WO1996030517A1 - Genetic sequences conferring nematode resistance in plants and uses therefor - Google Patents

Genetic sequences conferring nematode resistance in plants and uses therefor Download PDF

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Publication number
WO1996030517A1
WO1996030517A1 PCT/AU1996/000181 AU9600181W WO9630517A1 WO 1996030517 A1 WO1996030517 A1 WO 1996030517A1 AU 9600181 W AU9600181 W AU 9600181W WO 9630517 A1 WO9630517 A1 WO 9630517A1
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Prior art keywords
seq
leu
plant
nucleic acid
sequence
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PCT/AU1996/000181
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French (fr)
Inventor
Evans Sylvanus Lagudah
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Commonwealth Scientific And Industrial Research Organisation
Grains Research And Development Corporation
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Application filed by Commonwealth Scientific And Industrial Research Organisation, Grains Research And Development Corporation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to EP96907218A priority Critical patent/EP0817849A4/en
Priority to NZ303905A priority patent/NZ303905A/en
Priority to AU50966/96A priority patent/AU710189B2/en
Publication of WO1996030517A1 publication Critical patent/WO1996030517A1/en

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material 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
    • C12N15/8285—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 nematode resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to genetic sequences, and more particularly to genetic sequences which confer, or otherwise facilitate or enhance, resistance in plants to plant parasitic nematodes, such as cyst nematodes and root knot nematodes.
  • the present invention further provides for plants into which the subject genetic sequences have been introduced, generating enhanced resistance qualities to plant parasitic nematodes.
  • the present invention is particularly useful in the development of plants resistant to plant parasitic nematodes such as food, fibre and ornamental plants.
  • plant pests such as plant parasitic nematodes
  • productivity is the effect of plant pests, such as plant parasitic nematodes.
  • plant parasitic nematodes invade a wide range of food, fibre and ornamental plants, causing damage to different plant tissues with varying severity on productivity.
  • Parasitic nematodes cost the agriculture and horticulture industries approximately US$78 billion per annum.
  • Plant parasitic nematodes are broadly classified as either migratory ectoparasites, sedentary ectoparasites, migratory ectoendoparasites, migratory endoparasites, or sedentary endoparasites, on the basis of their feeding patterns. Most crop damage is caused by sedentary endoparasites, for example the cyst nematodes Heterodera sp. and Globodera sp. and the root knot nematodes Meloidogyne sp., through their devastating effect on root structures. Juvenile nematodes invade the plant root and migrate to the vascular tissue where they induce a multinucleate feeding structure or syncitium from which the nematode feeds.
  • sedentary endoparasites for example the cyst nematodes Heterodera sp. and Globodera sp. and the root knot nematodes Meloidogyne sp.,
  • genetic sequences conferring resistance to a plant pathogen preferably a plant parasitic nematode
  • a plant pathogen preferably a plant parasitic nematode
  • the cloning of these sequences permits the generation of transgenic plants with de novo, improved or otherwise enhanced nematode resistance.
  • the present invention also permits the screening through genetic or immunological means, similar nematode resistance genes in other plants for use in developing or enhancing nematode resistance in commercially and economically important species.
  • one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a nucleic acid molecule which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a nematode in a plant.
  • the present invention provides an isolated DNA molecule comprising a sequence of nucleotides which: (i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and
  • (ii) has at least about 40% nucleotide sequence similarity to any one or more of the sequences set forth in SEQ ID NOS: 1, 3, 5 or 7 or a part thereof.
  • the present invention provides an isolated nucleic acid molecule which:
  • the invention provides an isolated nucleic acid molecule which is substantially the same as any one or more of the sequences set forth in SEQ ID NOS: 1, 3, 5 or 7 or is at least 40% identical thereto.
  • nucleic acid molecule which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a nematode in a plant.
  • the nucleic acid molecule is operably linked to a promoter sequence, thereby regulating expression of said nucleic acid molecule in a eukaryotic cell, for example a plant cell, or a prokaryotic cell.
  • the present invention provides a genetic construct comprising an isolated promoter sequence from a gene which when expressed encodes a polypeptide that confers, enhances, or otherwise facilitates nematode resistance in a cell, or a functional part, derivative, fragment, homologue or analogue thereof, wherein said promoter is operably linked to the coding region isolated from a second genetic sequence.
  • the invention extends to the recombinant polypeptide product of said genetic construct.
  • the present invention also provides an oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7.
  • nucleic acid molecule and/or oligonucleotide of the present invention are useful in the isolation of nematode resistance or nematode resistance-like genetic sequences from other plants, using hybridisation and/or PCR-based approaches.
  • a method of identifying a nematode resistance genetic sequence or nematode resistance-like genetic sequence comprises contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a genetic sequence encoding, or complementary to a genetic sequence encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance, or a part thereof, and then detecting said hybridisation.
  • a method of identifying a nematode resistance genetic sequence or a nematode resistance-like genetic sequence in a plant cell comprises contacting genomic DNA, mRNA, or cDNA with one or more oligonucleotide molecules to a genetic sequence from said plant for a period of time and under conditions sufficient to form a double- stranded nucleic acid molecule and amplifying copies of the said genetic sequence in a polymerase chain reaction.
  • this invention also provides an isolated polypeptide which comprises an amino acid sequence which confers, enhances, or otherwise facilitates resistance to a nematode in a plant cell, or a functional mutant, derivative part, fragment, or analogue of said polypeptide.
  • the present invention extends to a synthetic peptide comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or having at least 40% similarity to all or a part thereof.
  • polypeptide and synthetic peptides of the present invention may be used to generate specific immuno-interactive molecules. Accordingly, the present invention also provides an antibody that binds to a polypeptide which confers, enhances or otherwise facilitates resistance to a nematode in a plant or a part or fragment thereof, wherein said polypeptide, part or fragment threof further comprises an amino acid sequence which is substantially the same as the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or is at least 40% similar to all or a part thereof.
  • a method of identifying a nematode resistance gene product or nematode resistance-like gene product in a plant cell comprises contacting the antibody with an antigen from said plant for a period of time and under conditions sufficient to form an antibody-antigen complex and measuring the amount of said antibody-antigen complex formed.
  • the present invention is useful for the generation of plants with enhanced nematode resistance or nematode resistance-like characteristics and there is also provided a plant carrying a non- endogenous nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances, or otherwise facilitates nematode resistance in said plant.
  • the present invention extends to the progeny derived from said plant.
  • Figure 1 is a graphical representation of an RFLP linkage and physical map of chromosome 2D produced from Triticum tauschii F2 progeny of the genetic crosses (a) CPI 110813 X CPI 110795; (b) AUS 18913 x CPI 110856. The map location of the nematode resistance genes Ccn- Dl and Ccn-D2 are indicated.
  • Figure 2 is a photographic representation of an agarose gel showing PCR amplification products obtained from a survey of hydroxylapatite-fractionated DNA enriched for low copy sequences.
  • DNA samples were from bulked segregants of the Triticum tauschii cross CPI 110810 (resistant) x CPI 110825 (susceptible). Odd-numbered lanes contain DNA from resistant bulked segregants. Even-numbered lanes contain DNA from susceptible bulked segregants.
  • Random primers used for each pair were OPF12 (lanes 1,2), OPF13 (lanes 3,4), OPG2 (lanes 5,6), OPG3 (lanes 7,8), OPG6 (lanes 9,10), OPG6 (lanes 11,12) and OPG13 (lanes 13,14).
  • the arrow indicates the presence of a polymorphic PCR fragment present in lane 13 but absent from lane 14.
  • Lane 15 is a size marker.
  • Figure 3 is a photographic representation of an agarose gel showing PCR amplification products obtained from total genomic DNA and hydroxylapatite-fractionated DNA enriched for low copy sequences.
  • DNA samples were from parental and bulked segregants of the Triticum tauschii cross AUS 188913 (resistant) x CPI 110856 (susceptible). Amplification products were obtained using the random primers OPE20 (lanes 1-6) and OPF12 (lanes 7,8).
  • Templates were from low copy bulked resistant segregant (lanes 1,7), low copy bulked susceptible segregant (lanes 2,8), total genomic bulked resistant segregant (lane 3), total genomic bulked susceptible segregant (lane 4), total genomic from AUS 188913 (lane 5), and total genomic from CPI 110856 (lane 6).
  • the arrow indicates the presence of a polymorphic PCR fragment present in resistant bulked segregant (lane 1) but not in the susceptible bulked segregant (lane 2).
  • the converse type of polymorphism is shown with primer OPF 12 in lanes 7 and 8.
  • Figure 4 is a photographic representation showing an autoradiograph of one euploid (lane 1) and several nullitetrasomic lines (lanes 2-6) of Triticum aestivum cv Chinese Spring, showing RFLP patterns assayed with the cloned PCR fragment csE20-2. Cytogenetic stocks missing chromosome 2D are present in lanes 8 (nulli 2D tetra 2A) and 9 (nulli 2D tetra 2B).
  • Figure 5 is a photographic representation showing an autoradiograph of Triticum tauschii F2 individuals from the cross AUS 188913 (resistant) x CPI 110856 (susceptible) assayed with the cloned PCR fragment csE20-2. Ccn-DI resistance (R) and susceptibility (S) are indicated.
  • Figure 6 is a photographic representation showing linkage between the XcsE20 RFLP marker and CreS (Ccn-DI) in Triticum aestivum resistant (R) and susceptible (S) backcross individuals.
  • Figure 7 is a schematic representation of a lambda clone containing the Cre3 gene.
  • the positions of the 6.5kb Ec ā‡ RV RFLP fragment and PCR fragment csE20-2 are indicated.
  • the position of the Cre3 gene within the lambda clone is also indicated. Restriction enzyme sites BamHi B) and EcoRV(E) are indicated.
  • One aspect of the present invention comprises an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a protein or derivative thereof, which confers, enhances or otherwise facilitates resistance to a nematode in a plant.
  • nematode resistance gene or ā€œresistance-like geneā€, or similar term shall be used to define a nucleic acid molecule which upon expression confers, enhances, or otherwise facilitates resistance of a cell and/or organism to one or more plant parasitic pathogens.
  • nematode resistance gene further defines a nucleic acid molecule which upon expression confers, enhances, or otherwise facilitates resistance to one or more plant parasitic nematode pathogens.
  • a "geneā€ is to be taken in its broadest context and includes: (i) a classical genomic gene consisting of a coding region optionally together with transcriptional and/or translational regulatory sequences and a coding region with or without non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and optionally 5'- and 3'- untranslated sequences of the gene.
  • a classical genomic gene consisting of a coding region optionally together with transcriptional and/or translational regulatory sequences and a coding region with or without non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and optionally 5'- and 3'- untranslated sequences of the gene.
  • the term "geneā€ is also used to describe a synthetic or fusion molecule, or derivative which encodes, or is complementary to a molecule which encodes, all or part of a functional product.
  • a functional product is one which confers, enhances or otherwise facilitates resistance of a cell to a parasitic nematode.
  • Preferred nematode resistance-like genes are derived from a naturally occurring nematode resistance gene by standard recombinant techniques. Generally, a nematode resistance gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions.
  • Nucleotide insertional derivatives of the nematode resistance gene of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides.
  • Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterised by the removal of one or more nucleotides from the sequence.
  • Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place.
  • substitution may be "silent" in that the substitution does not change the amino acid defined by the codon.
  • substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.
  • the present invention extends to the isolated nucleic acid when integrated into a plant genome and to propagated plants containing same nucleic acid molecule.
  • nucleic acid molecule which comprises a sequence of nucleotides corresponding or complementary to the nucleotide sequence set forth in any one or more of SEQ ID NOS: 1, 3, 5 or 7, or having at least 40% similarity to all or a part thereof and wherein said nucleic acid molecule encodes a protein that confers, enhances, or otherwise facilitates resistance to a nematode in a plant.
  • the percentage similarity to a sequence set forth in SEQ ID NOS:l, 3, 5 or 7 is at least 50%. Even more preferably, the percentage similarity is at least 60-65%. Still more preferably, the percentage similarity is at least 70-75%. Yet still more preferably, the percentage similarity as at least 80-90%, including at least 91% or 93% or 95%.
  • SEQ ID NOS: 1, 3, 5 and 7 relate to the CRE 3 resistance gene of Triticum tauschii which controls resistance to cyst nematodes Heterodera sp. More preferably, SEQ ID NOS: 1 and 3 are nucleotide sequences of a genomic clone isolated from Triticum tauschii, containing the Cre 3 gene.
  • the nucleotide sequence set forth in SEQ ID NO: 5 is a cDNA clone encoding CRE 3, which was isolated from T. tauschii AUS 18913 seedlings using genomic clone sequences.
  • SEQ ID NO: 7 shows the nucleotide sequence of the promoter and complete open reading frame of the Cre gene, without introns.
  • the amino acid sequence of the complete CRE3 polypeptide is presented in SEQ ID NO: 8.
  • the cyst nematode is the cereal cyst nematode (CCN) Heterodera avenae. More preferably, the cyst nematode is the Australian pathotype of H. avenae.
  • CRE 3 is also synonymous with the designation "Ccn-DIā€ referred to by Eastwood et al. (1993), among others.
  • a further aspect of the present invention contemplates a nucleic acid molecule which encodes a protein that confers or otherwise facilitates nematode resistance in a plant and which is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in any one or more of SEQ ID NOS: 1, 3 , 5 or 7 or to a derivative homologue or analogue thereof.
  • a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28 ā‡ C.
  • the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.
  • genetic analysis indicates that specific interactions may occur between resistance genes and gene products of the parasitic nematode.
  • the genetic sequences of the present invention control host range via specific recognition of the gene products of the nematode pest, in a "gene-for-gene" interaction that is understood by one normally skilled in the art. Accordingly, the genetic sequences are useful in increasing the range of resistance of a plant to nematode pests, by providing de novo the required nematode resistance gene, or being introduced together with the corresponding nematode gene or genes, on, for example, a single genetic cassette. Accordingly, these aspects of the invention are covered by the expression ā€œconferring, improving, or otherwise enhancing nematode resistanceā€ or other similar expression.
  • the present invention is particularly directed to resistance that is conferred, enhanced, or facilitated against a nematode, preferably a cereal cyst nematode, more preferably Heterodera avenae, even more preferably the Australian pathotype of H. avenae by a polypeptide encoded by genetic sequences from Triticum tauschii.
  • a nematode preferably a cereal cyst nematode, more preferably Heterodera avenae, even more preferably the Australian pathotype of H. avenae by a polypeptide encoded by genetic sequences from Triticum tauschii.
  • Examples of genetic sequences in Triticum tauschii which confer resistance to a nematode include, but are not limited to the Ccn genes, Ccn-DI and Ccn-D2 and the Cre 1.
  • the subject invention clearly contemplates other sources of nematode resistance genes, such as but not limited to, other monocotyledonous plants, other Triticum sp., barley, maize, rye, oats, and rice, amongst others.
  • the genetic sequences which encode a protein which confers, enhances, or otherwise facilitates nematode resistance may correspond to the naturally occurring sequence or may differ by one or more nucleotide substitutions, deletions and/or additions. Accordingly, the present invention extends to nematode resistance genes and any functional genes, mutants, derivatives, parts, fragments, homologues or analogues thereof or non-functional molecules but which are at least useful as, for example, genetic probes, or primer sequences in the enzymatic or chemical synthesis of said gene, or in the generation of immunologicaily interactive recombinant molecules.
  • the nematode resistance genetic sequences or like genetic sequences are employed to identify and isolate similar genes, or nematode resistance- like genes from other plants.
  • the present invention extends to the use of said genetic sequence, or a part thereof to detect polymorphisms of a nematode resistance genetic sequence or nematode resistance-like genetic sequence.
  • an oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7.
  • a method for identifying a related nematode resistance genetic sequence or nematode resistance-like genetic sequence comprising contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a genetic sequence encoding or complementary to a genetic sequence encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance, or a part thereof, and then detecting said hybridisation.
  • the related nematode resistance genetic sequence or like sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell.
  • the related genetic sequence originates from Triticum aestivum or similar plant such as maize, barley, rye, oats, or rice and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
  • the related genetic sequence may be bound to a support matrix, for example nylon, nitrocellulose, polyacrylamide, agarose, amongst others.
  • the genetic sequence which encode a polypeptide which confers, enhances, or otherwise facilitates nematode resistance is from Triticum sp., or similar plant such as maize, barley, rye, oats, or rice.
  • the latter comprises a sequence of nucleotides set forth in any one or more of SEQ ID NOS: 1, 3, 5 or 7 or a homologue, derivative or analogue thereof.
  • the latter genetic sequence is labelled with a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32 P or 35 S or a biotintylated molecule).
  • a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32 P or 35 S or a biotintylated molecule).
  • An alternative method contemplated in the present invention involves hybridising a nucleic acid primer molecule of at least 10 nucleotides in length to a nucleic acid "template molecule", said template molecule herein defined as a nematode resistance genetic sequence, or resistance-like genetic sequence, or a functional part thereof, or its complementary sequence.
  • template molecule herein defined as a nematode resistance genetic sequence, or resistance-like genetic sequence, or a functional part thereof, or its complementary sequence.
  • Specific nucleic acid molecule copies of the template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art.
  • the nucleic acid primer molecule or molecule effective in hybridisation is contained in an aqueous mixture of other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form. In a preferred embodiment, the nucleic acid primer molecule is from Triticum sp., or similar plant such as maize, barley, rye, oats, or rice. In a most preferred embodiment, (he nucleic acid primer molecule is any nucleotide sequence of at least 10 nucleotides in length derived from, or contained within any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7.
  • the nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell.
  • the related genetic sequence originates from Triticum aestivum or similar plant such as maize, barley, rye, oats, or rice and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
  • a further aspect of the present invention is directed to a genetic construct comprising an isolated nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes a protein, or derivative thereof, that confers, enhances, or otherwise facilitates resistance against a nematode in a plant cell.
  • the gene sequence is related to or a functional derivative, part fragment, homologue, or analogue of the nucleotide sequence defined by any one or more of SEQ ID NOS: 1, 3, 5 or 7. More preferably, the genetic construct comprises the entire open reading frame of the Cre 3 gene sequence.
  • the present invention extends to genetic constructs designed to assist expression of a nucleic acid molecule that confers, enhances or facilitates nematode resistance in a cell.
  • the genetic construct comprises in addition to the subject nucleic acid molecule, a promoter and optional other regulatory sequences that modulate expression of the nucleic acid molecule.
  • the promoter may be the CRE 3 gene promoter, or a promoter from another genetic source.
  • the promoter is capable of expression in a plant cell, in particular a root cell.
  • the subject nucleic acid molecule may be genomic DNA or cDNA and may correspond in sequence exactly with the nucleotide sequence as set forth in any one or more of SEQ ID NOS : 1, 3, 5 or 7 or it may contain one or more nucleotide substitutions, additions and/or deletions, either dispersed throughout, or clustered.
  • an isolated promoter sequence from a gene which, when expressed encodes a polypeptide that confers, enhances or otherwise facilitates nematode resistance in a cell, or a functional part, derivative, fragment, homologue or analogue thereof, is operably linked to the coding region of a second genetic sequence, for example the ā‡ - glucuronidase gene, or the chloramphenicol acetyltransferase gene, or the firefly luciferase gene, amongst others.
  • the promoter sequence is contained within nucleotides 1 to 1138 of the sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 7.
  • Yet another aspect of the present invention provides for the expression of the subject genetic sequence in a suitable host (e.g. a prokaryote or eukaryote) to produce full length or non-full length recombinant nematode resistance gene products.
  • a suitable host e.g. a prokaryote or eukaryote
  • the nematode resistance gene product has a sequence that is identical to, or contained within an amino acid sequence set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. More preferably, the nematode resistance gene product has a sequence that is identical to or contained within the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8.
  • the present invention extends also to a synthetic peptide fragment of a nematode resistance gene product, preferably the resistance gene product set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
  • the present invention provides an isolated polypeptide which comprises an amino acid sequence which confers, enhances, or otherwise facilitates resistance to a nematode in a plant cell, or a functional mutant, derivative part, fragment, or analogue of said polypeptide.
  • the present invention also extends to the protein or polypeptide product of the Triticum tauschii nematode resistance gene Cre3 and the weaker resistance gene Ccn-D2. This is done, however, with the understanding that the subject invention extends to a range of resistance genes for nematode and other pathogens.
  • the present invention extends to a nematode resistance gene characterised by said gene encoding a product having at least one imperfect leucine rich repeat region.
  • the leucine rich repeat region is located at the C-terminal end of the protein molecule and has at least 60% similarly to amino acid residues 185 to 412 of the amino acid sequence set forth in SEQ ED NO: 4, or residues 308 to 768 of SEQ ID NO: 6 and even more preferably is at least 80% similar thereto. Still more preferably, the leucine rich region corresponds to amino acid residues 185 to 412 of the amino acid sequence set forth in SEQ ID NO: 4 or residues 308 to 768 of SEQ ID NO: 6.
  • the nematode resistance gene product further contains a p-Loop, or kinase- la motif, having the sequence: GV(G/S)GSGKST and more particularly
  • GfflGV(G/S)GSGKST or having one or more amino acid substitutions, insertions and/or deletions thereto provided that such derivatives still function as a p-Loop in conferring nematode resistance in a cell.
  • a p- Loop is involved in ATP/GTP binding.
  • the nematode resistance gene product further contains a kinase-2 motif, having the sequence:
  • X is any hydrophobic amino acid residue, and more particularly:
  • KLDGKRFLL(I/V)LDDVWC or having one or more amino acid substitutions, deletions, and/or insertions thereto provided that such derivatives still function as a kinase-2 motif.
