.
  • Seeds of the susceptible tomato line 52201 were surface-sterilized in 2% sodium hypochlorite for 10 min, rinsed three times in sterile distilled water, and placed on germination medium (consisting of half-strength MS medium according to Murashige and Skoog (1962, Physiol. Plant. 15, 473 ⁇ 497), with 1% (w/v) sucrose and 0.8% agar) in glass jars or polypropylene culture vessels. They were left to germinate for 8 days. Culture conditions were 25°C, a photon flux density of 30 ⁇ mol.m "2 .s '1 and a photoperiod of 16 /24 h.
  • Transformation of tomato was performed according to Koornneef ef al. (1987, In: Tomato Biotechnology, 169-178, Alan R. Liss, Inc.), and is described briefly below.
  • Eight day old cotyledon explants were precultured for 24 h in Petri dishes containing a feeder layer of Petunia hybrida suspension cells plated on MS20 medium (culture medium according to Murashige and Skoog (1962, Physiol. Plant.
  • the cotyledon explants were transferred to Petri dishes with selective medium consisting of MS20 supplemented with 4.56 ⁇ M zeatin, 67.3 ⁇ M vancomycin, 418.9 ⁇ M cefotaxime and 171.6 ⁇ M kanamycin sulphate, and cultured under the culture conditions described above.
  • the explants were subcultured every 3 weeks onto fresh medium. Emerging shoots were dissected from the underlying callus and transferred to glass jars with selective medium without zeatin to form roots. The formation of- roots in a medium containing kanamycin sulphate was regarded as an indication of the transgenic nature of the shoot in question.
  • Truly transgenic regenerants were propagated in vitro by subculturing the apical meristem and auxiliary buds into glass jars with fresh selective medium without zeatin.
  • the transformed plants (Ro plants) of the transformation experiments were grown in the greenhouse for seed set as described in Example 8.
  • cosmid inserts Since the size of the cosmid inserts is within the range of 13-24 kb, standard techniques such as single, double and partial digestion analysis with restriction enzymes which are present in the polylinker sequence of the cosmid vector were performed as described by Sambrook ef al. (in: Molecular cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press). A physical map of cosmid A52, B22 and partially A55, was constructed and the overlap between the three cosmids giving rise to a resistant phenotype (A52.B22 and A55) with respect to the adjacent cosmid clone CC16 revealing a susceptible phenotype, was determined and is depicted in Figure 5.
  • cosmids A52 and B22 could be calculated and amount 23 and 17 kb, respectively. It appeared that the plant insert of cosmid B22 completely fits within cosmid A52.
  • the minimal DNA segment containing the 1-2 resistance gene was defined by the left-end of cosmid A55 until the right-end of cosmid B22 encompassing a region of approximately 8 kb in size.
  • EXAMPLE11 NUCLEOTIDE SEQUENCE AND DEDUCED AMINO ACID SEQUENCEOFTHE 1-2RESISTANCEGENE Various fragments of the 8 kb DNA segment of cosmid B22 were subcloned into the E coli vector pBluescript (Stratagene, La Jolla, CA, USA) using standard techniques as described by Sambrook ef al. (in: Molecular cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press) and used for sequence analysis making use of the Pharmacia Autoread Sequencing Kit and the Pharmacia LKB A.L.F. DNA Sequencer device (Pharmacia LKB, Uppsala,
  • Transcript mapping studies were performed to map the 5' and 3' end of the 1-2 resistance gene and to determine whether the 1-2 resistance gene comprises any introns.
  • the polymerase chain reaction to amplify parts of the transcripts from the /- 2 resistance gene was used for this pu ⁇ ose.
  • RNA from leaf tissue of the resistant tomato cultivar E22 was isolated using the hot phenol method as described by Sambrook ef al. (in: Molecular cloning: a laboratory manual, 1989, Cold Spring Harbor Laboratory Press). Poly A+ RNA was isolated using biotinylated oligo(dT) bound to Dynabeads M-280 Streptavidin (DYNAL A.S., Oslo, Norway) according to the instructions of the manufacturer. A cDNA library was constructed using the Superscript RNase H Reverse Transcriptase cDNA kit from Life technologies, Inc. (Gaithersburg, MD, USA) and the protocol supplied by the manufacturer. The cDNA was used as template for oligonucleotide primers in various PCR reactions.
  • primers were designed based on the nucleotide sequence provided in Figure 6, and six primer sets were used covering the coding sequence of the 1-2 resistance gene.
  • 5' and 3' RACE products were obtained using the Marathon cDNA amplification kit from Clontech (Paolo Alto, CA, USA). Subsequently, the various PCR fragments obtained with the 6 internal primer sets, and the 5' and 3'-RACE fragments were cloned into the TA cloning vector pCRII
  • the DNA sequence of the intemal PCR products derived from cDNA as template indicated that the 1-2 resistance gene does not contain any introns within the coding region.
  • the DNA segment comprising the 1-2 resistance gene was defined by the left end of cosmid A55 until the right end of cosmid B22 encompassing a region of approximately 8 kb in size.
  • One large open reading frame with a size of 3798 nucleotides encoding a protein of 1266 amino acids could be deduced from this 8 kb region. Part of this 8 kb DNA segment was retransformed into the susceptible tomato line 52201.
  • the cointegrate vector pKG1505 was used as starting vector for subcloning of the DNA segment.
  • This vector is a derivative of plasmid pGV1500, the prototype cointegrate vector described by Deblaere ef al. (1987, Methods in Enzymology, 153, 277-292).
  • the vector is based on the common cloning vector pBR322. It contains a streptomycin/spectinomycin resistance gene to be used as selection marker in Agrobacterium tumefaciens and maintenance of the cointegrate structure.
  • the vector contains the left (LB) and right (RB) border repeat sequences of the octopine T L -DNA.
  • Vector pKG1505 differs from pGV1500 in that it contains no residual T-DNA between the border repeat sequences, but a nos-nptll-nos cassette for kanamycin resistance in plants instead and a synthetic polylinker sequence.
  • Plasmid pKG6016 comprises a 6.2 kb Scal/Sa I fragment from cosmid
  • B22 comprising the coding sequence of the 1-2 resistance gene and a 1.3 kb upstream and 1.1 downstream DNA region.
  • a 3.8 kb SamHI/Sa/l fragment from cosmid B22 was cloned into pKG1505 resulting in plasmid pKG6014.
  • This 3.8 kb DNA segment contains the second half of the 1-2 resistance gene and the 1.1 kb 3' untranslated region.
  • pKG6015 was cut with Xhol and BamHI and the 2.4 kb DNA segment containing the first half of the 1-2 resistance gene including a
  • Plasmid pKG6016 was introduced into Agrobacterium tumefaciens strain C ⁇ CIRif (pGV2260) (Deblaere et al., 1987, Methods in Enzymology, 153, 277-
  • the Agrobacterium tumefaciens transconjugants were characterized by Southern blot analysis of chromosomal DNA using the Sm gene as a probe. Therefore, chromosomal DNA was isolated according to the method described by Lichtenstein and Draper (in: DNA cloning, 1985, volume II, 67-119, D.M. Glover edit., IRL Press), digested with the restriction enzymes EcoRI or Hin ⁇ .W and blotted onto a nitrocellulose membrane (Gene Screen Plus, DuPont NEN, Boston, MA, USA). Those transconjugants containing one copy of the integrative plasmid were selected for transformation to the susceptible tomato line 52201 using the protocol as described before.

