WO2001018221A1 - Gene de resistance de plante - Google Patents

Gene de resistance de plante Download PDF

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Publication number
WO2001018221A1
WO2001018221A1 PCT/GB2000/003438 GB0003438W WO0118221A1 WO 2001018221 A1 WO2001018221 A1 WO 2001018221A1 GB 0003438 W GB0003438 W GB 0003438W WO 0118221 A1 WO0118221 A1 WO 0118221A1
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Prior art keywords
nucleic acid
plant
sequence
lml
polypeptide
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PCT/GB2000/003438
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WO2001018221A8 (fr
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Ella Mary Christina Dixelius
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Plant Bioscience Limited
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Priority to CA002384014A priority Critical patent/CA2384014A1/fr
Priority to AU70240/00A priority patent/AU7024000A/en
Publication of WO2001018221A1 publication Critical patent/WO2001018221A1/fr
Publication of WO2001018221A8 publication Critical patent/WO2001018221A8/fr

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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to methods and materials for use in manipulating the resistance of plants against plant diseases such as blackleg disease.
  • Blackleg is judged to be the fungal disease which causes the highest degree of damage to oilseed rape (B . napus) and turnip rape in Central and Southern Europe, Canada and Australia.
  • Two major symptoms are produced: leaf lesions, which arise after infection of leaves by both wind dispersed ascospores and splash dispersed conidia, and stem cankers, which arise by the direct infection of stems or by the systemic growth of the fungus from leaf lesions through the vascular tissue (Hammond et al . 1985; Ansah-Melayah et al, 1998, Plant Breeding 117, 373-378) .
  • B . nigra, B . carinata and B . j uncea exhibit a high level of resistance to blackleg (Roy 1978;
  • the present invention makes available nucleic acids which encode blackleg resistance, and other materials, which can be used to manipulate this trait in plants.
  • an isolated nucleic acid molecule encoding a polypeptide which is capable of altering an L . maculans- mediated-disease resistance phenotype of a plant into which the nucleic acid is introduced and expressed.
  • the alteration in resistance may be assessed by complementation i.e. a comparison with a corresponding ( . maculans susceptible) plant in which the nucleic acid is not present (or not transcribed, or not expressed) . It may be preferable to use a sample of plants in each case.
  • the resistance may be measured directly by spread of the disease (e.g. scoring leaf lesions on adult leaves) . Apart from the change in disease resistance (e.g. to blackleg) it is preferred that other characteristics of the plant are substantially unchanged by the expression of the polypeptide. It will be understood that when the terms 'resistant' and 'susceptible' are used herein, they are used relatively i.e.
  • a resistant plant will have enhanced resistance, even though it may not be fully resistant to all L . maculans isolates at all concentrations under all conditions. Altering the disease resistance in this context may be by way of a defence response to L . maculans (or an elicitor associated therewith) involving multiple stages or components, of which the polypeptide is only a part.
  • Nucleic acids of this aspect of the invention may be obtainable from Brassica B genome species (e.g. B . nigra, B . carinata and B . j uncea, most preferably B . nigra) .
  • the sequence encoding the L . maculans resistance may map to the linkage group 3 and/or 8 of the genome, in accordance with the linkage map established by Lagercrantz and Lydiate (1995) .
  • Nucleic acid according to the present invention may include, comprise, consist, or consist essentially of any of the sequences disclosed herein, whether as cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid) . Where a DNA sequence is specified, e.g. with reference to a Figure, unless context requires otherwise, the RNA equivalent, with U substituted for T where it occurs, is encompassed. Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term 'isolated' encompasses all of these possibilities.
  • the nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser. Where a sequence is disclosed, its complement will also form part of the invention.
  • One embodiment of the invention is concerned with a gene isolatable from a B . nigra cDNA library, which is associated with clone 27:8 in the Examples below (and named Lml herein) .
  • This clone is believed to map to a position consistent with known blackleg resistance markers, and has been shown to complement L . maculans susceptibility.
  • Fig 1 The nucleotide sequence of clone 27:8 is shown in Fig 1 (2435 base pairs) . It is believed that the complementary sequence of bp 1-1008 represents an ORF (Fig 2) encoding an active, but possibly truncated, L . maculans resistance polypeptide as shown in Fig 4 (336 amino acids) . However it will be understood that, should other ORFs within the sequence turn out to have a role in L . maculans resistance, then the following applies correspondingly to these.
  • an isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide of Fig 4.
  • the nucleic acid may comprise the complementary sequence of bp 1-1008 shown in Fig 1, or be degeneratively equivalent thereto.
  • the nucleotide sequence may be extended at its 5 ' or 3 ' end such as to encode a longer polypeptide than is shown in Fig 4.
  • the clone 27:8 sequence has no homology to any known, cloned, resistance genes. However it is highly homologous to an uncharacterised Arabidopsis thaliana gene. The amino acid sequence encoded by this gene (from BAC T19F6_16) is shown alongside the 27:8 1-1008 ORF in Fig 3. The A . thaliana sequence is considerably longer, and it may be that remaining sequence of the 27:8 clone is correspondingly homologous to the rest of BAC T19F6_16.