  • a kinase-2 motif functions in nucleotide binding, preferably in binding of ATP/GTP.
  • the present invention extends to a recombinant gene product that contains the p-Loop, and/or kinase-2, and/or imperfect leucine-rich repeat sequence in any relative combination, or frequency, provided that said recombinant gene product confers, enhances, or facilitates nematode resistance in a cell.
  • the present invention also extends to a synthetic peptide comprising any part of the amino acid sequence set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or having at least 40% similarity to all or a part thereof.
  • the recombinant nematode resistance gene product, nematode resistance-like gene product, or functional derivative thereof may be used to produce immunologicaily interactive molecules, such as antibodies, or functional derivatives thereof, the only requirement being that the recombinant products are immunologicaily interactive with antibodies to all or part of said gene product.
  • an antibody that binds to a polypeptide comprising an amino acid sequence which:
  • Antibodies to a recombinant nematode resistance gene product are particularly useful in the screening of plants for the presence of said gene product.
  • Another aspect of the present invention is, therefore, directed to antibodies to a recombinant nematode resistance gene product or part or fragment thereof.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to a nematode resistance gene product or may be specifically raised to a recombinant nematode resistance gene product. In the case of the latter, the nematode resistance gene product may first need to be associated with a carrier molecule. Alteraatively, fragments of antibodies may be used such as Fab fragments.
  • the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
  • a "synthetic antibodyā€ is considered herein to include fragments and hybrids of antibodies.
  • the antibodies and/or the recombinant nematode resistance gene products of the present invention are particularly useful for the immunological screening of nematode resistance gene products in various plants, in monitoring expression of nematode resistance genetic sequences in transgenic plants and as a proprietary tagging system.
  • specific antibodies are used to screen for nematode resistance gene products or nematode resistance-like gene products in plants.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of a recombinant nematode resistance gene product.
  • Both polyclonal and monoclonal antibodies are obtainable by immunisation with a recombinant nematode resistance gene product and either type is utilisable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of recombinant nematode resistance gene product, or antigenic or immunointeractive parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techmques.
  • antibodies produced by this method are utilisable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitised against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art (see, for example, Douillard and Hoffman, 1981; Kohler and Milstein, 1975; Kohler and Milstein, 1976).
  • nematode resistance gene product or nematode resistance-like gene product in a plant or more commonly plant extract may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non- competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
  • an unlabelled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the antigen labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
  • the first antibody is raised to a recombinant nematode resistance gene product and the antigen is a nematode resistance gene product in a plant.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
  • the sample is one which might contain nematode resistance gene product and include crude or purified plant extract such as extracts of leaves, roots and stems.
  • a first antibody raised against a recombinant nematode resistance gene product is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking, covalent binding or physically adsorption, the polymer-antibody complex is washed in preparation for the test sample.
  • An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25 Ā°C) to allow binding of any antigen present in the sample to the antibody.
  • the reaction locus is washed and dried and incubated with a second antibody specific for a portion of the first antibody.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
  • An alternative method involves immobilising the target molecules in the biological sample and then exposing the immobilised target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detected by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target- first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognised, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • the enzyme-labelled antibody is added to the first antibody-hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • enzyme immunoassays EIA
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofiuorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method.
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed
  • the present invention further extends to a plant such as a crop plant carrying a non-endogenous nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances, or otherwise facilitates nematode resistance in said plant.
  • a plant such as a crop plant carrying a non-endogenous nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances, or otherwise facilitates nematode resistance in said plant.
  • the plant is a monocot plant. More preferably the transgenic plant is one or more of the following: Triticum aestivum, Triticum tauschii, maize, barley, rye, oats, rice, sorghum, amongst others. Other species are not excluded.
  • the non-endogenous genetic sequence or transgene may originate from any plant species.
  • said genetic sequence is identical to any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7, or a functional derivative, fragment, part, complement, homologue, or analogue thereof.
  • said genetic sequence or transgene is a cDNA molecule such as set forth in SEQ ID NO: 5 or other nucleic acid molecule which lacks a functional promoter, it may be placed operably under control of the Cre3 promoter sequence, or under the control of a heterologous promoter sequence.
  • the expression of the transgene may be constitutive or inducible by an external stimulus such as physiological stress, or by addition of a chemical compound, or the expression may be developmentally-regulated, or expressed in a tissue- or cell-specific pattern.
  • the transgene may be inserted into or fused to a particular endogenous genetic sequence. Methods for placing a structural gene operably under the control of a promoter sequence are well-known to those skilled in the art.
  • a non-endogenous nucleic acid molecule encoding, or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance in a recipient plant may be introduced into said plant by any one, or a combination of procedures, including Agrobacterium-medi&ted transformation, microparticle bombardment, PEG fusion, electroporation, introgression via conventional breeding program, amongst others. It will be readily apparent to one skilled in the art how to produce plants carrying a non- endogenous genetic sequence and perform variations to said procedures.
  • the present invention extends to the progeny and clonal derivatives of said plant.
  • Heterodera avenae Segregation for resistance to the nematode Heterodera avenae was determined for the F2 progeny of each cross and for 10-12 individuals within each F3 family. A total of 2472 individuals were assessed for reactions to Heterodera avenae. Fifty eight to sixty two F2 plants from each cross were chosen on the basis of availability of F3 data and recovery of sufficient DNA to determine marker segregation. Chromosomal locations of polymorphic DNA markers were determined by nuUitetrasomic and ditelocentric lines of Triticum aestivum cv. Chinese Spring and wheat-barley addition lines (Islam et al., 1981; Sears, 1966).
  • the F3 families in crosses 2,3,4 segregated for reaction to Heterodera avenae at the Ccn-DI locus in the ratio 1 :2: 1 (homozygous resistant: heterozygous resistant, homozygous susceptible), consistent with the ratio expected for Mendelian inheritance of a single dominant autosomal gene.
  • cysts An average of 0.14 +/- 0.1 white females (cysts) were produced per homozygous resistant Ccn- DI plant, compared to 1.38 +/- 0.46 (range 0-8) per resistant line homozygous for the weaker Ccn-D2 gene.
  • the susceptible lines carried a significantly greater number of cysts, in the range of 10-90 cysts.
  • a total of 35 RFLP loci were analysed for linkage to Ccn-D2 using the segregants from cross 1, and 34 loci for linkage to Ccn-DI, using the segregants from cross 2.
  • a total of 17 polymorphic loci were identified on groups 2 and 6 chromosomes, of which 11 segregated with the Ccn-DI locus and 6 segregated with the Ccn-D2 locus.
  • Multipoint analysis of joint F2/F3 segregation of RFLP loci and Ccn resistance revealed a loose linkage between Ccn-D2 and chromosome 2 markers ( Figure 1). No methods used were able to identify polymorphisms to map further, the Ccn-D2 locus.
  • Two-point RFLP linkage data set for cross 1 showed 5cM map units between Ccn-D2 resistance and the RFLP markers ksuH9 (Gill et al. , 1991 ) and csIH52 (Lagudah et al. , 1993 ), where 1 cM is herein defined as 1% recombination between two genetic loci or markers, in a randomly segregating population.
  • Ccn-DI was linked to ksuH9 only ( Figure 1).
  • Molecular markers were generated from bulked homozygous resistant and susceptible F2 DNA pools of cross 2 (Table 2), by PCR amplification of genomic DNA using 260 random 10-mer oligonucleotides (Operon Technologies, Alameda, California) (OPA-01 to OPM 20). Other oligonucleotides included were 4 semirandom primers of 15-18 nucleotides in length, based on the consensus nucleotide sequences of intron-exon splice junctions for plant genes (Weining and Langridge, 1991), designated ISJR1, ISJR2, ISJE3 and ISJE4.
  • PCRs were performed in 10 ā‡ L of a reaction mix containing about 30 ng of template DNA, 0.5 units of Taq polymerase (Boehringer Mannheim GMBH, Germany), 15 ng of primer, 1.5 mM MgCI 2 and 1 x reaction buffer (0.2 mM dNTPs, 67 mM Tris-HCl (pH 8.8), 16 nM (NH,) 2 SO 4 0.01% (w/v) gelatin, and 0.45% (v/v) Triton X-100). Samples were loaded into capillary tips and run on a thermocycler (FTS-1 Thermal Sequencer, Corbett Research, Sydney, Australia) under the following conditions: 1.
  • reaction products were visualised in 1.5% (w/v) agarose gels containing ethidium bromide, using UV light.
  • Wheat genomic DNA has a large proportion of highly-repeated DNA sequences, which may reduce the probability of detecting low-copy sequences in the genome, using PCR
  • DNA was fractionated on hydroxylapatite to remove highly repetitive DNA sequences.
  • the average proportion of DNA recovered after hydroxylapatite fractionation in bulked segregants from cross 2 and cross 3 was 17% and 25%, respectively.
  • Polymorphic amplification products were obtained using four random 10-mer primers, including ISJE3, in the bulked segregants of cross 2 (see Table 2), an increase in the detectable level of polymorphism from 0.45% to 2%.
  • the polymorphic PCR product obtained was associated with the presence of a DNA band in the resistant bulk, that was absent from the susceptible bulk ( Figure 2).
  • Polymorphic amplification products were also obtained using eight random 10-mer primers, in the bulked segregants of cross 3 (Table 2).
  • the polymorphic PCR product was associated with the susceptible bulk and only one (OPE-20) was associated with the resistant bulk ( Figure 3).
  • OPE-20 produced a consistent polymorphisms in DNA enriched for low-copy nucleotide sequences, but not for total wheat genomic DNA of either the parental genotypes, or the bulked segregating progeny ( Figure 3).
  • polymorphic PCR products present in the resistant bulk plus the DNA amplified in the susceptible bulk using primer OPF12 were excised from low-melting agarose gels, radiolabelled and hybridised to membrane filters containing DNA of parental lines from all populations that had been digested with restriction enzymes.
  • E-20 lkb amplified DNA fragment
  • All Ccn- DI resistant parents showed one to two major hybridising DNA fragments, while the susceptible lines were characterised by a single minor hybridising fragment.
  • the E-20 fragment was subsequently cloned into a "T-overhang" pUCl 18 plasmid vector, to produce the recombinant plasmid csE20-2.
  • Genomic and cDNA clones used in the construction of Triticum tauschii, wheat, and barley genetic maps were used as RFLP markers to analyse joint segregation with CCN resistance susceptibility in the F 2 progenies. Procedures for RFLP analysis were as described by Lagudah et al. (199 la). As an aid in selecting potential markers to target the CCN resistance region, the genetic map of Triticum tauschii produced from the main mapping population (cross F) reported by Lagudah et al.
  • the csE20-2 RFLP patterns of parental genotypes of Triticum tauschii and resistance susceptibility to Heterodera avenae infestation reveal a complete linkage between Ccn-DI an the csE20-2 RFLP marker, for segregating progeny of crosses 2,3, and 4 (Table 2, see Figur 5). Pooled crosses were based on the RFLP analysis of 178 F2 lines and the Heterodera avena reactions observed for 2020 individual F3 plants.
  • both Ccn-DI and csE20-2 are approximately 13.8cM from the RFLP marker WG645 (Kleinho et al., 1993), 26.3cM from csIH57-l (Lagudah et al., 1991b), and 46. lcM from ksu H9 (Gi et al. , 1991), which have previously been localised on the long arm of chromosome 2.
  • the Cre3 gene from Triticum Wilmingtonii was introgressed into bread wheat Triticum aestivum b repeated backcrossing to produce backcross one F4 lines.
  • the csE20-2 RFLP marker was use as a probe to check for linkage among 30 progeny lines, between csE-20 and reaction t Heterodera avenae ( Figure 6).
  • the introgressed Cre3 gene was able to confer nematode resistance in bread wheat, Triticum aestivum.
  • Data from bulked segregant analysis provide indications of the genetic distance between loci, but no indication of the physical distance, in kb, which may be in the order of several megabases in a highly recombinogenic region such as the 2DL chromosome of Triticum tauschii.
  • kb physical size per unit of genetic recombination
  • cM genetic recombination
  • DNA from Triticum tauschii line AUS 18913 carrying the Cre3 gene was size-fractionated to isolate fragments in the range 15-20kb in length, and used to construct a lambda genomic library.
  • the insert from csE20-2 was radioactively labelled and used to isolate three genomic clones, which were sub-cloned for further analysis.
  • One sub-clone, designated CCN4 was shown to overlap 4kb at the 5' end of the 6.5kb EcoRV RFLP band ( Figure 7). This subclone was also shown to cosegregate with Ccn resistance.
  • the sequenced region of CCN4 clone contains the nucleotide sequences of the 889bp PCR amplification product E-20, between nucleotide positions 194 and 1082 of the nucleotide sequence set forth in SEQ ID NO: 1.
  • the sequenced region of CCN4 also contains two overlapping reading frames (exons) from nucleotides 1138 to 1614 of the nucleotide sequence set forth in SEQ ID NO: 1 and from nucleotides 1 to 1238 of the nucleotide sequence set forth in SEQ ID NO: 3, with no stop codon at the end of the second exon, suggesting that the clone contains a partial Cre3 gene sequence.
  • the first exon encodes a p-Loop Ā» (or kinase- la) motif between nucleotides 1414 and 1437 of the nucleotide sequence set forth in SEQ ID NO: 1, with the amino acid sequence GVGGSGKS.
  • the second exon encodes another nucleotide binding site, kinase-2, between nucleotide positions 73 and 87 and an imperfect leucine-rich repeat sequence from nucleotides 682 to 1238 of the nucleotide sequence set forth in SEQ ID NO: 3.
  • the T. tauschii AUS 18913 cDNA library was screened using the genomic clone containing the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3.
  • the 2.6kb cDNA sequence set forth as SEQ ID NO: 5 represents a partial gene and encodes a reading frame of 768 amino acids in addition to 324 baes of the 3 ' untranslated region.
  • the 768 amino acids contains a P loop (base positions 191 to 217) or ATP/GTP binding site, kinase 2 domain (base positions 443 to 457) and a leucine rich region (base positions 1202 to 2260).
  • SEQ ID NO: 5 sequence as identical to SEQ ED NO: 1 and SEQ ID NO: 3.
  • the SEQ ID NO: 1 and SEQ ID NO: 3 sequences cosegregates with cereal cyst nematode resistance at the Cre3 locus in 178 F2 families of 7. tauschii.
  • the root expressed gene sequence, SEQ ID NO: 5 is clustered with the genomic sequence, SEQ ID NO: 1 and SEQ ID NO: 3 at the Cre3 locus.
  • Scutellar tissue of immature wheat embryos derived from the cereal cyst nematode susceptible cultiva Gabo are co-transformed with the Cre3 gene construct and the plasmid pEmuKON (Chamberlain etal, 1994) which comprises an efficient promoter for gene expression in cereal cells operably linked to the i ā‡ t ā‡ gene (conferring resistance to aminoglycoside antibiotics) and a termination signal.
  • Stable tansformants are selected on paromomycin-containing media and the presence of the Cre3 gene construct verified subsequently by standard procedures (polymerase chain reaction Northern blotting and Southern blotting using re3-derived nucleotide sequences; Ausubel et al, 1987).
  • Transformed tissue containing the introduced Cre3 gene sequences are placed on regeneration medium and regenerated into whole plants. Transformed plants are retained for further analysis. The roots of transformed plants are assayed for expression of the introduced Cre3 gene, using northern blot hybridisation, reverse-transcription PCR or other procedure suitable for the detection of Cre3 gene transcripts in root tissue. Such methods are well-known to those skilled in the art.
  • Roots expressing the Cre3 gene are inoculated with juvenile cereal cyst nematodes of the Australian pathotype, essentially as described by Eastwood et al. (1991).
  • Non-transformed isogenic wheat are similarly infected in a parallel experiment.
  • Juveniles of cyst nematodes normally invade plant roots and migrate to the vascular tissue where they induce syncytia formation in a compatible host plant interaction.
  • host plant mechanisms lead to breakdown of syncytia with the production of large, vacuolated syncytia which possesses degenerated membranes in the roots of infected plants, at about 15 days post- invasion of the roots by the juvenile nematode.
  • Antibodies are raised against an E. coli fusion protein composed of the carboxy-terminal part of glutathione S-transferase and the 768 amino acids of the CRE3 protein set forth in SEQ ID NO: 6 or alternatively, the 797 amino acids of the full-length CRE 3 protein set forth in SEQ ED NO: 8.
  • the vectors encoding this construct are generated by cloning the 2654 bp fragment of the cDNA shown in SEQ ID NO: 5 or the entire Cre3 open reading frame of SEQ ID NO: 7, into a pGEX plasmid (Pharmacia, Uppsala) producing an in-frame fusion with a partial cDNA encoding about 250 amino acids (27.5 kD) of glutathione S-transferase, placed operably under the control of the lac promoter.
  • the construct is transformed into E. coli cells. After induction with IPTG (lmM final concentration) the expressed fusion protein is purified on Glutathione-Sepharose 4B (Pharmacia, Uppsala) according to the manufacturer's instruction.
  • the apparent molecular weight of the fusion polypeptide in SDS-PAGE is approximately 110- 115kD.
  • the purified fusion protein (100 ā‡ g) is subcutaneously injected into a female rabbit using Freund's adjuvant as described by Harlow and Lane (1988). After the second boost, antiserum is collected and used in Western blotting and ELISA tests.
  • Antisera are screened by ELISA (Enzyme-Linked Immunosorbent Assay) using purified CRE- GST fusion polypeptide.
  • the ELISA is performed as follows: partially purified CRE-GST fusion polypeptide in coating buffer (Na 2 CO 3 , 15 mM; NaHCO 3 , 35 mM, CaCl 2 , 0.1 mM; final pH 9.2) is incubated overnight at 4 ā‡ C in Nunc-Immuno Plate Maxisorb (Nunc-Kamstrup, Denmark).
  • washing butter Tris/HCl, 20 mM; NaCl, 120 mM; Tween 20, 0.05%; final pH 7.4
  • the primary antibody diluted serially between 1 :250 and 1 : 5000
  • wells are washed 4 times with washing buffer, and then incubated with peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO) at a 1:1000 dilution for an additional 1 hour at 37Ā°C.
  • washing buffer substrate solution O-phenylenediamine dihydrochloride, 0.05 ml, 0.4 mg/ml, Sigma
  • 0.1 M sodium citrate pH 4.5
  • H202 0.0006%
  • the reaction is stopped by adding H 2 SO 4 (0.025 ml; 8N), and absorbance at 490 mm is then measured.
  • a second antibody (horseradish peroxidase coupled goat anti- rabbit IgG (BioRad, Kunststoff)) is diluted 1:20000 fold in PBS 0.02% Tween 20, 0.2% gelatin. Peroxidase reaction is performed using the ECL Kit (Amersham International) to detect bound antibody. The antisenim strongly recognizes a band at ā€”110-115 kD corresponding to the molecular weight of the CRE3-GST fusion protein, which is not detected by the preimmune serum. There is no cross reactivity of the anti-CRE3-GST antisenim with the recombinant GST protein.
  • Root protein extracts are obtained from infected plants of the wheat cultivar Gabo, transformed with the Cre3 gene as described in Example 11 and which has improved resistance to the Australian pathotype of the cereal cyst nematode compared to untransfo ā‡ ned Triticum aestivum cv Gabo. Root protein extracts are also obtained from infected untransformed Gabo plants. Plants are infected with juvenile nematodes, according to Eastwood et al (1991). Soluble protein is fractionated on SDS-PAGE, transferred to nitrocellulose and probed with antisera to CRE3, as described in Example 12, to identify the CRE3 polypeptide. A cross-reactive band of approximately 80kDa molecular weight, corresponding to the CRE3 polypeptide, is only observed in protein extracts obtained from transformed plants which express the introduced Oe3 gene.
  • Triticum tauschii A novel source of resistance to cereal cyst nematode ā‡ Heterodera avenae). Aust. J. Agric. Res. 42:69-77. 9. Eastwood, R F., Lagudah, E. S., Halloran, G. M., Brown, J. S., Kollmorgen, J. F. and Appels, R (1993). Resistance to cereal cyst nemotodes in Triticum tauschii. In: Proceedings of the 10th Australian Plant Breeding Conference. (B.C. Imrie and J.B. hacker eds) pp. 7-9.
  • MAPMAKER an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1: 174-181.
  • MOLECULE TYPE DNA (genomic)
  • ATA AAT GAA GCA CAC CAA ATT CTG GAT AAG CTT AAC TTG TCA AGC ATA 1233 lie Asn Qlu Ala His Gin lie Leu Asp Lys Leu Asn Leu Ser Ser lie 20 25 30
  • GAG GAG ATA GCG AAA AAG CTG AAG GGG TCA CCT CTA GCT GCC AGA ACA 384 Glu Glu He Ala Lys Lys Leu Lys Gly Ser Pro Leu Ala Ala Arg Thr 115 120 125
  • CAC GAG AAA AAT GAC AAG CAA GAC AAC AAG GAA GAC CAT TTC GAC CTT 286 His Glu Lys A ā‡ n Asp Lys Gin Asp Asn Lys Qlu Asp Hie Phe A ā‡ p Leu 80 85 90 95
  • AAA AAG CTG AAG GGG TCA CCT TTA GCA GCT AGA ACA GTG GGT GGA AAT 766 Lys Lys Leu Lys Gly Ser Pro Leu Ala Ala Arg Thr Val Gly Gly Asn 240 245 250 255
  • GGT CTG GGA ATA TTT AAT GAA CTA TTG TCG ATA TCA TTT CTT CAA CCA 1054 Gly Leu Gly He Phe A ā‡ n Glu Leu Leu Ser He Ser Phe Leu Gin Pro 340 345 350
  • GCA TTC AAA GTA AGT CAT GAA CAA GGA CAT GAG GCA AAG CAG TTG AGG 1726 Ala Phe Lys Val Ser His Glu Gin Gly Hie Glu Ala Lys Gin Leu Arg 560 565 570 575

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Abstract

The present invention relates generally to a nucleic acid molecule encoding, or complementary to a nucleic acid molecule encoding, a polypeptide which confers, enhances, or otherwise facilitates resistance to a nematode in a plant cell. The nucleic acid molecule of the present invention is useful in the isolation of related nematode resistance, or nematode resistance-like genetic sequences, from other plants. Furthermore, the present invention provides for the generation of plants carrying non-endogenous nematode resistance, or nematode resistance-like genetic sequences, said plants exhibiting enhanced tolerance to parasitic nematodes and related pathogens.