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Abstract

Cette invention se rapporte à un acide nucléique comportant le gène de résistance DOLLAR I(I-2) qui, lorsqu'il est présent et exprimé dans une plante, peut conférer à ladite plante une certaine résistance aux champignons provoquant la flétrissure de ladite plante. La séquence d'ADN fait au minimum partie de la séquence d'ADN présentée dans la figure ou est constituée de toute séquence d'ADN homologue à celle-ci. L'invention se rapporte également à un produit génique codé par ce gène I-2.
PCT/EP1996/003480 1995-08-07 1996-08-06 Resistance aux champignons provoquant la fletrissure Ceased WO1997006259A2 (fr)

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IL12297396A IL122973A0 (en) 1995-08-07 1996-08-06 Nucleic acid and its use in conferring resistance against wilt inducing fungi
AU68710/96A AU722863B2 (en) 1995-08-07 1996-08-06 Resistance against wilt inducing fungi
EP96929215A EP0843727A2 (fr) 1995-08-07 1996-08-06 Resistance aux champignons provoquant la fletrissure
JP9508127A JP2000507082A (ja) 1995-08-07 1996-08-06 萎凋を誘発する真菌に対する抵抗性
CA002227524A CA2227524A1 (fr) 1995-08-07 1996-08-06 Resistance aux champignons provoquant la fletrissure