  • An extended sequence could be obtained if appropriate by probing or amplifying regions of cDNA or genomic libraries of (e.g. B . nigra) using probes or primers based on the presently disclosed sequences as set out in more detail below.
  • variants of the sequences provided will be homologous to part, and preferably all, of the sequences discussed above .
  • sequence comparisons are made using FASTA and FASTP (see Pearson & ipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows:
  • Gapopen (penalty for the first residue in a gap) : -12 for proteins / -16 for DNA
  • Gapext (penalty for additional residues in a gap) : -2 for proteins / -4 for DNA
  • KTUP word length 2 for proteins / 6 for DNA.
  • Homology may be at the nucleotide sequence and/or encoded amino acid sequence level.
  • the nucleic acid and/or amino acid sequence shares at least about 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology with the 27:8 1-1008 ORF in Fig 1.
  • Homology may be over the full-length of the relevant sequence shown herein, or may be over a part of it, preferably over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333 or more amino acids or codons, compared with the 27:8 1-1008 ORF.
  • variant polypeptide in accordance with the present invention may include within the sequence shown in Fig 4, a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes.
  • Variant nucleic acids may be derivatives (e.g. mutants) which may include changes to the nucleotide sequence by way of one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, which may lead to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Changes may be by way of conservative variation, i.e.
  • substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Also included are variants having non-conservative substitutions.
  • Particular modifications may include mixing or incorporating sequences from related genes (e.g the Arabidopsis genes disclosed herein) or sequences related thereto.
  • a method of producing a derivative nucleic acid comprising the step of modifying any of the sequences disclosed above, particularly the coding sequence of Fig 1.
  • Changes may be desirable for a number of reasons. For instance they may introduce or remove restriction endonuclease sites or alter codon usage.
  • changes may modify sites which are required for post translation modification such as cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide for glycosylation, lipoylation etc.
  • Leader or other targeting sequences e.g. membrane or golgi locating sequences
  • these variants will encode polypeptides which are capable of altering the L . maculans disease resistance phenotype of a plant into which the nucleic acid is introduced and expressed as described above.
  • Mutation e.g. by random or site directed mutagenesis
  • variants may be used to decrease L . maculans disease resistance in a resistant plant e.g. by down-regulating native sequences (as described hereinafter) .
  • the variants will retain the LMl activity, which is to say that L. maculans disease resistance is enhanced in a susceptible plant.
  • variants comprises allelic variants, pseudoalleles (from the same plant species, but having a different locus) or other resistance gene homologues from the same or other plant species, preferably Brassica B genome species. These can be identified by using the sequences disclosed herein. In a further aspect of the present invention there is provided a method of identifying and/or cloning a nucleic acid variant from a plant which method employs a sequence described above.
  • distinctive portions of the nucleotide sequence information provided herein may be used to design probes and primers for probing or amplification.
  • An oligonucleotide for use in probing or PCR may consist of about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) . Distinctive portions are those which are peculiar to the sequence in question i.e. not based on previously known motifs.
  • Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use in processes such as PCR.
  • probing can be done with entire restriction fragments of the gene disclosed herein which may be 100 's or even 1000 's of nucleotides in length.
  • Naturally sequences may be based on Fig 1 , or the complement thereof .
  • the complement of a nucleic acid (or nucleotide sequence) of the invention is referred to herein, this describes a nucleic acid (or nucleotide sequence) which in each case is the same length as the reference, but is 100% complementary thereto whereby by each nucleotide is base paired to its counterpart i.e. G to C, and A to T or U.
  • Such probes and primers form one aspect of the present invention.
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells . Probing may optionally be done by means of so-called 'nucleic acid chips' (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27- 31, for a review) .
  • a variant in accordance with the present invention is obtainable by means of a method which includes:
  • Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector,
  • probes may be radioactively, fluorescently or enzymatically labeled.
  • Other methods not employing labeling of probe include amplification using PCR (see below), RN'ase cleavage and allele specific oligonucleotide probing.
  • the identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host.
  • Preliminary experiments may be performed by hybridising under low stringency conditions.
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes - 1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • the T m is 57 °C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42 °C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 55 °C in 0. IX SSC, 0.1% SDS .
  • suitable conditions include hybridization overnight at 65 °C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS, 10% dextran sulfate and a final wash at 60 °C in 0.1X SSC, 0.1% SDS.
  • hybridisation of nucleic acid molecule to a variant may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) .
  • PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of LMl are employed.
  • RACE PCR only one such primer may be needed (see "PCR protocols,- A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990)) .
  • a method involving use of PCR in obtaining nucleic acid according to the present invention may include:
  • clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence. Although genomic sequences may be used directly, cDNA clones of smaller size may be preferable to transgenic studies. By use of suitable restriction sites in identified clones, the LMl homolog sequence can be inserted into e.g. a binary vector suitable for the plant in question as discussed below.
  • the nucleic acids described above is in the form of a recombinant and preferably replicable vector.
  • Vector' is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Molecular Cloning a Laboratory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . , John Wiley & Sons, 1992.