Description

GENETIC SEQUENCES CONFERRING NEMATODE RESISTANCE IN PLANTS AND USES THEREFOR
FIELD OF THE INVENTION
The present invention relates generally to genetic sequences, and more particularly to genetic sequences which confer, or otherwise facilitate or enhance, resistance in plants to plant parasitic nematodes, such as cyst nematodes and root knot nematodes. The present invention further provides for plants into which the subject genetic sequences have been introduced, generating enhanced resistance qualities to plant parasitic nematodes. The present invention is particularly useful in the development of plants resistant to plant parasitic nematodes such as food, fibre and ornamental plants.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence identity numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography.
Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
BACKGROUND TO THE INVENTION
Improvements in recombinant DNA technology have produced dramatic changes to the agricultural industry, in particular the approaches taken to improve crop productivity. A major concern is the effect of plant pests, such as plant parasitic nematodes, on productivity. Generically, plant parasitic nematodes invade a wide range of food, fibre and ornamental plants, causing damage to different plant tissues with varying severity on productivity. Parasitic nematodes cost the agriculture and horticulture industries approximately US$78 billion per annum.
Plant parasitic nematodes are broadly classified as either migratory ectoparasites, sedentary ectoparasites, migratory ectoendoparasites, migratory endoparasites, or sedentary endoparasites, on the basis of their feeding patterns. Most crop damage is caused by sedentary endoparasites, for example the cyst nematodes Heterodera sp. and Globodera sp. and the root knot nematodes Meloidogyne sp., through their devastating effect on root structures. Juvenile nematodes invade the plant root and migrate to the vascular tissue where they induce a multinucleate feeding structure or syncitium from which the nematode feeds.
The most cost-effective and sustainable method for control of plant pests is the development of resistant plants. However, the development of this method of control in relation to parasitic nematodes has faced many difficulties. For example, bioassays for nematodes, such as the cereal cyst nematode, are long and labour intensive. Although natural resistance to plant parasitic nematodes occurs in certain plant genotypes, the molecular basis of resistance was hitherto unknown. In particular, the molecular characterisation of genetic sequences encoding a polypeptide which confers nematode resistance on a plant, has not been a straightforward procedure. Furthermore, until the present invention, the chromosomal localisation of nematode resistance genes and genetic markers for nematode resistance in Triticum tauschii, were unknown.
SUMMARY OF THE INVENTION
In accordance with the present invention, genetic sequences conferring resistance to a plant pathogen, preferably a plant parasitic nematode, have been cloned from Triticum tauschii. The cloning of these sequences permits the generation of transgenic plants with de novo, improved or otherwise enhanced nematode resistance. The present invention also permits the screening through genetic or immunological means, similar nematode resistance genes in other plants for use in developing or enhancing nematode resistance in commercially and economically important species.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a nucleic acid molecule which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a nematode in a plant.
In another embodiment, the present invention provides an isolated DNA molecule comprising a sequence of nucleotides which: (i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and
(ii) has at least about 40% nucleotide sequence similarity to any one or more of the sequences set forth in SEQ ID NOS: 1, 3, 5 or 7 or a part thereof.
In yet another embodiment, the present invention provides an isolated nucleic acid molecule which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NOS: 1, 3, 5 or 7 or to a complementary strand thereof.
In yet another embodiment, the invention provides an isolated nucleic acid molecule which is substantially the same as any one or more of the sequences set forth in SEQ ID NOS: 1, 3, 5 or 7 or is at least 40% identical thereto.
Another aspect of the invention provides a genetic construct comprising a sequence of nucleotides which encodes or is complementary to a nucleic acid molecule which encodes a protein or derivative thereof, which confers, enhances, or otherwise facilitates resistance to a nematode in a plant. According to one embodiment, the nucleic acid molecule is operably linked to a promoter sequence, thereby regulating expression of said nucleic acid molecule in a eukaryotic cell, for example a plant cell, or a prokaryotic cell.
In yet another aspect, the present invention provides a genetic construct comprising an isolated promoter sequence from a gene which when expressed encodes a polypeptide that confers, enhances, or otherwise facilitates nematode resistance in a cell, or a functional part, derivative, fragment, homologue or analogue thereof, wherein said promoter is operably linked to the coding region isolated from a second genetic sequence.
The invention extends to the recombinant polypeptide product of said genetic construct.
The present invention also provides an oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7.
The nucleic acid molecule and/or oligonucleotide of the present invention are useful in the isolation of nematode resistance or nematode resistance-like genetic sequences from other plants, using hybridisation and/or PCR-based approaches.
Accordingly, there is provided a method of identifying a nematode resistance genetic sequence or nematode resistance-like genetic sequence which method comprises contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a genetic sequence encoding, or complementary to a genetic sequence encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance, or a part thereof, and then detecting said hybridisation.
There is also provided a method of identifying a nematode resistance genetic sequence or a nematode resistance-like genetic sequence in a plant cell, which method comprises contacting genomic DNA, mRNA, or cDNA with one or more oligonucleotide molecules to a genetic sequence from said plant for a period of time and under conditions sufficient to form a double- stranded nucleic acid molecule and amplifying copies of the said genetic sequence in a polymerase chain reaction.
In another aspect, this invention also provides an isolated polypeptide which comprises an amino acid sequence which confers, enhances, or otherwise facilitates resistance to a nematode in a plant cell, or a functional mutant, derivative part, fragment, or analogue of said polypeptide.
The present invention extends to a synthetic peptide comprising at least 10 contiguous amino acids of the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or having at least 40% similarity to all or a part thereof.
The polypeptide and synthetic peptides of the present invention may be used to generate specific immuno-interactive molecules. Accordingly, the present invention also provides an antibody that binds to a polypeptide which confers, enhances or otherwise facilitates resistance to a nematode in a plant or a part or fragment thereof, wherein said polypeptide, part or fragment threof further comprises an amino acid sequence which is substantially the same as the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or is at least 40% similar to all or a part thereof.
In yet another aspect of the present invention, there is provided a method of identifying a nematode resistance gene product or nematode resistance-like gene product in a plant cell, which method comprises contacting the antibody with an antigen from said plant for a period of time and under conditions sufficient to form an antibody-antigen complex and measuring the amount of said antibody-antigen complex formed.
The present invention is useful for the generation of plants with enhanced nematode resistance or nematode resistance-like characteristics and there is also provided a plant carrying a non- endogenous nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances, or otherwise facilitates nematode resistance in said plant. The present invention extends to the progeny derived from said plant. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphical representation of an RFLP linkage and physical map of chromosome 2D produced from Triticum tauschii F2 progeny of the genetic crosses (a) CPI 110813 X CPI 110795; (b) AUS 18913 x CPI 110856. The map location of the nematode resistance genes Ccn- Dl and Ccn-D2 are indicated.
Figure 2 is a photographic representation of an agarose gel showing PCR amplification products obtained from a survey of hydroxylapatite-fractionated DNA enriched for low copy sequences. DNA samples were from bulked segregants of the Triticum tauschii cross CPI 110810 (resistant) x CPI 110825 (susceptible). Odd-numbered lanes contain DNA from resistant bulked segregants. Even-numbered lanes contain DNA from susceptible bulked segregants. Random primers used for each pair were OPF12 (lanes 1,2), OPF13 (lanes 3,4), OPG2 (lanes 5,6), OPG3 (lanes 7,8), OPG6 (lanes 9,10), OPG6 (lanes 11,12) and OPG13 (lanes 13,14).The arrow indicates the presence of a polymorphic PCR fragment present in lane 13 but absent from lane 14. Lane 15 is a size marker.
Figure 3 is a photographic representation of an agarose gel showing PCR amplification products obtained from total genomic DNA and hydroxylapatite-fractionated DNA enriched for low copy sequences. DNA samples were from parental and bulked segregants of the Triticum tauschii cross AUS 188913 (resistant) x CPI 110856 (susceptible). Amplification products were obtained using the random primers OPE20 (lanes 1-6) and OPF12 (lanes 7,8). Templates were from low copy bulked resistant segregant (lanes 1,7), low copy bulked susceptible segregant (lanes 2,8), total genomic bulked resistant segregant (lane 3), total genomic bulked susceptible segregant (lane 4), total genomic from AUS 188913 (lane 5), and total genomic from CPI 110856 (lane 6). The arrow indicates the presence of a polymorphic PCR fragment present in resistant bulked segregant (lane 1) but not in the susceptible bulked segregant (lane 2). The converse type of polymorphism is shown with primer OPF 12 in lanes 7 and 8.
Figure 4 is a photographic representation showing an autoradiograph of one euploid (lane 1) and several nullitetrasomic lines (lanes 2-6) of Triticum aestivum cv Chinese Spring, showing RFLP patterns assayed with the cloned PCR fragment csE20-2. Cytogenetic stocks missing chromosome 2D are present in lanes 8 (nulli 2D tetra 2A) and 9 (nulli 2D tetra 2B).
Figure 5 is a photographic representation showing an autoradiograph of Triticum tauschii F2 individuals from the cross AUS 188913 (resistant) x CPI 110856 (susceptible) assayed with the cloned PCR fragment csE20-2. Ccn-DI resistance (R) and susceptibility (S) are indicated.
Figure 6 is a photographic representation showing linkage between the XcsE20 RFLP marker and CreS (Ccn-DI) in Triticum aestivum resistant (R) and susceptible (S) backcross individuals.
Figure 7 is a schematic representation of a lambda clone containing the Cre3 gene. The positions of the 6.5kb EcĻŒRV RFLP fragment and PCR fragment csE20-2 are indicated. The position of the Cre3 gene within the lambda clone is also indicated. Restriction enzyme sites BamHi B) and EcoRV(E) are indicated.
Single letter abbreviations used for amino acid residues in the specification are defined in Table 1.
IĪ”ELHI
Amino Acid Three-letter One-letter Abbreviation Symbol
Alanine Ala A
Arginine Arg R
Asparagine Asn N Aspartic acid Asp D
Cysteine Cys C
Glutamine Gin Q
Glutamic acid Glu E
Glycine Gly G Histidine His H
Isoleucine lie I
Leucine Leu L
Lysine Lys K
Methionine Met M Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y
Valine Val V
Any amino acid Xaa X DETAD ED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention comprises an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a protein or derivative thereof, which confers, enhances or otherwise facilitates resistance to a nematode in a plant.
Hereinafter the term nematode "resistance gene" or "resistance-like gene", or similar term shall be used to define a nucleic acid molecule which upon expression confers, enhances, or otherwise facilitates resistance of a cell and/or organism to one or more plant parasitic pathogens. The term "nematode resistance gene" further defines a nucleic acid molecule which upon expression confers, enhances, or otherwise facilitates resistance to one or more plant parasitic nematode pathogens. Reference herein to a "gene" is to be taken in its broadest context and includes: (i) a classical genomic gene consisting of a coding region optionally together with transcriptional and/or translational regulatory sequences and a coding region with or without non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and optionally 5'- and 3'- untranslated sequences of the gene.
The term "gene" is also used to describe a synthetic or fusion molecule, or derivative which encodes, or is complementary to a molecule which encodes, all or part of a functional product. A functional product is one which confers, enhances or otherwise facilitates resistance of a cell to a parasitic nematode. Preferred nematode resistance-like genes are derived from a naturally occurring nematode resistance gene by standard recombinant techniques. Generally, a nematode resistance gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions. Nucleotide insertional derivatives of the nematode resistance gene of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be "silent" in that the substitution does not change the amino acid defined by the codon. Alternatively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.
The present invention extends to the isolated nucleic acid when integrated into a plant genome and to propagated plants containing same nucleic acid molecule.
Another aspect of the present invention is directed to a nucleic acid molecule which comprises a sequence of nucleotides corresponding or complementary to the nucleotide sequence set forth in any one or more of SEQ ID NOS: 1, 3, 5 or 7, or having at least 40% similarity to all or a part thereof and wherein said nucleic acid molecule encodes a protein that confers, enhances, or otherwise facilitates resistance to a nematode in a plant.
Preferably, the percentage similarity to a sequence set forth in SEQ ID NOS:l, 3, 5 or 7 is at least 50%. Even more preferably, the percentage similarity is at least 60-65%. Still more preferably, the percentage similarity is at least 70-75%. Yet still more preferably, the percentage similarity as at least 80-90%, including at least 91% or 93% or 95%.
For the purposes of nomenclature, the sequences shown in SEQ ID NOS: 1, 3, 5 and 7 relate to the CRE 3 resistance gene of Triticum tauschii which controls resistance to cyst nematodes Heterodera sp. More preferably, SEQ ID NOS: 1 and 3 are nucleotide sequences of a genomic clone isolated from Triticum tauschii, containing the Cre 3 gene. The nucleotide sequence set forth in SEQ ID NO: 5 is a cDNA clone encoding CRE 3, which was isolated from T. tauschii AUS 18913 seedlings using genomic clone sequences. SEQ ID NO: 7 shows the nucleotide sequence of the promoter and complete open reading frame of the Cre gene, without introns. The amino acid sequence of the complete CRE3 polypeptide is presented in SEQ ID NO: 8.
Preferably, the cyst nematode is the cereal cyst nematode (CCN) Heterodera avenae. More preferably, the cyst nematode is the Australian pathotype of H. avenae. The designation "CRE 3" is also synonymous with the designation "Ccn-DI" referred to by Eastwood et al. (1993), among others.
A further aspect of the present invention contemplates a nucleic acid molecule which encodes a protein that confers or otherwise facilitates nematode resistance in a plant and which is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in any one or more of SEQ ID NOS: 1, 3 , 5 or 7 or to a derivative homologue or analogue thereof.
For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28Ī²C. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.
Conditions for hybridisations and washes are well understood by one normally skilled in the art.
For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference can conveniently be made to pages 2.10.8 to 2.10.16. of Ausubel et al.
(1987), which is herein incorporated by reference.
Genetic analysis indicates that specific interactions may occur between resistance genes and gene products of the parasitic nematode. Although not intending to limit the present invention to any one theory or mode of action, it is proposed that the genetic sequences of the present invention control host range via specific recognition of the gene products of the nematode pest, in a "gene-for-gene" interaction that is understood by one normally skilled in the art. Accordingly, the genetic sequences are useful in increasing the range of resistance of a plant to nematode pests, by providing de novo the required nematode resistance gene, or being introduced together with the corresponding nematode gene or genes, on, for example, a single genetic cassette. Accordingly, these aspects of the invention are covered by the expression "conferring, improving, or otherwise enhancing nematode resistance" or other similar expression.
The present invention is particularly directed to resistance that is conferred, enhanced, or facilitated against a nematode, preferably a cereal cyst nematode, more preferably Heterodera avenae, even more preferably the Australian pathotype of H. avenae by a polypeptide encoded by genetic sequences from Triticum tauschii. Examples of genetic sequences in Triticum tauschii which confer resistance to a nematode include, but are not limited to the Ccn genes, Ccn-DI and Ccn-D2 and the Cre 1. The subject invention clearly contemplates other sources of nematode resistance genes, such as but not limited to, other monocotyledonous plants, other Triticum sp., barley, maize, rye, oats, and rice, amongst others.
The genetic sequences which encode a protein which confers, enhances, or otherwise facilitates nematode resistance may correspond to the naturally occurring sequence or may differ by one or more nucleotide substitutions, deletions and/or additions. Accordingly, the present invention extends to nematode resistance genes and any functional genes, mutants, derivatives, parts, fragments, homologues or analogues thereof or non-functional molecules but which are at least useful as, for example, genetic probes, or primer sequences in the enzymatic or chemical synthesis of said gene, or in the generation of immunologicaily interactive recombinant molecules.
In a particularly preferred embodiment, the nematode resistance genetic sequences or like genetic sequences are employed to identify and isolate similar genes, or nematode resistance- like genes from other plants. The present invention extends to the use of said genetic sequence, or a part thereof to detect polymorphisms of a nematode resistance genetic sequence or nematode resistance-like genetic sequence.
In this aspect of the invention, there is provided an oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7.
Accordingly there is contemplated a method for identifying a related nematode resistance genetic sequence or nematode resistance-like genetic sequence, said method comprising contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a genetic sequence encoding or complementary to a genetic sequence encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance, or a part thereof, and then detecting said hybridisation.
The related nematode resistance genetic sequence or like sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from Triticum aestivum or similar plant such as maize, barley, rye, oats, or rice and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same. In addition, the related genetic sequence may be bound to a support matrix, for example nylon, nitrocellulose, polyacrylamide, agarose, amongst others.
Preferably, the genetic sequence which encode a polypeptide which confers, enhances, or otherwise facilitates nematode resistance (i.e latter genetic sequence) is from Triticum sp., or similar plant such as maize, barley, rye, oats, or rice. In a most preferred embodiment, the latter comprises a sequence of nucleotides set forth in any one or more of SEQ ID NOS: 1, 3, 5 or 7 or a homologue, derivative or analogue thereof.
Preferably, the latter genetic sequence is labelled with a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as 32P or 35S or a biotintylated molecule).
An alternative method contemplated in the present invention involves hybridising a nucleic acid primer molecule of at least 10 nucleotides in length to a nucleic acid "template molecule", said template molecule herein defined as a nematode resistance genetic sequence, or resistance-like genetic sequence, or a functional part thereof, or its complementary sequence. Specific nucleic acid molecule copies of the template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art.
Preferably, the nucleic acid primer molecule or molecule effective in hybridisation is contained in an aqueous mixture of other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form. In a preferred embodiment, the nucleic acid primer molecule is from Triticum sp., or similar plant such as maize, barley, rye, oats, or rice. In a most preferred embodiment, (he nucleic acid primer molecule is any nucleotide sequence of at least 10 nucleotides in length derived from, or contained within any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7.
The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from Triticum aestivum or similar plant such as maize, barley, rye, oats, or rice and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
A further aspect of the present invention is directed to a genetic construct comprising an isolated nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes a protein, or derivative thereof, that confers, enhances, or otherwise facilitates resistance against a nematode in a plant cell. Preferably, the gene sequence is related to or a functional derivative, part fragment, homologue, or analogue of the nucleotide sequence defined by any one or more of SEQ ID NOS: 1, 3, 5 or 7. More preferably, the genetic construct comprises the entire open reading frame of the Cre 3 gene sequence.
The present invention extends to genetic constructs designed to assist expression of a nucleic acid molecule that confers, enhances or facilitates nematode resistance in a cell. Generally, the genetic construct comprises in addition to the subject nucleic acid molecule, a promoter and optional other regulatory sequences that modulate expression of the nucleic acid molecule. The promoter may be the CRE 3 gene promoter, or a promoter from another genetic source. Preferably, however, the promoter is capable of expression in a plant cell, in particular a root cell.
The subject nucleic acid molecule may be genomic DNA or cDNA and may correspond in sequence exactly with the nucleotide sequence as set forth in any one or more of SEQ ID NOS : 1, 3, 5 or 7 or it may contain one or more nucleotide substitutions, additions and/or deletions, either dispersed throughout, or clustered.
In an alternative embodiment, an isolated promoter sequence from a gene which, when expressed encodes a polypeptide that confers, enhances or otherwise facilitates nematode resistance in a cell, or a functional part, derivative, fragment, homologue or analogue thereof, is operably linked to the coding region of a second genetic sequence, for example the Ī²- glucuronidase gene, or the chloramphenicol acetyltransferase gene, or the firefly luciferase gene, amongst others. Preferably, the promoter sequence is contained within nucleotides 1 to 1138 of the sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 7.
Yet another aspect of the present invention provides for the expression of the subject genetic sequence in a suitable host (e.g. a prokaryote or eukaryote) to produce full length or non-full length recombinant nematode resistance gene products. Preferably, the nematode resistance gene product has a sequence that is identical to, or contained within an amino acid sequence set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. More preferably, the nematode resistance gene product has a sequence that is identical to or contained within the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8. The present invention extends also to a synthetic peptide fragment of a nematode resistance gene product, preferably the resistance gene product set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.
The present invention provides an isolated polypeptide which comprises an amino acid sequence which confers, enhances, or otherwise facilitates resistance to a nematode in a plant cell, or a functional mutant, derivative part, fragment, or analogue of said polypeptide. According to this aspect, the present invention also extends to the protein or polypeptide product of the Triticum tauschii nematode resistance gene Cre3 and the weaker resistance gene Ccn-D2. This is done, however, with the understanding that the subject invention extends to a range of resistance genes for nematode and other pathogens. In fact, the present invention extends to a nematode resistance gene characterised by said gene encoding a product having at least one imperfect leucine rich repeat region. Preferably, the leucine rich repeat region is located at the C-terminal end of the protein molecule and has at least 60% similarly to amino acid residues 185 to 412 of the amino acid sequence set forth in SEQ ED NO: 4, or residues 308 to 768 of SEQ ID NO: 6 and even more preferably is at least 80% similar thereto. Still more preferably, the leucine rich region corresponds to amino acid residues 185 to 412 of the amino acid sequence set forth in SEQ ID NO: 4 or residues 308 to 768 of SEQ ID NO: 6.