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EP0820220A4 (fr) * 1995-04-13 1998-07-15 Yeda Res & Dev Plants de tomates transgeniques contenant un gene de resistance au fusarium
WO1999045118A1 (fr) * 1998-03-06 1999-09-10 Commonwealth Scientific And Industrial Research Organisation Sequences genetiques conferant aux plantes des proprietes de resistance aux pathogenes et leur utilisation
EP1024196A1 (fr) * 1999-01-29 2000-08-02 Keygene N.V. Séquence nucléotidique ayant une activité régulatrice de transcription des gènes de plantes spécifique pour un tissu
US6100449A (en) * 1995-04-13 2000-08-08 Yeda Research And Development Co. Ltd. Transgenic tomato plants containing a fusarium resistance gene
FR2795094A1 (fr) * 1999-06-21 2000-12-22 Inst Rech Developpement Ird Moyens pour l'identification du locus d'un gene majeur de la resistance au virus de la panachure jaune du riz et leurs applications
EP1156116A1 (fr) * 2000-05-19 2001-11-21 Keygene N.V. Séquences nucleotidiques codant pour composants de la transduction de signaux impliqués en la défense des plantes contre les pathogènes
WO2005028651A1 (fr) * 2003-09-25 2005-03-31 Queensland University Of Technology Genes de resistance de la banane et utilisations de ces genes
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WO2019118553A1 (fr) * 2017-12-13 2019-06-20 Seminis Vegetable Seeds, Inc. Plants de tomate présentant une résistance améliorée aux maladies
EP3584253A1 (fr) * 2018-06-18 2019-12-25 KWS SAAT SE & Co. KGaA Résistance équilibrée et expression de gène d'avirulence
CN113234804A (zh) * 2021-06-07 2021-08-10 华南农业大学 一种用于香蕉枯萎病菌milRNA检测的内参基因及其应用

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EP0820220A4 (fr) * 1995-04-13 1998-07-15 Yeda Res & Dev Plants de tomates transgeniques contenant un gene de resistance au fusarium
US6100449A (en) * 1995-04-13 2000-08-08 Yeda Research And Development Co. Ltd. Transgenic tomato plants containing a fusarium resistance gene
WO1999045118A1 (fr) * 1998-03-06 1999-09-10 Commonwealth Scientific And Industrial Research Organisation Sequences genetiques conferant aux plantes des proprietes de resistance aux pathogenes et leur utilisation
EP1024196A1 (fr) * 1999-01-29 2000-08-02 Keygene N.V. Séquence nucléotidique ayant une activité régulatrice de transcription des gènes de plantes spécifique pour un tissu
WO2000044916A1 (fr) * 1999-01-29 2000-08-03 Keygene N.V. Sequence de nucleotides ayant une activite regulatrice specifique des tissus sur la transcription des genes chez les plantes
FR2795094A1 (fr) * 1999-06-21 2000-12-22 Inst Rech Developpement Ird Moyens pour l'identification du locus d'un gene majeur de la resistance au virus de la panachure jaune du riz et leurs applications
WO2000079002A1 (fr) * 1999-06-21 2000-12-28 Institut De Recherche Pour Le Developpement (I.R.D.) Moyens pour l'identification du locus d'un gene majeur de la resistance au virus de la panachure jaune du riz et leurs applications
EP1156116A1 (fr) * 2000-05-19 2001-11-21 Keygene N.V. Séquences nucleotidiques codant pour composants de la transduction de signaux impliqués en la défense des plantes contre les pathogènes
WO2001088165A1 (fr) * 2000-05-19 2001-11-22 Keygene N.V. Sequences nucleotidiques codant pour des composants de transduction de signal dans des strategies de resistance durable et a large spectre fondees sur la defense vegetale
US7205451B2 (en) 2000-05-19 2007-04-17 Keygene N.V. Nucleotide sequences coding signal transduction components in durable and broad-range resistance strategies based on plant defence
EP1670916A4 (fr) * 2003-09-25 2007-03-14 Univ Queensland Genes de resistance de la banane et utilisations de ces genes
WO2005028651A1 (fr) * 2003-09-25 2005-03-31 Queensland University Of Technology Genes de resistance de la banane et utilisations de ces genes
US7601887B2 (en) 2003-09-25 2009-10-13 Queensland University Of Technology Banana resistance genes and uses thereof
CN102321162A (zh) * 2011-10-08 2012-01-18 左开井 抗植物枯萎病和黄萎病的GbRPS1基因及其应用
WO2019118553A1 (fr) * 2017-12-13 2019-06-20 Seminis Vegetable Seeds, Inc. Plants de tomate présentant une résistance améliorée aux maladies
US11479788B2 (en) 2017-12-13 2022-10-25 Seminis Vegetable Seeds, Inc. Tomato plants with improved disease resistance
EP3584253A1 (fr) * 2018-06-18 2019-12-25 KWS SAAT SE & Co. KGaA Résistance équilibrée et expression de gène d'avirulence
WO2019243370A3 (fr) * 2018-06-18 2020-03-12 KWS SAAT SE & Co. KGaA Expression équilibrée entre gène de résistance et d'avirulence
CN113234804A (zh) * 2021-06-07 2021-08-10 华南农业大学 一种用于香蕉枯萎病菌milRNA检测的内参基因及其应用
CN113234804B (zh) * 2021-06-07 2022-11-29 华南农业大学 一种用于香蕉枯萎病菌milRNA检测的内参基因及其应用

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JP2000507082A (ja) 2000-06-13
AU722863B2 (en) 2000-08-10
IL122973A0 (en) 1998-08-16
AU6871096A (en) 1997-03-05

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