Alternatively or in addition to, the nematode resistance gene product further contains a p-Loop, or kinase- la motif, having the sequence: GV(G/S)GSGKST and more particularly
GfflGV(G/S)GSGKST, or having one or more amino acid substitutions, insertions and/or deletions thereto provided that such derivatives still function as a p-Loop in conferring nematode resistance in a cell. A p- Loop is involved in ATP/GTP binding.
Alternatively, or in addition to, the nematode resistance gene product further contains a kinase-2 motif, having the sequence:
XXXD, where X is any hydrophobic amino acid residue, and more particularly:
KLDGKRFLL(I/V)LDDVWC, or having one or more amino acid substitutions, deletions, and/or insertions thereto provided that such derivatives still function as a kinase-2 motif. A kinase-2 motif functions in nucleotide binding, preferably in binding of ATP/GTP. The present invention extends to a recombinant gene product that contains the p-Loop, and/or kinase-2, and/or imperfect leucine-rich repeat sequence in any relative combination, or frequency, provided that said recombinant gene product confers, enhances, or facilitates nematode resistance in a cell.
The present invention also extends to a synthetic peptide comprising any part of the amino acid sequence set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or having at least 40% similarity to all or a part thereof.
The recombinant nematode resistance gene product, nematode resistance-like gene product, or functional derivative thereof, may be used to produce immunologicaily interactive molecules, such as antibodies, or functional derivatives thereof, the only requirement being that the recombinant products are immunologicaily interactive with antibodies to all or part of said gene product.
According to this aspect, there is provided an antibody that binds to a polypeptide comprising an amino acid sequence which:
(i) confers, enhances, or otherwise facilitates resistance to a nematode in a plant; or (ii) is substantially the same as the amino acid sequence set forth in any one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or having at least 40% similarity to all or a part thereof.
Antibodies to a recombinant nematode resistance gene product are particularly useful in the screening of plants for the presence of said gene product. Another aspect of the present invention is, therefore, directed to antibodies to a recombinant nematode resistance gene product or part or fragment thereof. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to a nematode resistance gene product or may be specifically raised to a recombinant nematode resistance gene product. In the case of the latter, the nematode resistance gene product may first need to be associated with a carrier molecule. Alteraatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies and/or the recombinant nematode resistance gene products of the present invention are particularly useful for the immunological screening of nematode resistance gene products in various plants, in monitoring expression of nematode resistance genetic sequences in transgenic plants and as a proprietary tagging system.
In one embodiment, specific antibodies are used to screen for nematode resistance gene products or nematode resistance-like gene products in plants. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of a recombinant nematode resistance gene product.
Both polyclonal and monoclonal antibodies are obtainable by immunisation with a recombinant nematode resistance gene product and either type is utilisable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of recombinant nematode resistance gene product, or antigenic or immunointeractive parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techmques. Although antibodies produced by this method are utilisable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product. The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitised against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art (see, for example, Douillard and Hoffman, 1981; Kohler and Milstein, 1975; Kohler and Milstein, 1976).
The presence of a nematode resistance gene product or nematode resistance-like gene product in a plant or more commonly plant extract may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non- competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilised on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time and under conditions sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
In this case, the first antibody is raised to a recombinant nematode resistance gene product and the antigen is a nematode resistance gene product in a plant. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain nematode resistance gene product and include crude or purified plant extract such as extracts of leaves, roots and stems.
In the typical forward sandwich assay, a first antibody raised against a recombinant nematode resistance gene product is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking, covalent binding or physically adsorption, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25 Ā°C) to allow binding of any antigen present in the sample to the antibody. Following the incubation period, the reaction locus is washed and dried and incubated with a second antibody specific for a portion of the first antibody. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
An alternative method involves immobilising the target molecules in the biological sample and then exposing the immobilised target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detected by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target- first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta- galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. The term "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in enzyme immunoassays (EIA), the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofiuorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
It will be readily apparent to the skilled technician how to vary the above assays and all such variations are encompassed by the present invention.
The present invention further extends to a plant such as a crop plant carrying a non-endogenous nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances, or otherwise facilitates nematode resistance in said plant. Preferably, the plant is a monocot plant. More preferably the transgenic plant is one or more of the following: Triticum aestivum, Triticum tauschii, maize, barley, rye, oats, rice, sorghum, amongst others. Other species are not excluded.
The non-endogenous genetic sequence or transgene may originate from any plant species. Preferably, said genetic sequence is identical to any one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5 or 7, or a functional derivative, fragment, part, complement, homologue, or analogue thereof.
Further, where said genetic sequence or transgene is a cDNA molecule such as set forth in SEQ ID NO: 5 or other nucleic acid molecule which lacks a functional promoter, it may be placed operably under control of the Cre3 promoter sequence, or under the control of a heterologous promoter sequence. The expression of the transgene may be constitutive or inducible by an external stimulus such as physiological stress, or by addition of a chemical compound, or the expression may be developmentally-regulated, or expressed in a tissue- or cell-specific pattern. Furthermore the transgene may be inserted into or fused to a particular endogenous genetic sequence. Methods for placing a structural gene operably under the control of a promoter sequence are well-known to those skilled in the art.
A non-endogenous nucleic acid molecule encoding, or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance in a recipient plant may be introduced into said plant by any one, or a combination of procedures, including Agrobacterium-medi&ted transformation, microparticle bombardment, PEG fusion, electroporation, introgression via conventional breeding program, amongst others. It will be readily apparent to one skilled in the art how to produce plants carrying a non- endogenous genetic sequence and perform variations to said procedures.
The present invention extends to the progeny and clonal derivatives of said plant.
The present invention is further described in the following Examples. The embodiments exemplified hereinafter are in no way to be taken as limiting the subject invention.
EXAMPLE 1
PLANT MATERIAL
Experiments were conducted in four resistant and three susceptible accessions of Triticum tauschii, as indicated in Table 2. TABLE 2. Parental Triticum tauschii lines used to create crosses for bulked segregant analysis
Accession Taxon Reaction to Heterodera avenae
AUS 18912 ssp. eusquarrosa var. resistant {Ccn-DI) meyeri AUS 18913 ssp. eusquarrosa var. resistant {Ccn-DI) meyeri
CPI 110810 ssp. eusquarrosa var. resistant {Ccn-DI) typica, intermediate
CPI 110813 ssp. eusquarrosa var. resistant {Ccn-D2) typica, intermediate
CPI 110856 ssp. eusquarrosa var. susceptible typica
CPI 110825 Intermediate susceptible CPI 110795 Intermediate susceptible
The four segregating progeny analysed were from the following crosses: 1. CPI 110813 x CPI 110795 2. CPI 110810 x CPI 110825
3. AUS 18913 x CPI 110856
4. AUS 18912 x CPI 110856
Segregation for resistance to the nematode Heterodera avenae was determined for the F2 progeny of each cross and for 10-12 individuals within each F3 family. A total of 2472 individuals were assessed for reactions to Heterodera avenae. Fifty eight to sixty two F2 plants from each cross were chosen on the basis of availability of F3 data and recovery of sufficient DNA to determine marker segregation. Chromosomal locations of polymorphic DNA markers were determined by nuUitetrasomic and ditelocentric lines of Triticum aestivum cv. Chinese Spring and wheat-barley addition lines (Islam et al., 1981; Sears, 1966).
EXAMPLE 2
PLANT RESISTANCE
Resistance to the nematode Heterodera avenae was assessed using the method of Eastwood et al. (1991), except that the white female nematodes were washed from the roots onto a 300 micron sieve, decanted from the sand and counted under a magnifying lamp.
The F3 families in crosses 2,3,4 (see Table 2) segregated for reaction to Heterodera avenae at the Ccn-DI locus in the ratio 1 :2: 1 (homozygous resistant: heterozygous resistant, homozygous susceptible), consistent with the ratio expected for Mendelian inheritance of a single dominant autosomal gene.
An average of 0.14 +/- 0.1 white females (cysts) were produced per homozygous resistant Ccn- DI plant, compared to 1.38 +/- 0.46 (range 0-8) per resistant line homozygous for the weaker Ccn-D2 gene. The susceptible lines carried a significantly greater number of cysts, in the range of 10-90 cysts.
EXAMPLE 3
RFLP SEGREGATION ANALYSIS
The prior art teachings of Andersen and Andersen (1973), Slootmaker et al. (1974), Rivoal et al. (1986), and Aseidu et al. (1990) indicated the presence of resistance loci to Heterodera avenae on group 2 and group 6 homeologous chromosomes of bread wheat Triticum aestivum. One-half of the 60 RFLP markers used were selected because they map to these chromosome locations, thus maximising the probability of selecting an RFLP linked to the Ccn loci.
A total of 35 RFLP loci were analysed for linkage to Ccn-D2 using the segregants from cross 1, and 34 loci for linkage to Ccn-DI, using the segregants from cross 2. A total of 17 polymorphic loci were identified on groups 2 and 6 chromosomes, of which 11 segregated with the Ccn-DI locus and 6 segregated with the Ccn-D2 locus.
Multipoint analysis of joint F2/F3 segregation of RFLP loci and Ccn resistance revealed a loose linkage between Ccn-D2 and chromosome 2 markers (Figure 1). No methods used were able to identify polymorphisms to map further, the Ccn-D2 locus.
Two-point RFLP linkage data set for cross 1 showed 5cM map units between Ccn-D2 resistance and the RFLP markers ksuH9 (Gill et al. , 1991 ) and csIH52 (Lagudah et al. , 1993 ), where 1 cM is herein defined as 1% recombination between two genetic loci or markers, in a randomly segregating population. Ccn-DI was linked to ksuH9 only (Figure 1). EXAMPLE 4
PCR AMPLIFICATION OF DNA FROM BULKED F2 SEGREGANTS TO IDENTIFY DNA PRODUCTS LINKED TO Ccn-DI
Parental lines and individuals F2 plants were processed for the isolation of leaf DNA as described in Lagudah et al. (1991). Bulked DNA pools for resistance and susceptibility to Heterodera avenae were generated from F2 populations from cross 2 and cross 3 (Table 2). Pooled samples from cross 2 were created by bulking together DNA from 12 homozygous resistant and 13 homozygous susceptible F2 lines, and in cross 3 from 10 resistant and 13 susceptible homozygotes. Two hundred micrograms of genomic DNA from each sample was sonicated for 6 seconds to give a size range of 0.5-6 kb (Clarke et al. 1992). A second set of unsonicated bulked DNA segregants from population 3 was included in the study. Each sample was ethanol precipitated and resuspended in 400 Ī¼L of 0.12 M phosphate buffer (pH 6.8).
Molecular markers were generated from bulked homozygous resistant and susceptible F2 DNA pools of cross 2 (Table 2), by PCR amplification of genomic DNA using 260 random 10-mer oligonucleotides (Operon Technologies, Alameda, California) (OPA-01 to OPM 20). Other oligonucleotides included were 4 semirandom primers of 15-18 nucleotides in length, based on the consensus nucleotide sequences of intron-exon splice junctions for plant genes (Weining and Langridge, 1991), designated ISJR1, ISJR2, ISJE3 and ISJE4.
PCRs were performed in 10 Ī¼L of a reaction mix containing about 30 ng of template DNA, 0.5 units of Taq polymerase (Boehringer Mannheim GMBH, Germany), 15 ng of primer, 1.5 mM MgCI2 and 1 x reaction buffer (0.2 mM dNTPs, 67 mM Tris-HCl (pH 8.8), 16 nM (NH,)2 SO4 0.01% (w/v) gelatin, and 0.45% (v/v) Triton X-100). Samples were loaded into capillary tips and run on a thermocycler (FTS-1 Thermal Sequencer, Corbett Research, Sydney, Australia) under the following conditions: 1. Five cycles of denaturation at 93Ā°C for 30 seconds, annealing at 35 Ī²C for 120 seconds, and extension at 72Ā°C for 90 seconds;and 2. Thirty five cycles of denaturation at 92Ī²C for 5 seconds, annealing at 40Ī²C for 20 seconds, and extension at 72 Ī²C for 90 seconds; and
3. One cycle of denaturation at 92 DC for 10 seconds, annealing at 40 Ā°C for 20 seconds, and extension at 72Ā°C for 5 minutes. When longer and specific oligonucleotide (24 bases) primer pairs were used the annealing temperature was 55 Ī²C.
The reaction products were visualised in 1.5% (w/v) agarose gels containing ethidium bromide, using UV light.
EXAMPLE 5
FRACTIONATION OF DNA TO ENRICH FOR LOW-COPY SEQUENCES
Wheat genomic DNA has a large proportion of highly-repeated DNA sequences, which may reduce the probability of detecting low-copy sequences in the genome, using PCR To improve the intensity of the PCR band generated using ISJE3, DNA was fractionated on hydroxylapatite to remove highly repetitive DNA sequences.
Enrichment for low copy sequences from total genomic DNA was achieved by reannealing the heat denatured DNA (100Ā°C for 10 minutes) at 61 Ā°C for at least 20 hours to a Cj (=moles nucleotide/litre x incubation time [seconds]) value of greater than 100 (Smith and Flavell,
1975). Resistant and susceptible bulks in segregants of cross 2 (Table 2) were annealed to a Cj value of 145, while those in bulked segregants of cross 3 were annealed for a Cj value of 120.
The samples were then loaded into a 10 mm diameter hydroxylapatite (Biorad DNA grade, Bio- gel HTP) column maintained at 60Ī²C that had been prewashed with several volumes of 0.0 IM phosphate buffer (pH 6.8). The column was rinsed with 3 mL of 0.01M phosphate buffer and the single-stranded DNA was eluted with one column volume of 0.15M phosphate buffer
(60Ī²C) and collected in 15 x 0.5 mL aliquots. The DNA concentration in each aliquot was determined with UV (260 nm) spectrophotometry and three to four of the 0.5 mL aliquotes that contained most of the DNA were further concentrated with butan-2-ol extractions (Sambrook et al. 1989) and each sample of three to four tubes finally reduced to a single sample of 100-120 Ī¼L. Sodium phosphates were removed from the DNA using a Sephadex 50 column equilibrated with TEN buffer (10 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8.0). DNA was recovered by ethanol precipitation and diluted to a concentration of 300 ng/Ī¼L in TE ready for use in PCR amplification reactions.
The average proportion of DNA recovered after hydroxylapatite fractionation in bulked segregants from cross 2 and cross 3 was 17% and 25%, respectively.
EXAMPLE 6
PCR AMPLIFICATION OF DNA ENRICHED FOR LOW-COPY SEQUENCES FROM BULKED F2 SEGREGANTS
Polymorphic amplification products were obtained using four random 10-mer primers, including ISJE3, in the bulked segregants of cross 2 (see Table 2), an increase in the detectable level of polymorphism from 0.45% to 2%. In each case, the polymorphic PCR product obtained was associated with the presence of a DNA band in the resistant bulk, that was absent from the susceptible bulk (Figure 2).
Polymorphic amplification products were also obtained using eight random 10-mer primers, in the bulked segregants of cross 3 (Table 2). In seven of the polymorphisms, the polymorphic PCR product was associated with the susceptible bulk and only one (OPE-20) was associated with the resistant bulk (Figure 3). Primer OPE-20 produced a consistent polymorphisms in DNA enriched for low-copy nucleotide sequences, but not for total wheat genomic DNA of either the parental genotypes, or the bulked segregating progeny (Figure 3). EXAMPLE 7
CHROMOSOME LOCATION AND GENETIC LINKAGE OF POLYMORPHIC
PCR PRODUCTS
The polymorphic PCR products present in the resistant bulk plus the DNA amplified in the susceptible bulk using primer OPF12 (Figure 2, Figure 3) were excised from low-melting agarose gels, radiolabelled and hybridised to membrane filters containing DNA of parental lines from all populations that had been digested with restriction enzymes.
Polymorphism was observed using a lkb amplified DNA fragment, designated E-20. All Ccn- DI resistant parents showed one to two major hybridising DNA fragments, while the susceptible lines were characterised by a single minor hybridising fragment. The E-20 fragment was subsequently cloned into a "T-overhang" pUCl 18 plasmid vector, to produce the recombinant plasmid csE20-2.
Genomic and cDNA clones used in the construction of Triticum tauschii, wheat, and barley genetic maps (Sharp etal., 1989; Gill et al, 1991; Lagudah et al, 1991a; Heun et al, 1991), as well as the csE20-2 clone were used as RFLP markers to analyse joint segregation with CCN resistance susceptibility in the F2 progenies. Procedures for RFLP analysis were as described by Lagudah et al. (199 la). As an aid in selecting potential markers to target the CCN resistance region, the genetic map of Triticum tauschii produced from the main mapping population (cross F) reported by Lagudah et al. (1991a, 1993) was aligned with common reference RFLP loci mapped in the CCN population (Figure 1). Linkage analysis of segregating RFLP loci and CCN resistance derived from all F2 progenies were carried out using the MAPMAKER program (Lander et al, 1987).
Digested DNA from nuUitetrasomic and ditelocentric lines of Triticum aestivum cv Chinese
Spring were hybridised with the csE20-2 fragment, to determine its chromosome location. The csE20-2 hybridising band was present in all lines except those deleted for chromosome 2D (Figure 4), including the ditelo 2DS line, indicating that the csE20-2 clone maps to the long ar of chromosome 2D.
The csE20-2 RFLP patterns of parental genotypes of Triticum tauschii and resistance susceptibility to Heterodera avenae infestation reveal a complete linkage between Ccn-DI an the csE20-2 RFLP marker, for segregating progeny of crosses 2,3, and 4 (Table 2, see Figur 5). Pooled crosses were based on the RFLP analysis of 178 F2 lines and the Heterodera avena reactions observed for 2020 individual F3 plants.
Further RFLP markers were mapped on chromosome 2D, in cross 3. As shown in Figure l both Ccn-DI and csE20-2 are approximately 13.8cM from the RFLP marker WG645 (Kleinho et al., 1993), 26.3cM from csIH57-l (Lagudah et al., 1991b), and 46. lcM from ksu H9 (Gi et al. , 1991), which have previously been localised on the long arm of chromosome 2. The Ccn DI parent CPI 110813 was mapped in cross 1, and the RFLP variant of csE20-2 shown als to be linked distally, 25cM from csIH57-l, but independent of the Ccn-D2 locus (Figure 1 suggesting that Ccn-DI and Ccn-D2 are non-allelic nematode resistance genes.
EXAMPLE 8
INTROGRESSION OF THE Cre3 GENE INTO BREAD WHEAT. Triticum tauschii
The Cre3 gene from Triticum tauschii was introgressed into bread wheat Triticum aestivum b repeated backcrossing to produce backcross one F4 lines. The csE20-2 RFLP marker was use as a probe to check for linkage among 30 progeny lines, between csE-20 and reaction t Heterodera avenae (Figure 6). The 6.5kb RFLP fragment detectable by hybridisation with csE 20 in resistant parental lines of Triticum tauschii, was observed in all resistant homozygous an heterozygous progeny of Triticum aestivum (Figure 6, lanes 4,7,9,14,15). In contrast, brea wheat lines that were susceptible to infestation with Heterodera avenae all lacked the 6.5k RFLP fragment and exhibited identical RFLP patterns to the parental susceptible line (Figur 6, lanes 3,5,6, 8,10,11,12,13,16,17).
Thus, the introgressed Cre3 gene was able to confer nematode resistance in bread wheat, Triticum aestivum.
EXAMPLE 9
POSITIONAL CLONING OF THE Crg3GENE
Data from bulked segregant analysis provide indications of the genetic distance between loci, but no indication of the physical distance, in kb, which may be in the order of several megabases in a highly recombinogenic region such as the 2DL chromosome of Triticum tauschii. To determine the physical size (kb) per unit of genetic recombination (cM) in the Cre3 gene region, a series of chromosome deletion lines for the long arm of chromosome 2D (Endo, 1990) were employed. Genetic data from one deletion line, with 24% of the 2DL chromosome deleted, indicated that there were approximately 300kb of DNA per cM in the Cre3 region of 2DL. Southern analysis on 178 F2 f-unilies, using the csE20-2 RFLP marker as a probe, showed complete cosegregation between Cre3 and the 6.5kb EcoRV RFLP band that hybridises to csE20-2 (0.0% recombination, p=0.05). Thus, the Cre3 gene was estimated to be within 15kb of csE20-2.
DNA from Triticum tauschii line AUS 18913 carrying the Cre3 gene, was size-fractionated to isolate fragments in the range 15-20kb in length, and used to construct a lambda genomic library. The insert from csE20-2 was radioactively labelled and used to isolate three genomic clones, which were sub-cloned for further analysis. One sub-clone, designated CCN4, was shown to overlap 4kb at the 5' end of the 6.5kb EcoRV RFLP band (Figure 7). This subclone was also shown to cosegregate with Ccn resistance.
The sequenced region of CCN4 clone contains the nucleotide sequences of the 889bp PCR amplification product E-20, between nucleotide positions 194 and 1082 of the nucleotide sequence set forth in SEQ ID NO: 1. The sequenced region of CCN4 also contains two overlapping reading frames (exons) from nucleotides 1138 to 1614 of the nucleotide sequence set forth in SEQ ID NO: 1 and from nucleotides 1 to 1238 of the nucleotide sequence set forth in SEQ ID NO: 3, with no stop codon at the end of the second exon, suggesting that the clone contains a partial Cre3 gene sequence.
The first exon encodes a p-LoopĀ»(or kinase- la) motif between nucleotides 1414 and 1437 of the nucleotide sequence set forth in SEQ ID NO: 1, with the amino acid sequence GVGGSGKS. The second exon encodes another nucleotide binding site, kinase-2, between nucleotide positions 73 and 87 and an imperfect leucine-rich repeat sequence from nucleotides 682 to 1238 of the nucleotide sequence set forth in SEQ ID NO: 3.
EXAMPLE 10
ISOLATION OF A ROOT-EXPRESSED NEMATODE RESISTANCE cDNA CLONE
Total RNA was extracted from pooled root samples taken from seedlings of T. tauschii, AUS 18913, (resistant source of Cre3 gene) grown for 10, 15 and 25 days. Polyadenylated mRNA was prepared using an oligodT sequence coupled to a magnetic bead (DynalĀ®). First and second cDNA strand synthesis was performed using manufacturers instructions (StratageneĀ®) and cloned into a lambda ZAP vector. The T. tauschii AUS 18913 cDNA library was screened using the genomic clone containing the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3. Eight size classes of cDNA clones ranging from 1.7 to 2.6 kb with strong hybridising intensity to SEQ ID NO: 1 and SEQ ID NO: 3 sequences were identified. The complete nucleotide sequence of the 2.6kb cDNA was determined and compared with sequences from the remaining seven clones. The sequence comparison revealed that all seven clones were identical and represented shorter fragments of the 2.6kb cDNA clone. Nucleotide sequence comparison between the 2.6kb cDNA and the SEQ ID NO: 1 and SEQ ID NO: 3 sequences revealed 87% identity. The regions of sequence identity to the 2.6kb cDNA was localised to the reading frames present in SEQ ID NO: 1 and SEQ ID NO: 3. Thus the csE20 fragment which lacks an open reading frame was incapable of detecting candidate Cre3 nematode resistance genes expressed in roots.
The 2.6kb cDNA sequence set forth as SEQ ID NO: 5 represents a partial gene and encodes a reading frame of 768 amino acids in addition to 324 baes of the 3 ' untranslated region. The 768 amino acids contains a P loop (base positions 191 to 217) or ATP/GTP binding site, kinase 2 domain (base positions 443 to 457) and a leucine rich region (base positions 1202 to 2260). When the 2.6kb root cDNA clone was used as a hybridisation probe to Dra I restricted genomic DNA of the bread wheat variety, Chinese Spring, and its nulliitetrasomic chromosome stocks, an identical hybridisation pattern to SEQ ID NO: 1 and SEQ ID NO: 3 occurred. This analysis confirmed the chromosome 2D location of SEQ ID NO: 5 sequence as identical to SEQ ED NO: 1 and SEQ ID NO: 3. The SEQ ID NO: 1 and SEQ ID NO: 3 sequences cosegregates with cereal cyst nematode resistance at the Cre3 locus in 178 F2 families of 7. tauschii. Thus the root expressed gene sequence, SEQ ID NO: 5, is clustered with the genomic sequence, SEQ ID NO: 1 and SEQ ID NO: 3 at the Cre3 locus.
EXAMPLE 11
COMPLEMENTATION ANALYSIS
Wheat are transformed with a genetic construct comprising the complete open reading frame of the Cre3 gene, essentially according to the established transformation and regeneration procedures of Weeks et al, 1993, Nehra et al, 1994 and Becker et al, 1994. The complete genomic sequence of the Cre3 gene including at least lkb of 5' untranslated sequence comprising nucleotide sequence information required for efficient transcription and the 3' untranslated region, are cloned into a plasmid vector. Scutellar tissue of immature wheat embryos derived from the cereal cyst nematode susceptible cultiva Gabo are co-transformed with the Cre3 gene construct and the plasmid pEmuKON (Chamberlain etal, 1994) which comprises an efficient promoter for gene expression in cereal cells operably linked to the iĻˆtĻ‹ gene (conferring resistance to aminoglycoside antibiotics) and a termination signal.
Stable tansformants are selected on paromomycin-containing media and the presence of the Cre3 gene construct verified subsequently by standard procedures (polymerase chain reaction Northern blotting and Southern blotting using re3-derived nucleotide sequences; Ausubel et al, 1987). Transformed tissue containing the introduced Cre3 gene sequences are placed on regeneration medium and regenerated into whole plants. Transformed plants are retained for further analysis. The roots of transformed plants are assayed for expression of the introduced Cre3 gene, using northern blot hybridisation, reverse-transcription PCR or other procedure suitable for the detection of Cre3 gene transcripts in root tissue. Such methods are well-known to those skilled in the art.
Roots expressing the Cre3 gene are inoculated with juvenile cereal cyst nematodes of the Australian pathotype, essentially as described by Eastwood et al. (1991). Non-transformed isogenic wheat are similarly infected in a parallel experiment. Juveniles of cyst nematodes normally invade plant roots and migrate to the vascular tissue where they induce syncytia formation in a compatible host plant interaction. Alternatively, in resistance plants, host plant mechanisms lead to breakdown of syncytia with the production of large, vacuolated syncytia which possesses degenerated membranes in the roots of infected plants, at about 15 days post- invasion of the roots by the juvenile nematode.
Significantly fewer cysts (0-8) are observed on the roots of wheat plants expressing the introduced Cre3 gene sequence, compared to high cyst counts (>40) on untransformed wheat lines, confirming the ability of the introdued Cre3 gene to confer resistance to the cereal cyst nematode in a compatible interaction. EXAMPLE 12
PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST CRE3
Antibodies are raised against an E. coli fusion protein composed of the carboxy-terminal part of glutathione S-transferase and the 768 amino acids of the CRE3 protein set forth in SEQ ID NO: 6 or alternatively, the 797 amino acids of the full-length CRE 3 protein set forth in SEQ ED NO: 8. The vectors encoding this construct are generated by cloning the 2654 bp fragment of the cDNA shown in SEQ ID NO: 5 or the entire Cre3 open reading frame of SEQ ID NO: 7, into a pGEX plasmid (Pharmacia, Uppsala) producing an in-frame fusion with a partial cDNA encoding about 250 amino acids (27.5 kD) of glutathione S-transferase, placed operably under the control of the lac promoter. The construct is transformed into E. coli cells. After induction with IPTG (lmM final concentration) the expressed fusion protein is purified on Glutathione-Sepharose 4B (Pharmacia, Uppsala) according to the manufacturer's instruction. The apparent molecular weight of the fusion polypeptide in SDS-PAGE is approximately 110- 115kD.
The purified fusion protein (100 Ī¼g) is subcutaneously injected into a female rabbit using Freund's adjuvant as described by Harlow and Lane (1988). After the second boost, antiserum is collected and used in Western blotting and ELISA tests.
Antisera are screened by ELISA (Enzyme-Linked Immunosorbent Assay) using purified CRE- GST fusion polypeptide. The ELISA is performed as follows: partially purified CRE-GST fusion polypeptide in coating buffer (Na2CO3, 15 mM; NaHCO3, 35 mM, CaCl2, 0.1 mM; final pH 9.2) is incubated overnight at 4Ī²C in Nunc-Immuno Plate Maxisorb (Nunc-Kamstrup, Denmark). After 4 rinses in washing butter (Tris/HCl, 20 mM; NaCl, 120 mM; Tween 20, 0.05%; final pH 7.4), the primary antibody (diluted serially between 1 :250 and 1 : 5000) is added for 1 hour at 37Ī²C, wells are washed 4 times with washing buffer, and then incubated with peroxidase-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO) at a 1:1000 dilution for an additional 1 hour at 37Ā°C. Wells are washed 4 times using washing buffer substrate solution (O-phenylenediamine dihydrochloride, 0.05 ml, 0.4 mg/ml, Sigma) dissolved in 0.1 M sodium citrate (pH 4.5) containing H202 (0.0006%) is added, and color is allowed to develop. The reaction is stopped by adding H2SO4 (0.025 ml; 8N), and absorbance at 490 mm is then measured.
For Western blotting, total lysate of E. coli clones expressing either the pGEX encoded GST protein alone or the CRE3-GST fusion protein before and after induction with IPTG are separated on SDS-PAGE. The bacteria are harvested by centrifugation and resuspended in Laemmli sample buffer (4% SDS, 125 mM Tris pH 6.8, 10% Ī²-mercapto ethanol, 10% glyceol, 0.02% bromphenol blue) to a concentration of 2xl07 cells/Ī¼l of sample buffer. After boiling for 5 min, 10 Ī¼l of this SDS lysate are applied to SDS-PAGE (10% polyacrylamide), transferred to nitrocellulose using standard techniques (Sambrook et al., (1989), 200 mA, 40 min), blocked with PBS containing 2% nonfat dry milk, 0.02% Tween 20, washed (PBS 0.02% Tween 30, 0.2% gelatin) and probed using polyclonal antisenim raised against the CRE3-GST fusion protein preimmune serum (both sera diluted at least 1: 1000 in PBS containing 0.05% Tween 20 and 0.2% gelatine). A second antibody (horseradish peroxidase coupled goat anti- rabbit IgG (BioRad, Munich)) is diluted 1:20000 fold in PBS 0.02% Tween 20, 0.2% gelatin. Peroxidase reaction is performed using the ECL Kit (Amersham International) to detect bound antibody. The antisenim strongly recognizes a band at ā€”110-115 kD corresponding to the molecular weight of the CRE3-GST fusion protein, which is not detected by the preimmune serum. There is no cross reactivity of the anti-CRE3-GST antisenim with the recombinant GST protein.
EXAMPLE 13
WESTERN BLOT ANALYSIS OF THE CRE3 PROTEIN
Root protein extracts are obtained from infected plants of the wheat cultivar Gabo, transformed with the Cre3 gene as described in Example 11 and which has improved resistance to the Australian pathotype of the cereal cyst nematode compared to untransfoĻ€ned Triticum aestivum cv Gabo. Root protein extracts are also obtained from infected untransformed Gabo plants. Plants are infected with juvenile nematodes, according to Eastwood et al (1991). Soluble protein is fractionated on SDS-PAGE, transferred to nitrocellulose and probed with antisera to CRE3, as described in Example 12, to identify the CRE3 polypeptide. A cross-reactive band of approximately 80kDa molecular weight, corresponding to the CRE3 polypeptide, is only observed in protein extracts obtained from transformed plants which express the introduced Oe3 gene.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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17. Kohler and Milstein (1976). Eur.J. Immunology, 6: 511-519 18. Lagudah, E. S., Appels, R, Brown, A. H. D. and McNeil, D. (1991a). The molecular- genetic analysis of Triticum tauschii, the D-genome donor to hexaploid wheat. Genome, 34: 375-386.
19. Lagudah, E. S., Appels, R and McNeil, D. (1991b). The Nor-D3 locus of Triticum tauschii natural variation and linkage to chromosome 5 markers. Genome, 34: 387-395.
20. Lagudah, E. S., Appels, R, McNeil, D. and Schachtman, D. P. (1993). Exploiting the diploid D genome chromatin for wheat improvement. In Gene conservation and exploitation. (J.P. Gustafson, R. Appels and R. Raven eds) Plenum Press, New York pp. 87-107.
21. Lander, E. S., Green, P., Abrahamson, J., Barlow, A., Daly, M. J., Lincoln, S. E. and Newburg, L. (1987). MAPMAKER an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1: 174-181.
22. Nehra, N.S., Chibbar, RN., Leung, N., Caswell, K., Mallard, C, Steinhauer, L., Baga, M. And Kartha, K. (1994) Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs. The Plant J. 5: 285-297.
23. Rivoal, R., Dosba, F., Jabier, J. and Pierre, J. S. (1986). Les lignees d'addition ble- Aegilops ventricosa Tausch. VI. Etude de la localisation chromosomique de la resistance a l'egard d! Heterodera avenae. Woll. Agronomie, 6: 143-148.
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25. Sears, E. R. (1966). Nullisomic-tetrasomic combinations in hexaploid wheat. In Chromosome manipulations and plant genetics. (R. Riley and K.R. Lewis eds.) Oliver and Boyd, Edinburgh pp. 29-45. 26. Sharp, P. J., Chao, S., Desai, S. and Gale, M. D. (1989). The isolation, characterization and apphcation in the Triticeae of a set of wheat RFLP probes identifying each homoeologous chromosome arm. Theor. Appl. Genet. 78: 342-348.
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SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION
(ii) TITLE OF INVENTION: Genetic Sequences conferring nematode resistance in plants and uses therefor
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS*
(A) ADDRESSEE: DavieĪ² ColliĪ²on Cave
(B) STREET: 1, Little Collins Street
(C) CITY: Melbourne
(D) STATE: Victoria
(E) COUNTRY: Australia
(F) ZIP: 3000
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT International
(B) FILING DATE: 29-MAR-1996
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/414,938
(B) FILING DATE: 31-MAR-1995
(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Slattery, John M.
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 61-3-9254 2777
(B) TELEFAX: 61-3-92542770 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1614 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1138..1614
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATCCGGAGTC GTGGTTGTGG CCGCTGTCTT CCACTTQTGT ACGGTTTTCC CGTTGTCQAG 60
TGGCCTTCCT TCTTTCGGCC TTGCGGTTAA CAGQTTAGGG GCGCCGGTAG TGTTGTGGTG 120
TGTCATCTTT GGTGAGCTTA TTAATGTCGG CTTGTGGTGT CACATGGTGG TGGGTTCTCA 180
TTTAACGTCG AGGCGGTQAC CTCAGGTGGC GGTCCGTCAA GTCGGCTTCT CAACAAGCGT 240
CTTGGCGGCG GCCGTGGTGG CATTGTTTGG TCGTGTGGAC GGCGGAQGAT GCTAAGTTGG 300
GTGATCCTAG TGTCGGTGGT GCTTTGGCAC TGGCGGTGCC CAGATCGTGT TCTAGTGGTC 360
TGQCTTGGTA GTGACATGTT CACCTCGGTG TGGGCTGGTG CTCGGGAGGC CTAGTGTQGC 420
GTGGAAGAGT GCAACAAGGT CCGGCGATTT TCCTTGGAGC GAACTTTCAT CTTTGTTGGT 480
AGTTTAGGTA GCTTTGTGTT AGGGTGTGGT TCCTCCTATT TTCTTGTTTT TCTTTGATCT 540
GCTTTGTAAG AGGGTCTCCT CATCACCTTG TATCTCTTTG GTCGTGGTTC TTTATATATA 600
AAGCGGGGCC GAAGTAATTT TTGGTAGGAT TCACCAACAT CATGAOAACA AAGCACGAAA 660
ATATAGTAGT ACGGTAGTAG AGAATGTTAA TTCCTCTTGT ATCCAATGTT ATCTCTTGTA 720
TACCGTGATT CTTGCCCATC AGTATTCTCT TAGGCTTCTG TTAGCGAAAC AAAATTCCTT 780 CTTCCAAATT ACCAAACTTC TAGCTCATGA GTATGTTCAT ATAGTGCGCG GAGGATGTGC 840
GTGCCACATG CGTGCGCATG ATGGTGTTGA TAGACTAACA TGTQTGTGTG GTTTCTGTGT 900
GACTGCCTTG TGTTCTCTGC AAAACTAGGC TTTTGGCAAQ TCAGTCTAGA TCCCTCQQCG 960
TATTTTTTAG AAGTATACCG GAGAQTAGAC GAATTCCCTA TATTACATTA GTCTTTTTTC 1020
TTTATTTAGT GTCATGATAG TTTATGTGAA GATAAAATCT CTCTTCTGTA ATQGTCACCT 1080
ATAATTTATT TTTTAAAQAT TTCTCTCTTG TTATTTGGGQ TCTCGCAGGA GAGTGGC 1137
ATG TCA AAG AAA AAG TTG ATA GAC AGC CTG AAG AAG ATA GAA GAC AAT 1185 Met Ser Lys Lys Lys Leu lie Asp Ser Leu Lys Lys lie Glu Asp Asn
1 5 10 15
ATA AAT GAA GCA CAC CAA ATT CTG GAT AAG CTT AAC TTG TCA AGC ATA 1233 lie Asn Qlu Ala His Gin lie Leu Asp Lys Leu Asn Leu Ser Ser lie 20 25 30
AGT GAT GGA AAT AGA AGA CAT GTA ATG GAT GCT AAT CGT CCT ACT ACT 1281 Ser Asp Gly Asn Arg Arg His Val Met Asp Ala Asn Arg Pro Thr Thr 35 40 45
GCA GTT TCT CCG CAT AAA GTA CTT GGT CGA GAT AAT GAG CGC GAC AAG 1329 Ala Val Ser Pro His Lys Val Leu Gly Arg Asp Asn Glu Arg Asp Lys 50 55 60
ATC ATA AAA ATG CTT CAC AAA AAT GAA GGT GGT GTT CAA CCA AGC ACC 1377 lie lie Lys Met Leu His Lys Asn Glu Gly Gly Val Gin Pro Ser Thr 65 70 75 80
AGC AAC AGT CTA TGC TTT TCT GTA ATT GGC ATA CAT GGA GTT GGT GGG 1425 Ser Asn Ser Leu Cys Phe Ser Val He Gly He His Gly Val Gly Gly 85 90 95
TCA GGG AAA TCT ACC CTT GCA CAA TTG GTT TAT GCC CAT GAG GAA AAA 1473 Ser Gly Lys Ser Thr Leu Ala Gin Leu Val Tyr Ala His Glu Glu Lys 100 105 110 GAC AAG AAA GAC AAC AAG GAA GGT CAC TTC GAC CTG GTT ATG TGG GTC 1521 Asp Lys Lys Asp Asn Lys Glu Gly His Phe Asp Leu Val Met Trp Val 115 120 125
CAT GTC TCT CAG AAT TTT AGT QTG GGC QAC ATC TTC AAG GAG TTG TAT 1569 His Val Ser Gin Asn Phe Ser Val Gly Asp He Phe Lys Glu Leu Tyr 130 135 140
GAG GCA QCT TCA GAG CCT AAG GTT CCA TGC CAT TCA ATA ACA TG 1614
Glu Ala Ala Ser Glu Pro Lys Val Pro Cys His Ser He Thr 145 150 155
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 158 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Lys Lys Lys Leu He Asp Ser Leu Lys Lys He Glu Asp AĪ²n 1 5 10 15
He Asn Glu Ala His Gin He Leu Asp Lys Leu Asn Leu Ser Ser He 20 25 30
Ser Asp Gly Asn Arg Arg His Val Met Asp Ala Asn Arg Pro Thr Thr 35 40 45
Ala Val Ser Pro His Lys Val Leu Gly Arg Asp Asn Glu Arg Asp Lys 50 55 60
He He Lye Met Leu His Lys Asn Glu Gly Gly Val Gin Pro Ser Thr 65 70 75 80
Ser Asn Ser Leu Cys Phe Ser Val He Gly He His Gly Val Gly Gly 85 90 95 Ser Gly Lys Ser Thr Leu Ala Gin Leu Val Tyr Ala His Glu Glu Lys 100 105 110
Asp Lys Lys Asp AĪ²n Lys Glu Gly His Phe Asp Leu Val Met Trp Val
115 120 125
His Val Ser Gin Asn Phe Ser Val Gly Asp He Phe Lys Glu Leu Tyr 130 135 140
Glu Ala Ala Ser Glu Pro Lye Val Pro Cys His Ser He Thr 145 150 155
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1236 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1238
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGT TCC ATG CCA TTC AAT AAC ATG AAT TCC TTG GGA AAA GAA TTG GAA 48 Gly Ser Met Pro Phe Asn Asn Met Asn Ser Leu Gly Lys Glu Leu Glu
1 5 10 15
AGG AAA CTA GAT GGA AAG CGA TTC CTT CTG ATA CTG GAT GAT GTC TGG 96 Arg Lys Leu Asp Gly Lys Arg Phe Leu Leu He Leu Asp Asp Val Trp 20 25 30
TGC AAT AAG GAT GTC AGC GAT CAG AAT CTA CCA GAG TTA CTT TCT CCA 144 Cys Asn Lys Asp Val Ser Asp Gin Asn Leu Pro Glu Leu Leu Ser Pro 35 40 45 TTG AAG GTT GGA AAG AGA GGA AGC AAG ATC CTA GTG ACG ACT CGA AGT 192 Leu Lys Val Gly Lys Arg Qly Ser Lys He Leu Val Thr Thr Arg Ser 50 55 60
AAA TAT GCA TTA CCQ GTT CTA GGT CCT GGT GTG AGA TGT ACT GCC ATA 240 Lys Tyr Ala Leu Pro Val Leu Gly Pro Gly Val Arg Cya Thr Ala He 65 70 75 80
CCA GTA CCT GAG TTT GAT GAT ACC GCC TTC TTC GAQ CTA TTC ATG CAC 288 Pro Val Pro Glu Phe Asp Asp Thr Ala Phe Phe Glu Leu Phe Met His 85 90 95
TAT GCC CTG GAA GAA GGC CAA GAT CAG AGC CTG TTC TGT ATA ATT GGT 336 Tyr Ala Leu Glu Glu Gly Gin Asp Gin Ser Leu Phe Cys He He Gly 100 105 110
GAG GAG ATA GCG AAA AAG CTG AAG GGG TCA CCT CTA GCT GCC AGA ACA 384 Glu Glu He Ala Lys Lys Leu Lys Gly Ser Pro Leu Ala Ala Arg Thr 115 120 125
GTG GGA GGA AAT TTA CGT CGA CAA CCA GAT GTC GAC CAT TGG AGA AGA 432 Val Gly Gly Asn Leu Arg Arg Gin Pro Asp Val Asp His Trp Arg Arg 130 135 140
GTC AGA GAT CAA GAC CTT TTC AAG GTA TGG GGA GGG CCT CTG TGG TGG 480 Val Arg Asp Gin Asp Leu Phe Lys Val Trp Gly Gly Pro Leu Trp Trp 145 150 155 160
AGC TAC TAT CAG CTT GGT GAG CAG GCT AGG CGT TGC TTT GCT TAT TGC 528 Ser Tyr Tyr Gin Leu Qly Glu Gin Ala Arg Arg Cys Phe Ala Tyr Cys 165 170 175
AGT ATT TTT CCT AGG AGA CAT CGC CTG TAC CGT GAT GAC CTA GTT AGA 576 Ser He Phe Pro Arg Arg His Arg Leu Tyr Arg Asp Asp Leu Val Arg 180 185 190
CTT TGG GTT GCA GAA GGG TTC ATA AGA AGC ACA GAT GAA GGG GCG GAT 624 Leu Trp Val Ala Glu Gly Phe He Arg Ser Thr Asp Glu Gly Ala Asp 195 200 205 ATT GAA GAT GTT GGT CAQ GAA ATA TTT AAT GAA CTA TTG TCG ATC TCG 672 He Glu Asp Val Gly Gin Glu He Phe Asn Glu Leu Leu Ser He Ser 210 215 220
TTT CTT CAA CCA GGA GGC ACQ AAC AAC TCT TAT CTC GCC GGC ATT TAT 720 Phe Leu Gin Pro Gly Gly Thr Asn Asn Ser Tyr Leu Ala Gly He Tyr 225 230 235 240
TAT GGC AAG GAA TAC TAT TTA GTT CAT QAT CTG CTQ CAC GAT TTA GCA 768 Tyr Gly Lys Glu Tyr Tyr Leu Val His Asp Leu Leu His Asp Leu Ala 245 250 255
GAG GCA GTA GCT GGC AGT GAC TGC TTC AGA ATT GAC AAT AAC QCG AGC 816 Glu Ala Val Ala Gly Ser Asp Cys Phe Arg He Asp Asn Asn Ala Ser 260 265 270
CAG AAA GGA GGA GGA TGG ACA AGA GAT GTT CCC CGA GAC GTT CGG CAT 864 Gin Lys Gly Gly Gly Trp Thr Arg Asp Val Pro Arg Asp Val Arg His 275 280 285
CTT TTT GTT CAG AGT TAT GAT GCA ACA TTG ATT ACT GAA AAG ATT CTT 912 Leu Phe Val Gin Ser Tyr Asp Ala Thr Leu He Thr Glu Lys He Leu 290 295 300
GAA TTG AGA AAG TTA CAC ACT CTT ATC ATT TAT AGT GTT GGA GGG GAT 960 Glu Leu Arg Lys Leu His Thr Leu He He Tyr Ser Val Gly Gly Asp 305 310 315 320
ACA CCA GTT GAG GAA ATA GTC ATC AAG AAC ATA CTC AAG AGT CTG CCA 1008 Thr Pro Val Glu Glu He Val He Lys Asn He Leu Lys Ser Leu Pro 325 330 335
AAA CTG CGG GTA CTA GCA ATT GCT TCG AGT CTG GAG GAC AGT GCA TTT 1056 Lys Leu Arg Val Leu Ala He Ala Ser Ser Leu Glu Asp Ser Ala Phe 340 345 350
ATT TGG AAA CCA GAT ACA TTC TCT GTC CCA GAA TCT GTT GGT CAA TTG 1104 He Trp Lys Pro Asp Thr Phe Ser Val Pro Glu Ser Val Gly Gin Leu 355 360 365 AAA CAT CTG CGC TAT CTT GCT TTC COG ACA GAT AGA GGA TGC CGA GTA 1152 Lys His Leu Arg Tyr Leu Ala Phe Arg Thr Asp Arg Gly Cys Arg Val 370 375 380
ATT TTA CCA AGC AGT CTA AAC CAG CTT TAC CAG ATG CAA CTG CTA GAT 1200 He Leu Pro Ser Ser Leu Asn Gin Leu Tyr Gin Met Gin Leu Leu Asp 385 390 395 400
TTT QGT CAA TGC CAT GAT TTG GTA TTT TGC TGT GAT GA 1238
Phe Gly Gin Cys His Asp Leu Val Phe Cys Cys Asp 405 410
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 412 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Gly Ser Met Pro Phe Asn Asn Met Asn Ser Leu Gly Lys Glu Leu Glu 1 5 10 15
Arg Lys Leu Asp Gly Lys Arg Phe Leu Leu He Leu Asp Asp Val Trp 20 25 30
Cys Asn Lys Asp Val Ser Asp Gin Asn Leu Pro Glu Leu Leu Ser Pro 35 40 45
Leu Lys Val Gly Lys Arg Gly Ser Lys He Leu Val Thr Thr Arg Ser 50 55 60
Lys Tyr Ala Leu Pro Val Leu Gly Pro Gly Val Arg Cys Thr Ala He 65 70 75 80
Pro Val Pro Glu Phe Asp Asp Thr Ala Phe Phe Glu Leu Phe Met His 85 90 95 Tyr Ala Leu Glu Glu Gly Gin Asp Gin Ser Leu Phe Cys He He Qly 100 105 110
Glu Glu He Ala Lys Lys Leu Lys Gly Ser Pro Leu Ala Ala Arg Thr 115 120 125
Val Gly Qly Asn Leu Arg Arg Gin Pro Asp Val Asp His Trp Arg Arg 130 135 140
Val Arg Asp Gin Asp Leu Phe Lys Val Trp Qly Gly Pro Leu Trp Trp 145 150 155 160
Ser Tyr Tyr Gin Leu Gly Glu Gin Ala Arg Arg Cys Phe Ala Tyr Cys 165 170 175
Ser He Phe Pro Arg Arg His Arg Leu Tyr Arg Asp Asp Leu Val Arg 180 185 190
Leu Trp Val Ala Glu Gly Phe He Arg Ser Thr Asp Glu Gly Ala Asp 195 200 205
He Glu Asp Val Gly Qln Glu He Phe Asn Glu Leu Leu Ser He Ser 210 215 220
Phe Leu Gin Pro Gly Gly Thr Asn Asn Ser Tyr Leu Ala Gly He Tyr 225 230 235 240
Tyr Gly Lys Glu Tyr Tyr Leu Val His Asp Leu Leu His Asp Leu Ala 245 250 255
Glu Ala Val Ala Gly Ser Asp Cys Phe Arg He Asp Asn Asn Ala Ser 260 265 270
Gin Lys Gly Gly Gly Trp Thr Arg Asp Val Pro Arg Asp Val Arg His 275 280 285
Leu Phe Val Gin Ser Tyr Asp Ala Thr Leu He Thr Glu Lys He Leu 290 295 300
Glu Leu Arg Lys Leu His Thr Leu He He Tyr Ser Val Gly Gly Asp 305 310 315 320 Thr Pro Val Glu Glu He Val He Lys Asn He Leu Lys Ser Leu Pro 325 330 335
Lys Leu Arg Val Leu Ala He Ala Ser Ser Leu Glu Asp Ser Ala Phe 340 345 350
He Trp Lys Pro Asp Thr Phe Ser Val Pro Glu Ser Val Gly Gin Leu 355 360 365
Lys His Leu Arg Tyr Leu Ala Phe Arg Thr Asp Arg Qly Cys Arg Val 370 375 Ā«ā–  380
He Leu Pro Ser Ser Leu Asn Gin Leu Tyr Gin Met Gin Leu Leu Asp 385 390 395 400
Phe Gly Gin Cys His Asp Leu Val Phe Cys Cys Asp 405 410
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2627 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum tauschii
(B) STRAIN: AUS 18913 (F) TISSUE TYPE: Root
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..2305
(ix) FEATURE:
(A) NAME/KEY: 3'UTR
(B) LOCATION: 2306..2627 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
G TCA AGC ATA AGT GAT GGC AAT ATA CGA CAC ACA ATG GTT GTC AAT 46
Ser Ser He Ser Asp Qly Asn He Arg His Thr Met Val Val Asn 1 5 10 15
CCT ACQ ACT ACC GCA GTT TCC CCG CAA AAA GTT TTT GGT CGA GAT AAT 94 Pro Thr Thr Thr Ala Val Ser Pro Gin Lys Val Phe Gly Arg Asp AĪ²n 20 25 30
GAT CGC QAC AAG ATC ATA GCA ATG CTT CAT GAA AAG GAA GGT GGT CTT 142 Asp Arg Asp Lys He He Ala Met Leu His Glu Lys Glu Gly Qly Leu 35 40 45
GAT CCA AGC ACT AGC AAA GGT CTA TGT TTT TCT GTA ATT GGC ATA CAT 190 Asp Pro Ser Thr Ser Lys Gly Leu Cys Phe Ser Val He Gly He His 50 55 60
GGA GTC AGC GGG TCT GGG AAA TCT ACC CTT GCA CAG CTT GTT TAT GCC 238 Gly Val Ser Gly Ser Gly Lys Ser Thr Leu Ala Gin Leu Val Tyr Ala 65 70 75
CAC GAG AAA AAT GAC AAG CAA GAC AAC AAG GAA GAC CAT TTC GAC CTT 286 His Glu Lys AĪ²n Asp Lys Gin Asp Asn Lys Qlu Asp Hie Phe AĪ²p Leu 80 85 90 95
GTT ATG TGG GTT CAT GTC TCT CAG GAT TTT AGT GTG TGG GGC ATC TTC 334 Val Met Trp Val Hie Val Ser Gin AĪ²p Phe Ser Val Trp Gly He Phe 100 . 105 110
ANG GAG TTG TAT GAG GCA GCT TCA GAT CCT AAG GTT CCA TGC CCT CAA 382 Xaa Glu Leu Tyr Glu Ala Ala Ser AĪ²p Pro Lye Val Pro Cys Pro Gin 115 120 125
TTT AAT AAC TTG ANT GCC TTG QAA GAA GAA CTG GAG AGG AAA CTA GAT 430 Phe Asn Asn Leu Xaa Ala Leu Glu Glu Glu Leu Glu Arg Lys Leu Asp 130 135 140 GGA AAG CGA TTC CTT CTG GTA CTG GAT GAT GTC TGG TGC AAT GCG GAT 478 Gly Lys Arg Phe Leu Leu Val Leu Asp Asp Val Trp Cys AĪ²n Ala AĪ²p 145 150 155
GTT GGT AAC CAG GAG CTA CCA AAG TTA CTT TCT CCA CTG AAG AAA GGA 526 Val Gly AĪ²n Gin Glu Leu Pro Lys Leu Leu Ser Pro Leu Lys Lye Gly 160 165 170 175
AAG AAA GGA AGC AAG ATC CTA GTG ACA ACT CGA AGT AAA TAT GCA CTA 574 Lys Lys Gly Ser Lys He Leu Val Thr Thr Arg Ser Lys Tyr Ala Leu 180 185 190
CCG GAT CTA TGT CCT GGT GTG AGA TAT ACT GCC ATG CCG ATA ACT GAG 622 Pro Asp Leu CyĪ² Pro Gly Val Arg Tyr Thr Ala Met Pro He Thr Glu 195 200 205
GTT GAT GAT ACC GCC TTC TTT GAG TTG TTC ATG CAT TAT GCC CTC GAA 670 Val AĪ²p Asp Thr Ala Phe Phe Glu Leu Phe Met His Tyr Ala Leu Glu 210 215 220
GAT GGC CAA GAT CAA AGC ATG TTC CAG AAC ATT QQG GTT GAG ATT GCA 718 Asp Gly Gin Asp Gin Ser Met Phe Gin Asn He Gly Val Glu He Ala 225 230 235
AAA AAG CTG AAG GGG TCA CCT TTA GCA GCT AGA ACA GTG GGT GGA AAT 766 Lys Lys Leu Lys Gly Ser Pro Leu Ala Ala Arg Thr Val Gly Gly Asn 240 245 250 255
TTA CGT CGA CAG CAA GAT GTT GAC CAT TGG AGA AGA GTC GGA GAT CAA 814 Leu Arg Arg Gin Gin Asp Val Asp His Trp Arg Arg Val Gly Asp Gin 260 265 270
GAC CTT TTC AAG GTA TGG ACG GGA CCT CTG TGG TGG AGC TAC TAT CAG 862 Asp Leu Phe Lys Val Trp Thr Gly Pro Leu Trp Trp Ser Tyr Tyr Gin 275 280 285
CTT GGT GAG CAG GCT AGG CGT TGC TTT GCT TAC TGC AGT ATT TTT CCT 910 Leu Gly Glu Gin Ala Arg Arg Cys Phe Ala Tyr CyĪ² Ser He Phe Pro 290 295 300 AGG AGA CAT CGC TTG TAC CGY GAT GAA TTA GTT AGA CTC TGG ATG GCA 958 Arg Arg His Arg Leu Tyr Arg Asp Glu Leu Val Arg Leu Trp Met Ala 305 310 315
QAA GGG TTC ATA AQA AAC ACA GAT GAA GGG GCG GAT GCT GAA GAC GTT 1006 Glu Gly Phe He Arg Asn Thr Asp Glu Gly Ala Asp Ala Glu AĪ²p Val 320 325 330 335
GGT CTG GGA ATA TTT AAT GAA CTA TTG TCG ATA TCA TTT CTT CAA CCA 1054 Gly Leu Gly He Phe AĪ²n Glu Leu Leu Ser He Ser Phe Leu Gin Pro 340 345 350
GGA GGC CAG GAC TGG TAC AAT CAT GGC AAG GAA TAC TAT TTA GTT CAT 1102 Gly Gly Gin Asp Trp Tyr AĪ²n Hie Qly Lys Glu Tyr Tyr Leu Val His 355 360 365
GAT TTG CTG TAT GAT TTA GCA GGG GCA GYA GCT GGA ACT GAC TGC TTC 1150 Asp Leu Leu Tyr Asp Leu Ala Gly Ala Xaa Ala Gly Thr Asp CyĪ² Phe 370 375 380
AGA ATT GAC AAT AAC ATG ATC CAG ACA GGA GAA AGC TGG GCA AAA GAT 1198 Arg He Asp Asn Asn Met He Gin Thr Gly Glu Ser Trp Ala Lys Asp 385 390 395
GTT CCC AQA GAC GTT CGC CAT CTT TTT GTT CAG AGT TAT GAT GCA NCN 1246 Val Pro Arg Asp Val Arg Hie Leu Phe Val Gin Ser Tyr Asp Ala Xaa 400 405 410 415
TTG AGT ACA GGG AGA TTG CTT GTA TTG GAG GAN TTA CAC ACA CTC GTC 1294 Leu Ser Thr Gly Arg Leu Leu Val Leu Glu Xaa Leu His Thr Leu Val 420 425 430
ATT TAT AGT GTT GGA GGG GAT ACA ACA GTT GAG GAA ATA GTC ATC AAG 1342 He Tyr Ser Val Gly Gly AĪ²p Thr Thr Val Glu Glu He Val He Lys 435 440 445
AAC ATA CTC AAG AGT CTG CCT AAA CTG CGG GTA CTA GCA ATA GCT TTA 1390 Asn He Leu Lys Ser Leu Pro Lys Leu Arg Val Leu Ala He Ala Leu 450 455 460 TGT CTG GAA AAG GAT GGA TTT ATN TGT AGA CCA AAT ATA TTG TCT GTT 1438 Cys Leu Glu Lys AĪ²p Gly Phe Xaa Cys Arg Pro AĪ²n He Leu Ser Val 465 470 475
CCA GAA TCT ATT AGT CAA TTA AAA CAT CTA CGA TAT CTT GCT TTC CGG 1 86 Pro Glu Ser He Ser Qln Leu Lys Hie Leu Arg Tyr Leu Ala Phe Arg 480 485 490 495
ACA OAT ATT GAA TGC AGA GTA ATT TTA CCA AGC AGT CTA AAC CAG CTT 1534 Thr Asp He Glu CyĪ² Arg Val He Leu Pro Ser Ser Leu AĪ²n Gin Leu 500 505 510
TAC CAG ATG CAA CTG CTA GAT TTT GGT GTC TGC ATG AAT TTQ GTA TTT 1582 Tyr Gin Met Gin Leu Leu AĪ²p Phe Gly Val CyĪ² Met AĪ²n Leu Val Phe 515 520 525
TCC TGT GGT QAT CTT ATC AAC TTG CQQ CAT GTA TGC AGC GGT CCT QGA 1630 Ser CyĪ² Gly AĪ²p Leu He AĪ²n Leu Arg Hie Val CyĪ² Ser Gly Pro Gly 530 535 540
TTG CAA TTT TCA AAC ATC GGT AGO CTT GTC TCA CTC CAA ACA ATC CCA 1678 Leu Gin Phe Ser AĪ²n He Gly Arg Leu Val Ser Leu Gin Thr He Pro 545 550 555
GCA TTC AAA GTA AGT CAT GAA CAA GGA CAT GAG GCA AAG CAG TTG AGG 1726 Ala Phe Lys Val Ser His Glu Gin Gly Hie Glu Ala Lys Gin Leu Arg 560 565 570 575
TAC CTA AAC AGG CTC AGC GGC GAA CTG AGT ATA TAT GGT CTC CAA AGT 1774 Tyr Leu Asn Arg Leu Ser Qly Glu Leu Ser He Tyr Gly Leu Gin Ser 580 585 590
GTT GAA AGC AGA GAG GAA GCT CTT GCA TTC GAT CTA GCT GCC AAQ AAA 1822 Val Glu Ser Arg Glu Glu Ala Leu Ala Phe Asp Leu Ala Ala Lys Lye 595 600 605
CGG CTC GCA GAA CTA ACA CTA TCA TTC GGT GGA AGT TCA GAA GTT GCA 1870 Arg Leu Ala Glu Leu Thr Leu Ser Phe Gly Gly Ser Ser Glu Val Ala 610 615 620 QCA GAG GTA CTT GAG GGC CTT TGT CCT CCC GTG GGG CTT GTA ACA CTC 1918 Ala Glu Val Leu Glu Gly Leu Cys Pro Pro Val Gly Leu Val Thr Leu 625 630 635
GAC ATC CGT GAC TAC GAT GGT TTG GTA TAC CCA AAG TGG ATG GTG GGC 1966 AĪ²p He Arg Aep Tyr AĪ²p Qly Leu Val Tyr Pro Lye Trp Met Val Gly 640 645 650 655
AGG CAA AAT GGC GCA CCA GAG AAG CTG CAA CAA CTT GGT CTC TCA GGA 2014 Arg Gin AĪ²n Gly Ala Pro Glu Lye Leu Gin Gin Leu Gly Leu Ser Gly 660 665 670
TGG AGC CAG CCA GQA CCT GCT CCT GCA CTG AAG GCT TTC AAT CAT CTT 2062 Trp Ser Gin Pro Gly Pro Ala Pro Ala Leu Lye Ala Phe AĪ²n Hie Leu 675 680 685
CGT TGC CTC AAT CTG ATG CAC TGC AGC TGG AAC GCC TTG CCA TGC AAT 2110 Arg Cye Leu AĪ²n Leu Met His Cys Ser Trp Asn Ala Leu Pro CyĪ² AĪ²n 690 695 700
ATG GAG CAC CTC AGC TCG CTC GAA ACA GTA ATC ATT ATT AAA TGT TTG 2158 Met Glu Hie Leu Ser Ser Leu Glu Thr Val He He He Lye CyĪ² Leu 705 710 715
AAT ATC CGG TCG CTT CCA ACG CTG CCA CAG TCT CTT ACG TAT TTT TGG 2206 AĪ²n He Arg Ser Leu Pro Thr Leu Pro Gin Ser Leu Thr Tyr Phe Trp 720 725 730 735
CTC CTG AAG TGC GAC GAT GGG TTC ATG GAG TCT TGT CAA ACA GTT GGA 2254 Leu Leu Lys CyĪ² AĪ²p AĪ²p Gly Phe Met Glu Ser CyĪ² Gin Thr Val Gly 740 745 750
CAT CCA AAC TGG AAA AAG ATT CAA CAC ATC TGC AGG AAA TAT TTT AGT 2302 Hie Pro Asn Trp Lys Lys He Gin His He Cye Arg Lys Tyr Phe Ser 755 760 765
GAA TGACGCGGGC TTGGAATCGG AGTCAGAGTA CTTACTTATG GCCCCTAACT 2355
Glu
TGAGACCTGC ATGCCGCTGC AGCTATTTTA TTCCAATTGG AGTCAAQACA AGAGTATTTA 2415 CTCGAGATTC ATAATCTATT CCTGGGTQQA TCTTCTCTTG TGAGTCTGAA AACCTACCAG 2475
TGCCAGTCTG CAATATTGTA AGGAAAQGAG TACATCTATA GTGTCAGTGC ATATACAGTG 2535
TCTGAATCAT GCACTTCCQT TTCTGTATTT CACCGTATTA TTGATTAAAC AGTGCATQTG 2595
CACGTGCACA ATATATATTT CCCGAATCTT CT 2627
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 768 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Ser Ser He Ser Asp Gly Asn He Arg His Thr Met Val Val AĪ²n Pro
1 5 10 15
Thr Thr Thr Ala Val Ser Pro Gin Lye Val Phe Gly Arg Asp Asn Asp 20 25 30
Arg Asp Lys He He Ala Met Leu His Glu Lys Glu Gly Gly Leu Asp 35 40 45
Pro Ser Thr Ser Lys Qly Leu Cys Phe Ser Val He Gly He His Gly 50 55 60
Val Ser Gly Ser Gly Lye Ser Thr Leu Ala Gin Leu Val Tyr Ala His 65 70 75 80
Glu Lys Asn Asp Lye Gin AĪ²p Asn Lys Glu Asp His Phe Asp Leu Val 85 90 95
Met Trp Val His Val Ser Gin Asp Phe Ser Val Trp Gly He Phe Xaa
100 105 110 Glu Leu Tyr Glu Ala Ala Ser Asp Pro Lys Val Pro CyĪ² Pro Gin Phe 115 120 125
Asn Asn Leu Xaa Ala Leu Glu Glu Glu Leu Glu Arg Lys Leu Asp Gly 130 135 140
Lys Arg Phe Leu Leu Val Leu AĪ²p AĪ²p Val Trp Cye Asn Ala AĪ²p Val 145 150 155 160
Gly Asn Gin Glu Leu Pro Lys Leu Leu Ser Pro Leu Lys Lys Gly Lys 165 170 175
Lys Qly Ser Lye He Leu Val Thr Thr Arg Ser Lye Tyr Ala Leu Pro 180 185 190
Asp Leu Cys Pro Gly Val Arg Tyr Thr Ala Met Pro He Thr Glu Val 195 200 205
AĪ²p Asp Thr Ala Phe Phe Glu Leu Phe Met His Tyr Ala Leu Glu Asp 210 215 220
Qly Gin Asp Gin Ser Met Phe Gin Asn He Gly Val Glu He Ala Lye 225 230 235 240
Lye Leu Lys Gly Ser Pro Leu Ala Ala Arg Thr Val Gly Gly Asn Leu 245 250 255
Arg Arg Gin Gin Asp Val Asp His Trp Arg Arg Val Qly Asp Gin Asp 260 265 270
Leu Phe Lys Val Trp Thr Gly Pro Leu Trp Trp Ser Tyr Tyr Gin Leu 275 280 285
Gly Glu Gin Ala Arg Arg Cys Phe Ala Tyr CyĪ² Ser He Phe Pro Arg 290 295 300
Arg Hie Arg Leu Tyr Arg AĪ²p Glu Leu Val Arg Leu Trp Met Ala Glu 305 310 315 320
Gly Phe He Arg Aen Thr AĪ²p Glu Gly Ala AĪ²p Ala Glu AĪ²p Val Qly 325 330 335 Leu Gly He Phe Aen Glu Leu Leu Ser He Ser Phe Leu Gin Pro Gly 340 345 350
Gly Gin AĪ²p Trp Tyr Asn His Gly Lys Glu Tyr Tyr Leu Val Hie Aep 355 360 365
Leu Leu Tyr AĪ²p Leu Ala Gly Ala Xaa Ala Gly Thr AĪ²p CyĪ² Phe Arg 370 375 380
He AĪ²p AĪ²n AĪ²n Met He Gin Thr Gly Glu Ser Trp Ala Lye AĪ²p Val 385 390 395 400
Pro Arg AĪ²p Val Arg Hie Leu Phe Val Qln Ser Tyr AĪ²p Ala Xaa Leu
405 410 415
Ser Thr Qly Arg Leu Leu Val Leu Glu Xaa Leu Hie Thr Leu Val He 420 425 430
Tyr Ser Val Gly Gly AĪ²p Thr Thr Val Glu Glu He Val He Lye AĪ²n 435 440 445
He Leu Lye Ser Leu Pro Lye Leu Arg Val Leu Ala He Ala Leu CyĪ² 450 455 460
Leu Glu Lye AĪ²p Gly Phe Xaa CyĪ² Arg Pro AĪ²n He Leu Ser Val Pro 465 470 475 480
Glu Ser He Ser Gin Leu Lye Hie Leu Arg Tyr Leu Ala Phe Arg Thr 485 490 495
Asp He Glu Cys Arg Val He Leu Pro Ser Ser Leu Asn Gin Leu Tyr 500 505 510
Gin Met Gin Leu Leu Asp Phe Gly Val Cys Met Aen Leu Val Phe Ser 515 520 525
Cys Gly Aep Leu He Asn Leu Arg His Val Cys Ser Gly Pro Gly Leu 530 535 540
Gin Phe Ser Asn He Gly Arg Leu Val Ser Leu Gin Thr He Pro Ala 545 550 555 560 Phe Lys Val Ser His Glu Gin Gly His Glu Ala Lye Qln Leu Arg Tyr 565 570 575
Leu AĪ²n Arg Leu Ser Gly Glu Leu Ser He Tyr Gly Leu Gin Ser Val 580 585 590
Glu Ser Arg Glu Glu Ala Leu Ala Phe AĪ²p Leu Ala Ala Lye Lys Arg 595 600 605
Leu Ala Glu Leu Thr Leu Ser Phe Qly Qly Ser Ser Glu Val Ala Ala 610 615 Ā» 620
Glu Val Leu Glu Gly Leu Cys Pro Pro Val Gly Leu Val Thr Leu AĪ²p 625 630 635 640
He Arg Asp Tyr Asp Gly Leu Val Tyr Pro Lys Trp Met Val Gly Arg 645 650 655
Gin Asn Gly Ala Pro Glu Lys Leu Qln Qln Leu Gly Leu Ser Qly Trp 660 665 670
Ser Gin Pro Gly Pro Ala Pro Ala Leu Lye Ala Phe AĪ²n Hie Leu Arg 675 680 685
CyĪ² Leu AĪ²n Leu Met Hie Cye Ser Trp Asn Ala Leu Pro Cye Asn Met 690 695 700
Glu His Leu Ser Ser Leu Glu Thr Val He He He Lys Cys Leu Asn 705 710 715 720
He Arg Ser Leu Pro Thr Leu Pro Gin Ser Leu Thr Tyr Phe Trp Leu 725 730 735
Leu Lye Cye AĪ²p AĪ²p Gly Phe Met Glu Ser CyĪ² Gin Thr Val Gly Hie 740 745 750
Pro AĪ²n Trp Lye Lye He Gin His He Cys Arg Lys Tyr Phe Ser Glu 755 760 765 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3850 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Triticum tauschii
(B) STRAIN: AUS 18913 (F) TISSUE TYPE: Root
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1138..3528
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATCCGGAGTC GTGGTTGTGG CCGCTGTCTT CCACTTGTGT ACGGTTTTCC CGTTGTCGAG 60
TGGCCTTCCT TCTTTCGGCC TTGCGGTTAA CAGGTTAGGG GCGCCGGTAG TGTTGTGGTG 120
TGTCATCTTT GGTGAGCTTA TTAATGTCGG CTTGTGQTGT CACATGGTGG TGGQTTCTCA 180
TTTAACGTCG AGGCGGTGAC CTCAGGTGGC GGTCCGTCAA GTCGGCTTCT CAACAAGCGT 240
CTTGGCGGCG GCCGTGGTGG CATTGTTTGG TCGTGTGGAC GGCGGAGGAT GCTAAGTTGG 300
GTGATCCTAG TGTCGGTGGT GCTTTGGCAC TGGCGGTGCC CAGATCGTGT TCTAGTGGTC 360
TGGCTTGGTA GTGACATGTT CACCTCGGTG TGGGCTQGTG CTCGGGAGGC CTAGTGTGGC 420
GTGGAAGAGT GCAACAAGGT CCGGCGATTT TCCTTGGAGC GAACTTTCAT CTTTGTTGGT 480
AGTTTAGGTA GCTTTGTGTT AGGGTGTGGT TCCTCCTATT TTCTTGTTTT TCTTTGATCT 540 QCTTTGTAAQ AGGGTCTCCT CATCACCTTQ TATCTCTTTG GTCGTGGTTC TTTATATATA 600
AAGCGGGGCC GAAGTAATTT TTGGTAGGAT TCACCAACAT CATGAGAACA AAGCACGAAA 660
ATATAQTAGT ACGGTAGTAG AGAATQTTAA TTCCTCTTGT ATCCAATGTT ATCTCTTGTA 720
TACCGTGATT CTTGCCCATC AGTATTCTCT TAGGCTTCTG TTAQCQAAAC AAAATTCCTT 780
CTTCCAAATT ACCAAACTTC TAGCTCATGA GTATGTTCAT ATAGTGCQCG GAGGATGTQC 840
GTGCCACATG CGTGCGCATG ATGGTGTTGA TAGACTAACA TGTGTGTGTQ GTTTCTGTGT 900
GACTGCCTTG TGTTCTCTGC AAAACTAGGC TTTTGGCAAG TCAGTCTAGA TCCCTCGQCG 960
TATTTTTTAG AAGTATACCG GAGAGTAGAC GAATTCCCTA TATTACATTA GTCTTTTTTC 1020
TTTATTTAGT GTCATGATAG TTTATGTQAA GATAAAATCT CTCTTCTGTA ATGGTCACCT 1080
ATAATTTATT TTTTAAAGAT TTCTCTCTTG TTATTTGGQG TCTCGCAGGA GAQTGGC 1137
ATG TCA AAG AAA AAG TTG ATA GAC AGC CTG AAG AAG ATA GAA GAC AAT 1185 Met Ser Lys Lys Lys Leu He AĪ²p Ser Leu Lye Lye He Glu AĪ²p AĪ²n 1 5 10 15
ATA AAT GAA GCA CAC CAA ATT CTG GAT AAG CTT AAC TTG TCA AGC ATA 1233 He AĪ²n Glu Ala Hie Gin He Leu AĪ²p Lye Leu Asn Leu Ser Ser He 20 25 30
AGT GAT GGC AAT ATA CGA CAC ACA ATG GTT GTC AAT CCT ACG ACT ACC 1281 Ser Asp Gly Asn He Arg His Thr Met Val Val Asn Pro Thr Thr Thr 35 40 45
GCA GTT TCC CCG CAA AAA GTT TTT GGT CGA GAT AAT GAT CGC GAC AAG 1329 Ala Val Ser Pro Gin Lys Val Phe Qly Arg Asp Asn Asp Arg Asp Lys 50 55 60
ATC ATA GCA ATG CTT CAT GAA AAG GAA GGT GGT CTT GAT CCA AGC ACT 1377 He He Ala Met Leu His Glu Lys Glu Gly Gly Leu AĪ²p Pro Ser Thr 65 70 75 80 AGC AAA GGT CTA TGT TTT TCT GTA ATT GGC ATA CAT GGA GTC AGC QQG 1425
Ser Lye Gly Leu CyĪ² Phe Ser Val He Gly He His Gly Val Ser Gly 85 90 95
TCT GGG AAA TCT ACC CTT GCA CAG CTT GTT TAT GCC CAC GAG AAA AAT 1473
Ser Gly Lys Ser Thr Leu Ala Qln Leu Val Tyr Ala His Glu Lys Asn 100 105 110
GAC AAG CAA GAC AAC AAG GAA GAC CAT TTC QAC CTT GTT ATG TGG GTT 1521
Asp Lys Gin Asp Asn Lye Glu Aep Hie Phe Aep Leu Val Met Trp Val 115 120 125
CAT GTC TCT CAG GAT TTT AGT GTG TGG GGC ATC TTC ANG GAQ TTG TAT 1569
Hie Val Ser Gin Asp Phe Ser Val Trp Gly He Phe Xaa Glu Leu Tyr
130 135 140
GAG GCA GCT TCA GAT CCT AAG GTT CCA TGC CCT CAA TTT AAT AAC TTG 1617
Glu Ala Ala Ser AĪ²p Pro Lye Val Pro CyĪ² Pro Gin Phe Asn Asn Leu 145 150 155 160
ANT GCC TTG GAA GAA GAA CTG GAG AGG AAA CTA GAT GGA AAG CGA TTC 1665
Xaa Ala Leu Glu Glu Glu Leu Glu Arg Lys Leu Asp Gly Lye Arg Phe 165 170 175
CTT CTG GTA CTG GAT GAT GTC TGG TGC AAT GCG QAT GTT GOT AAC CAG 1713
Leu Leu Val Leu AĪ²p AĪ²p Val Trp Cys AĪ²n Ala Asp Val Gly Asn Gin 180 185 190
GAG CTA CCA AAG TTA CTT TCT CCA CTG AAG AAA GGA AAG AAA GGA AGC 1761
Glu Leu Pro Lys Leu Leu Ser Pro Leu Lys Lye Gly Lye Lye Gly Ser 195 200 205
AAG ATC CTA GTG ACA ACT CGA AGT AAA TAT QCA CTA CCG GAT CTA TGT 1809
Lye He Leu Val Thr Thr Arg Ser Lye Tyr Ala Leu Pro AĪ²p Leu CyĪ²
210 215 220
CCT GGT GTG AGA TAT ACT GCC ATG CCG ATA ACT GAG GTT GAT GAT ACC 1857
Pro Gly Val Arg Tyr Thr Ala Met Pro He Thr Glu Val AĪ²p Asp Thr 225 230 235 240 QCC TTC TTT QAG TTG TTC ATG CAT TAT GCC CTC GAA GAT GGC CAA GAT 1905 Ala Phe Phe Glu Leu Phe Met His Tyr Ala Leu Glu Asp Gly Gin Asp 245 250 255
CAA AGC ATG TTC CAG AAC ATT GGG GTT GAG ATT GCA AAA AAG CTG AAG 1953 Gin Ser Met Phe Gin Asn He Gly Val Qlu He Ala Lys Lys Leu Lys 260 265 270
QGG TCA CCT TTA GCA GCT AGA ACA GTG GGT GGA AAT TTA CGT CGA CAG 2001 Gly Ser Pro Leu Ala Ala Arg Thr Val Gly Gly Asn Leu Arg Arg Gin 275 280 285
CAA GAT GTT GAC CAT TGG AGA AGA GTC QGA GAT CAA GAC CTT TTC AAG 2049 Gin Asp Val AĪ²p Hie Trp Arg Arg Val Gly Asp Gin AĪ²p Leu Phe Lys 290 295 300
GTA TGG ACG GGA CCT CTG TGG TGG AGC TAC TAT CAG CTT GGT GAG CAG 2097 Val Trp Thr Gly Pro Leu Trp Trp Ser Tyr Tyr Qln Leu Qly Glu Gin 305 310 315 320
GCT AGG CGT TGC TTT GCT TAC TGC AGT ATT TTT CCT AGG AGA CAT CGC 2145 Ala Arg Arg CyĪ² Phe Ala Tyr CyĪ² Ser He Phe Pro Arg Arg His Arg 325 330 335
TTG TAC CGY GAT GAA TTA GTT AGA CTC TGG ATG GCA GAA GGG TTC ATA 2193 Leu Tyr Arg Asp Glu Leu Val Arg Leu Trp Met Ala Glu Gly Phe He 340 345 350
AGA AAC ACA GAT GAA GGG GCG GAT GCT GAA QAC GTT GGT CTG GGA ATA 22 1 Arg AĪ²n Thr Asp Glu Gly Ala Asp Ala Glu Asp Val Gly Leu Gly He 355 360 365
TTT AAT GAA CTA TTG TCG ATA TCA TTT CTT CAA CCA GGA GGC CAG GAC 2289 Phe AĪ²n Glu Leu Leu Ser He Ser Phe Leu Gin Pro Gly Gly Gin AĪ²p 370 375 380
TGG TAC AAT CAT GGC AAG GAA TAC TAT TTA GTT CAT GAT TTG CTG TAT 2337 Trp Tyr Asn His Gly Lys Glu Tyr Tyr Leu Val Hie AĪ²p Leu Leu Tyr 385 390 395 400 GAT TTA GCA GGG GCA GYA GCT QGA ACT GAC TGC TTC AGA ATT GAC AAT 2385 AĪ²p Leu Ala Gly Ala Xaa Ala Qly Thr AĪ²p CyĪ² Phe Arg He Asp Asn 405 410 415
AAC ATG ATC CAG ACA GGA GAA AGC TQQ GCA AAA GAT GTT CCC AGA GAC 2433 Asn Met He Qln Thr Qly Glu Ser Trp Ala Lys Asp Val Pro Arg AĪ²p 420 425 430
GTT CGC CAT CTT TTT GTT CAG AGT TAT QAT GCA NCN TTG AGT ACA GQQ 2481 Val Arg His Leu Phe Val Qln Ser Tyr AĪ²p Ala Xaa Leu Ser Thr Gly 435 440 445
AGA TTG CTT GTA TTG GAG GAN TTA CAC ACA CTC GTC ATT TAT AGT GTT 2529 Arg Leu Leu Val Leu Glu Xaa Leu Hie Thr Leu Val He Tyr Ser Val 450 455 460
GGA GGG GAT ACA ACA GTT GAG GAA ATA GTC ATC AAG AAC ATA CTC AAG 2577 Gly Gly AĪ²p Thr Thr Val Glu Qlu He Val He Lye Aen He Leu Lye 465 470 475 480
AGT CTG CCT AAA CTG CGG GTA CTA GCA ATA GCT TTA TGT CTG GAA AAG 2625 Ser Leu Pro Lye Leu Arg Val Leu Ala He Ala Leu Cye Leu Glu Lye 485 490 495
GAT GGA TTT ATN TGT AQA CCA AAT ATA TTG TCT GTT CCA GAA TCT ATT 2673 AĪ²p Gly Phe Xaa CyĪ² Arg Pro Aen He Leu Ser Val Pro Glu Ser He 500 505 510
AGT CAA TTA AAA CAT CTA CGA TAT CTT GCT TTC CGG ACA GAT ATT GAA 2721 Ser Gin Leu Lys His Leu Arg Tyr Leu Ala Phe Arg Thr Asp He Glu 515 520 525
TGC AGA GTA ATT TTA CCA AGC AGT CTA AAC CAG CTT TAC CAG ATG CAA 2769 Cys Arg Val He Leu Pro Ser Ser Leu AĪ²n Gin Leu Tyr Gin Met Gin 530 535 540
CTG CTA GAT TTT GGT GTC TGC ATG AAT TTG GTA TTT TCC TGT GGT GAT 2817 Leu Leu Asp Phe Gly Val Cys Met Asn Leu Val Phe Ser Cys Gly AĪ²p 545 550 555 560 CTT ATC AAC TTG CGG CAT GTA TGC AQC GGT CCT GGA TTG CAA TTT TCA 2865 Leu He Asn Leu Arg His Val Cys Ser Gly Pro Gly Leu Gin Phe Ser 565 570 575
AAC ATC GGT AGG CTT GTC TCA CTC CAA ACA ATC CCA GCA TTC AAA GTA 2913 AĪ²n He Gly Arg Leu Val Ser Leu Gin Thr He Pro Ala Phe Lys Val 580 585 590
AGT CAT GAA CAA GGA CAT GAG GCA AAG CAG TTG AGG TAC CTA AAC AGG 2961 Ser Hie Glu Gin Gly His Glu Ala Lye Gin Leu Arg Tyr Leu AĪ²n Arg 595 600 605
CTC AGC GGC GAA CTG AGT ATA TAT GGT CTC CAA AGT GTT GAA AGC AGA 3009 Leu Ser Gly Glu Leu Ser He Tyr Gly Leu Gin Ser Val Glu Ser Arg 610 615 620
GAG GAA GCT CTT GCA TTC QAT CTA GCT GCC AAG AAA CGG CTC GCA GAA 3057 Glu Glu Ala Leu Ala Phe AĪ²p Leu Ala Ala Lye Lye Arg Leu Ala Glu 625 630 635 640
CTA ACA CTA TCA TTC GGT GGA AGT TCA GAA GTT GCA GCA GAG GTA CTT 3105 Leu Thr Leu Ser Phe Gly Gly Ser Ser Glu Val Ala Ala Glu Val Leu 645 650 655
GAG GGC CTT TGT CCT CCC GTG GGG CTT GTA ACA CTC GAC ATC CGT GAC 3153 Glu Gly Leu CyĪ² Pro Pro Val Gly Leu Val Thr Leu AĪ²p He Arg Asp 660 665 670
TAC GAT GGT TTG GTA TAC CCA AAG TGG ATG GTG GGC AGG CAA AAT GGC 3201 Tyr Asp Gly Leu Val Tyr Pro Lye Trp Met Val Gly Arg Qln AĪ²n Qly 675 680 685
GCA CCA GAG AAG CTG CAA CAA CTT GGT CTC TCA GGA TGG AGC CAG CCA 3249 Ala Pro Glu Lye Leu Gin Gin Leu Gly Leu Ser Gly Trp Ser Gin Pro 690 695 700
GGA CCT GCT CCT GCA CTG AAG GCT TTC AAT CAT CTT CGT TGC CTC AAT 3297 Gly Pro Ala Pro Ala Leu Lys Ala Phe AĪ²n His Leu Arg Cys Leu Asn 705 710 715 720 CTG ATG CAC TGC AGC TGG AAC GCC TTG CCA TGC AAT ATG GAG CAC CTC 3345 Leu Met His Cys Ser Trp AĪ²n Ala Leu Pro Cye Aen Met Glu His Leu 725 730 735
AGC TCG CTC GAA ACA GTA ATC ATT ATT AAA TGT TTG AAT ATC CGG TCG 3393 Ser Ser Leu Glu Thr Val He He He Lys CyĪ² Leu AĪ²n He Arg Ser 740 745 750
CTT CCA ACG CTG CCA CAG TCT CTT ACG TAT TTT TGG CTC CTG AAG TGC 3441 Leu Pro Thr Leu Pro Gin Ser Leu Thr Tyr Phe Trp Leu Leu Lye CyĪ² 755 760 765
GAC GAT GGG TTC ATG GAG TCT TGT CAA ACA GTT QGA CAT CCA AAC TGG 3489 AĪ²p Aep Gly Phe Met Glu Ser CyĪ² Gin Thr Val Gly His Pro AĪ²n Trp 770 775 780
AAA AAG ATT CAA CAC ATC TGC AGG AAA TAT TTT AGT GAA TGACGCGGGC 3538 Lye Lye He Gin Hie He Cye Arg Lys Tyr Phe Ser Glu 785 790 795
TTGGAATCGG AGTCAGAGTA CTTACTTATG GCCCCTAACT TGAGACCTGC ATGCCGCTGC 3598
AGCTATTTTA TTCCAATTGG AGTCAAGACA AGAGTATTTA CTCGAGATTC ATAATCTATT 3658
CCTGQGTGGA TCTTCTCTTG TGAGTCTGAA AACCTACCAG TGCCAGTCTG CAATATTGTA 3718
AGGAAAGGAG TACATCTATA GTGTCAGTGC ATATACAGTG TCTGAATCAT GCACTTCCGT 3778
TTCTGTATTT CACCGTATTA TTGATTAAAC AGTGCATGTG CACGTGCACA ATATATATTT 3838
CCCGAATCTT CT 3850 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENQTH: 797 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Ser Lys Lys Lys Leu He Asp Ser Leu Lys Lye He Glu AĪ²p AĪ²n 1 5 10 15
He AĪ²n Glu Ala Hie Gin He Leu AĪ²p Lye Leu AĪ²n Leu Ser Ser He 20 25 30
Ser AĪ²p Gly AĪ²n He Arg Hie Thr Met Val Val AĪ²n Pro Thr Thr Thr 35 40 45
Ala Val Ser Pro Gin Lys Val Phe Gly Arg Asp Asn Asp Arg Asp Lys 50 55 60
He He Ala Met Leu His Glu Lys Glu Gly Gly Leu AĪ²p Pro Ser Thr 65 70 75 80
Ser Lye Gly Leu Cye Phe Ser Val He Gly He Hie Gly Val Ser Gly 85 90 95
Ser Gly Lys Ser Thr Leu Ala Gin Leu Val Tyr Ala His Glu Lye AĪ²n 100 105 110
AĪ²p Lys Gin Asp Asn Lys Glu Asp His Phe Asp Leu Val Met Trp Val 115 120 125
His Val Ser Qln Asp Phe Ser Val Trp Gly He Phe Xaa Glu Leu Tyr 130 135 140
Glu Ala Ala Ser Asp Pro Lys Val Pro Cys Pro Gin Phe Asn AĪ²n Leu 145 150 155 160 Xaa Ala Leu Glu Glu Glu Leu Glu Arg Lys Leu Asp Gly Lys Arg Phe 165 170 175
Leu Leu Val Leu Asp Asp Val Trp Cys Asn Ala AĪ²p Val Gly Asn Gin 180 185 190
Glu Leu Pro Lys Leu Leu Ser Pro Leu Lys Lye Gly Lye Lye Qly Ser 195 200 205
Lye He Leu Val Thr Thr Arg Ser Lye Tyr Ala Leu Pro AĪ²p Leu CyĪ² 210 215 * 220
Pro Gly Val Arg Tyr Thr Ala Met Pro He Thr Glu Val AĪ²p AĪ²p Thr 225 230 235 240
Ala Phe Phe Glu Leu Phe Met Hie Tyr Ala Leu Glu AĪ²p Gly Gin AĪ²p 245 250 255
Gin Ser Met Phe Gin AĪ²n He Gly Val Glu He Ala Lys Lys Leu Lys 260 265 270
Gly Ser Pro Leu Ala Ala Arg Thr Val Gly Gly Asn Leu Arg Arg Gin 275 280 285
Gin Asp Val Asp His Trp Arg Arg Val Gly AĪ²p Gin AĪ²p Leu Phe Lye 290 295 300
Val Trp Thr Gly Pro Leu Trp Trp Ser Tyr Tyr Gin Leu Gly Glu Gin 305 310 315 320
Ala Arg Arg Cys Phe Ala Tyr Cys Ser He Phe Pro Arg Arg His Arg 325 330 335
Leu Tyr Arg Asp Glu Leu Val Arg Leu Trp Met Ala Glu Gly Phe He 340 345 350
Arg AĪ²n Thr Aep Glu Gly Ala Asp Ala Glu Asp Val Gly Leu Gly He 355 360 365
Phe Asn Glu Leu Leu Ser He Ser Phe Leu Gin Pro Gly Gly Gin Asp 370 375 380 Trp Tyr Asn His Qly Lys Glu Tyr Tyr Leu Val His Asp Leu Leu Tyr 385 390 395 400
Asp Leu Ala Gly Ala Xaa Ala Gly Thr Asp Cys Phe Arg He Asp AĪ²n 405 410 415
Asn Met He Gin Thr Qly Glu Ser Trp Ala Lye AĪ²p Val Pro Arg Asp 420 425 430
Val Arg Hie Leu Phe Val Gin Ser Tyr AĪ²p Ala Xaa Leu Ser Thr Gly 435 440 - 445
Arg Leu Leu Val Leu Glu Xaa Leu His Thr Leu Val He Tyr Ser Val 450 455 460
Qly Gly AĪ²p Thr Thr Val Glu Glu He Val He Lys Asn He Leu Lys 465 470 475 480
Ser Leu Pro Lye Leu Arg Val Leu Ala He Ala Leu CyĪ² Leu Glu Lye 485 490 495
Asp Gly Phe Xaa Cys Arg Pro Asn He Leu Ser Val Pro Glu Ser He 500 505 510
Ser Gin Leu Lys His Leu Arg Tyr Leu Ala Phe Arg Thr Asp He Glu 515 520 525
Cys Arg Val He Leu Pro Ser Ser Leu Asn Gin Leu Tyr Gin Met Gin 530 535 540
Leu Leu Asp Phe Gly Val Cys Met Asn Leu Val Phe Ser Cys Gly AĪ²p 545 550 555 560
Leu He Asn Leu Arg Hie Val CyĪ² Ser Gly Pro Gly Leu Gin Phe Ser 565 570 575
Asn He Gly Arg Leu Val Ser Leu Gin Thr He Pro Ala Phe Lye Val 580 585 590
Ser Hie Glu Qln Gly His Glu Ala Lys Gin Leu Arg Tyr Leu Asn Arg 595 600 605 Leu Ser Gly Glu Leu Ser He Tyr Gly Leu Gin Ser Val Glu Ser Arg 610 615 620
Glu Glu Ala Leu Ala Phe AĪ²p Leu Ala Ala Lye Lys Arg Leu Ala Glu 625 630 635 640
Leu Thr Leu Ser Phe Gly Qly Ser Ser Glu Val Ala Ala Glu Val Leu 645 650 655
Glu Gly Leu CyĪ² Pro Pro Val Gly Leu Val Thr Leu Asp He Arg Asp 660 665 670
Tyr Asp Gly Leu Val Tyr Pro Lys Trp Met Val Gly Arg Gin AĪ²n Gly 675 680 685
Ala Pro Glu Lye Leu Gin Gin Leu Gly Leu Ser Qly Trp Ser Gin Pro 690 695 700
Gly Pro Ala Pro Ala Leu Lye Ala Phe AĪ²n Hie Leu Arg CyĪ² Leu AĪ²n 705 710 715 720
Leu Met His Cys Ser Trp Asn Ala Leu Pro Cys Asn Met Glu His Leu 725 730 735
Ser Ser Leu Glu Thr Val He He He Lye CyĪ² Leu Asn He Arg Ser 740 745 750
Leu Pro Thr Leu Pro Qln Ser Leu Thr Tyr Phe Trp Leu Leu Lys Cys 755 760 765
Asp Asp Gly Phe Met Glu Ser CyĪ² Gin Thr Val Gly Hie Pro Asn Trp 770 775 780
Lys Lys He Gin Hie He Cys Arg Lys Tyr Phe Ser Glu 785 790 795

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS :-
1. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to a sequence encoding a polypeptide which confers, enhances, or otherwise facilitates resistance to a nematode in a plant or a part or fragment of said polypeptide.
2. An isolated nucleic acid molecule according to claim 1, wherein said polypeptide encoded thereof comprises at least one amino acid sequence motif selected from the group consisting of p-Loop, kinase-2, or leucine-rich repeat motifs as hereinbefore defined.
3. An isolated nucleic acid molecule according to claim 1 or 2 wherein said nucleic acid is DNA.
4. An isolated nucleic acid molecule according to claim 1 or 2 or 3 wherein the polypeptide product of said nucleic acid is of plant origin.
5. An isolated nucleic acid molecule according to claim 4, wherein the plant is a monocotyledonous plant selected from the group consisting of Triticum tauschii, wheat, maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
6. An isolated nucleic acid molecule according to claim 5 wherein said nucleic acid molecule comprises a nucleotide sequence or complementary nucleotide sequence which is substantially the same as the nucleotide sequence set forth in SEQ ID NO: 1 or is at least about 40% similar to all or a part thereof.
7. An isolated nucleic acid molecule according to claim 5 wherein said nucleic acid molecule comprises a nucleotide sequence or complementary nucleotide sequence which is substantially the same as the nucleotide sequence set forth in SEQ ID NO: 3 or is at least about 40% similar to all or a part thereof.
8. An isolated nucleic acid molecule according to claim 5 wherein said nucleic acid molecule comprises a nucleotide sequence or complementary nucleotide sequence which is substantially the same as the nucleotide sequence set forth in SEQ ID NO: 5 or is at least about 40% similar to all or a part thereof.
9. An isolated nucleic acid molecule according to claim 5 wherein said nucleic acid molecule comprises a nucleotide sequence or complementary nucleotide sequence which is substantially the same as the nucleotide sequence set forth in SEQ ID NO: 7 or is at least about 40% similar to all or a part thereof.
10. An isolated DNA molecule comprising a sequence of nucleotides which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) has at least about 40% nucleotide sequence similarity to SEQ ID NO: 1 or a part thereof.
11. An isolated DNA molecule comprising a sequence of nucleotides which: (i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) has at least about 40% nucleotide sequence similarity to SEQ ID NO: 3 or a part thereof.
12. An isolated DNA molecule comprising a sequence of nucleotides which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) has at least about 40% nucleotide sequence similarity to SEQ ID NO: 5 or a part thereof.
13. An isolated DNA molecule comprising a sequence of nucleotides which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) has at least about 40% nucleotide sequence similarity to SEQ ID NO: 7 or a part thereof.
14. An isolated DNA molecule according to claim 10 comprising a nucleotide sequence substantially as the same as any one or more of SEQ ID NO: 1.
15. An isolated DNA molecule according to claim 11 comprising a nucleotide sequence substantially as the same as any one or more of SEQ ID NO: 3.
16. An isolated DNA molecule according to claim 12 comprising a nucleotide sequence substantially as the same as any one or more of SEQ ID NO: 5.
17. An isolated DNA molecule according to claim 13 comprising a nucleotide sequence substantially as the same as any one or more of SEQ ID NO: 7.
18. An isolated nucleic acid molecule which: (i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 1 or to a complementary strand thereof.
19. An isolated nucleic acid molecule which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 3 or to a complementary strand thereof.
20. An isolated nucleic acid molecule which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 5 or to a complementary strand thereof.
21. An isolated nucleic acid molecule which:
(i) encodes or is complementary to a sequence encoding a polypeptide of plant origin which confers, enhances, or otherwise facilitates nematode resistance in a plant; and (ii) hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 7 or to a complementary strand thereof.
22. An isolated nucleic acid molecule having a nucleotide sequence substantially the same as the sequence set forth in SEQ ID NO: 1 or a complementary strand thereof or at least 40% identical thereto.
23. An isolated nucleic acid molecule having a nucleotide sequence substantially the same as the sequence set forth in SEQ ID NO: 3 or a complementary strand thereof or at least 40% identical thereto.
24. An isolated nucleic acid molecule having a nucleotide sequence substantially the same as the sequence set forth in SEQ ID NO: 5 or a complementary strand thereof or at least 40% identical thereto.
25. An isolated nucleic acid molecule having a nucleotide sequence substantially the same as the sequence set forth in SEQ ID NO: 7 or a complementary strand thereof or at least 40% identical thereto.
26. A genetic construct comprising the nucleic acid molecule according to claim 1.
27. A genetic construct according to claim 26 wherein the nucleic acid molecule is operably linked to a promoter sequence.
28. A genetic construct according to claim 27 wherein the promoter sequence comprises a sequence of nucleotides having at least 40% nucleotide sequence similarity to nucleotides
1 to 1138 set forth in SEQ ID NO: 1, or a functional part thereof.
29. A genetic construct according to claim 26 or 27 or 28 capable of being expressed in a plant cell.
30. A genetic construct according to claim 29 wherein the plant cell is a root cell.
31. A genetic construct according to claim 26 or 27 or 28 capable of being expressed in a prokaryotic cell.
32. A genetic construct according to claim 29 or 30 wherein the plant cell is a monocotyledonous plant cell selected from the group consisting of Triticum tauschii, what, maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
33. A genetic construct comprising an isolated promoter sequence which originates from a gene which when expressed encodes a polypeptide that confers, enhances, or otherwise facilitates nematode resistance in a cell, or a functional part, derivative, fragment, homologue or analogue thereof, wherein said promoter is operably linked to the coding region isolated from a second genetic sequence.
34. A genetic construct according to claim 33 wherein the promoter sequence comprises a sequence of nucleotides having at least 40% nucleotide sequence similarity to nucleotides 1 to 1138 set forth in SEQ ID NO: 1, or a functional part thereof.
35. A genetic construct according to claim 33 or 34 wherein the plant cell is a monocotyledonous plant cell selected from the group consisting of Triticum tauschii, wheat, maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
36. An oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of the nucleotide sequence set forth in SEQ ID NO: 1.
37. An oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of the nucleotide sequence set forth in SEQ ID NO: 3.
38. An oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of the nucleotide sequence set forth in SEQ ID NO: 5.
39. An oligonucleotide molecule of at least 10 nucleotides in length capable of hybridising under low stringency conditions to part of the nucleotide sequence, or to a complement of the nucleotide sequence set forth in SEQ ID NO: 7.
40. A recombinant polypeptide product of the genetic construct according to any one of claims 26 to 35.
41. A recombinant polypeptide according to claim 40 wherein said polypeptide comprises at least one amino acid sequence motif selected from the group consisting of p- Loop, kinase-2 or leucine-rich repeat motifs as hereinbefore defined.
42. An isolated polypeptide which comprises an amino acid sequence which confers, enhances, or otherwise facilitates resistance to a nematode in a plant cell, or a functional mutant, derivative part, fragment, or analogue of said polypeptide.
43. An isolated polypeptide according to claim 42 being of plant origin.
44. An isolated polypeptide according to claim 43 wherein the plant is a monocotyledonous plant selected from the group consisting of Triticum tauschii, wheat maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
45. An isolated polypeptide according claim 42 or 43 or 44 having an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 2 or a part thereof.
46. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 4 or a part thereof.
47. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 6 or a part thereof.
48. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence substantially the same as the amino acid sequence set forth in SEQ ED NO: 8 or a part thereof.
49. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence which is at least 40% similar to the amino acid sequence set forth in SEQ ID NO: 2 or a part thereof.
50. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence which is at least 40% similar to the amino acid sequence set forth in SEQ ID NO: 4 or a part thereof
51. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence which is at least 40% similar to the amino acid sequence set forth in SEQ ID NO:
6 or a part thereof.
52. An isolated polypeptide according to claim 42 or 43 or 44 having an amino acid sequence which is at least 40% similar to the amino acid sequence set forth in SEQ ID NO: 8 or a part thereof.
53. A synthetic peptide comprising at least 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 2 or having at least 40% similarity to all or a part thereof.
54. A synthetic peptide comprising at least 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 4 or having at least 40% similarity to all or a part thereof.
55. A synthetic peptide comprising at least 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 6 or having at least 40% similarity to all or a part thereof
56. A synthetic peptide comprising at least 10 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 8 or having at least 40% similarity to all or a part thereof.
57. An antibody that binds to a polypeptide which confers, enhances, or otherwise facilitates resistance to a nematode in a plant or a part or fragment thereof, wherein said polypeptide further comprises a sequence of amino acids is substantially the same as the amino acid sequence set forth in SEQ ED NO: 2 or is at least 40% similar to all or a part thereof.
58. An antibody that binds to a polypeptide which confers, enhances, or otherwise facilitates resistance to a nematode in a plant or a part or fragment thereof, wherein said polypeptide further comprises a sequence of amino acids is substantially the same as the amino acid sequence set forth in SEQ ID NO: 4 or is at least 40% similar to all or a part thereof.
59. An antibody that binds to a polypeptide which confers, enhances or otherwise facilitates resistance to a nematode in a plant or a part or fragment thereof, wherein said polypeptide, part or fragment further comprises a sequence of amino acids is substantially the same as the amino acid sequence set forth in SEQ ID NO: 6 or is at least 40% similar to all or a part thereof.
60. An antibody that binds to a polypeptide which confers, enhances, or otherwise facilitates resistance to a nematode in a plant or a part or fragment thereof, wherein said polypeptide further comprises a sequence of amino acids is substantially the same as the amino acid sequence set forth in SEQ ED NO: 8 or is at least 40% similar to all or a part thereof.
61. An antibody according to claim 57 or 58 or 59 or 60 whiich is a polyclonal antibody.
62. An antibody according to claim 57 or 58 or 59 or 60 which is a monoclonal antibody.
63. A method of identifying a nematode resistance gene product or nematode resistanceĀ¬ like gene product in a plant cell, which method comprises contacting the antibody of any one of claims 57 to 62 with an antigen from said plant for a period of time and under conditions sufficient to form an antibody-antigen complex and measuring the amount of said antibody-antigen complex formed.
64. A method according to claim 63 wherein the antigen is obtained from the roots of a monocotyledonous plant selected from the group consisting of Triticum tauschii, wheat, maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
65. A method according to claim 63 or 64, wherein said step of measuring the amount of antibody-antigen complex formed is by an immunoassay.
66. A method according to claim 65, wherein said immunoassay is an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA).
67. A method of identifying a nematode resistance genetic sequence or nematode resistance-like genetic sequence which method comprises contacting genomic DNA, or mRNA, or cDNA, or parts, or fragments thereof, or a source thereof, with a hybridisation effective amount of a genetic sequence encoding, or complementary to a genetic sequence encoding a polypeptide which confers, enhances or otherwise facilitates nematode resistance, or a part thereof, and then detecting said hybridisation.
68. A method of identifying a nematode resistance genetic sequence or a nematode resistance-like genetic sequence in a plant cell, which method comprises contacting genomic DNA, mRNA, or cDNA with one or more oligonucleotide molecules of claim 36 or 37 or 38 or 39 to a genetic sequence from said plant for a period of time and under conditions sufficient to form a double-stranded nucleic acid molecule and amplifying copies of the said genetic sequence in a polymerase chain reaction.
69. A method according to claim 68 wherein the plant is a monocotyledonous plant selected from the group consisting of Triticum tauschii, wheat, maize, rice, oats, barley and rye, and/or wilde varieties and/or hybrids or derivatives and/or ancestral progenitors of same.
70. A plant carrying a non-endogenous nucleic acid molecule encoding or complementary to a nucleic acid molecule encoding a polypeptide which confers, enhances, or otherwise facilitates nematode resistance in said plant.
71. A plant according to claim 70 wherein said non-endogenous nucleic acid molecule comprises at least 40% nucleotide sequence similarity to all or a part of the nucleotide sequence set forth in SEQ ID NO: 1 or hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 1 or to a complementary strand thereof.
72. A plant according to claim 70 wherein said non-endogenous nucleic acid molecule comprises at least 40% nucleotide sequence similarity to all or a part of the nucleotide sequence set forth in SEQ ID NO: 3 or hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ED NO: 3 or to a complementary strand thereof.
73. A plant according to claim 70 wherein said non-endogenous nucleic acid molecule comprises at least 40% nucleotide sequence similarity to all or a part of the nucleotide sequence set forth in SEQ ID NO: 5 or hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ED NO:5 or to a complementary strand thereof.
74. A plant according to claim 70 wherein said non-endogenous nucleic acid molecule comprises at least 40% nucleotide sequence similarity to all or a part of the nucleotide sequence set forth in SEQ ED NO: 7 or hybridises under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO: 7 or to a complementary strand thereof.
75. A plant according to claim 70 or 71 or 72 or 73 or 74 wherein the non-endogenous nucleic acid molecule is introduced into the plant by any one or a combination of procedures selected from the group consisting of Agrobacterium-mediated transformation, microparticle bombardment, PEG fusion, electroporation or in .regression.
76. The progeny derived from the plant of any one of claims 70 to 75.
PCT/AU1996/000181 1995-03-31 1996-03-29 Genetic sequences conferring nematode resistance in plants and uses therefor WO1996030517A1 (en)

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EP0817849A1 (en) 1998-01-14
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US5994627A (en) 1999-11-30
AU710189B2 (en) 1999-09-16
NZ303905A (en) 1998-05-27

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