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
  • a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome .
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell.
  • a host cell such as a microbial, e.g. bacterial, or plant cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • the promoter is an inducible promoter, particularly one which is responsive to L. maculans infection.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is “switched on” or increased in response to an applied stimulus . The nature of the stimulus varies between promoters . Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus .
  • this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter (optionally inducible) operably linked to a nucleotide sequence provided by the present invention, such as the Lml gene or a variant thereof .
  • nucleic acid constructs which operate as plant vectors.
  • Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148) .
  • Suitable vectors may include plant viral-derived vectors (see e.g. EP-A-194809) .
  • Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S) .
  • CaMV 35S Cauliflower Mosaic Virus 35S
  • Other examples are disclosed at pg 120 of Lindsey & Jones (1989) 'Plant
  • the promoter may be selected to include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression.
  • Inducible plant promoters include the ethanol induced promoter of Caddick et al (1998) Nature Biotechnology 16: 177-180.
  • selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and gl phosate) .
  • antibiotics or herbicides e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and gl phosate
  • the present invention also provides methods comprising introduction of such a construct into a plant cell or a microbial cell and/or induction of expression of a construct within a plant cell, by application of a suitable stimulus e.g. an effective exogenous inducer.
  • a suitable stimulus e.g. an effective exogenous inducer.
  • a host cell containing a heterologous construct according to the present invention especially a plant or a microbial cell.
  • heterologous is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (e.g. encoding Lml or a variant thereof) have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention.
  • a heterologous gene may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence.
  • Nucleic acid heterologous to a plant cell may be non-naturally occurring in cells of that type, variety or species.
  • heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant.
  • a nucleic acid sequence may be placed within a cell in which it or a homologue is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
  • the host cell e.g. plant cell
  • Nucleic acid can be introduced into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718 , NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinj ection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al .
  • a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718 , NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinj ection (WO 92/09696, WO 94/00583, EP 331083, EP
  • Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Transformation of oilseed rape has been described by Schroder et al . (1994) . Transformation of cauliflower (Brassi ca oleracea Var. Botrytis) is disclosed by Bhalla and Smith (1998) Molecular Breeding 4: 531-541.
  • the invention embraces all of the nucleic acid sequences disclosed herein, and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells.
  • the product may be isolated from the expression system (e.g. microbial) and may be used as desired.
  • purified Lml protein, or variants (e.g. fragments) thereof may be used to raise antibodies employing techniques which are standard in the art.
  • Antibodies and polypeptides comprising antigen-binding fragments of antibodies may be used in identifying homologues from other species as discussed further below.
  • Methods of producing antibodies include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof .
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest.
  • Antibodies may be polyclonal or monoclonal.
  • Antibodies may be modified in a number of ways. Indeed the term 'antibody' should be construed as covering any specific (with respect to the LMl protein) binding substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic .
  • antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces,- for instance see WO92/01047.
  • Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of variant polypeptides, and then their encoding genes.
  • the present invention provides a method of identifying or isolating LMl or a variant thereof (as discussed above) , comprising screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind said LMl polypeptide or variant thereof, or preferably has binding specificity for such a polypeptide.
  • Candidate polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source.
  • a polypeptide found to bind the antibody may be isolated and then may be subject to amino acid sequencing.
  • the invention further provides a method of influencing or affecting the L . maculans disease resistance phenotype of a plant, the method comprising the step of causing or allowing expression of a heterologous nucleic acid sequence as discussed above within the cells of the plant.
  • Particular plants of interest include oilseed rape, mustards and different types of cabbages. Examples include B . Oleraceae e.g. brocollis, green and white cabbages, curly kale cauliflowers, brussels sprouts, Raphanus sativa (radish), B . Juncea, B . Sinapis such as Alba, and B . Napus .
  • the step may be preceded by the earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof .
  • nucleic acids of the present invention for increasing the L. maculans disease resistance phenotype of a plant
  • information disclosed herein may also be used to reduce the activity of L. maculans disease resistance in cells in which it is desired to do so.
  • down- regulation of expression of a target gene may be achieved using anti-sense technology.
  • anti-sense genes or partial gene sequences to down-regulate gene expression a homologous nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • Antisense technology is also reviewed in Bourque, (1995) , Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
  • nucleotide sequence which is complementary to any of those, coding sequences, disclosed above forms one part of the present invention.
  • An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression.
  • van der Krol et al . (1990) The Plant Cell 2, 291- 299; Napoli et al . , (1990) The Plant Cell 2, 279-289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588, and US-A- 5,231,020.
  • ribozymes e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) 'The new world of ribozymes' Curr Opin Struct Biol 7:324-335, or Gibson & Shillitoe (1997) 'Ribozymes : their functions and strategies form their use' Mol Biotechnol 7: 242-251.)
  • RNA such as mRNA
  • a further aspect of the invention is a nucleic acid molecule encoding the promoter of the LMl gene. This may have utility in the production of specifically L. maculans responsive constructs or plants.
  • Any promoter region sequence which is not explicitly set out herein may be readily identified using a probe or primer based on the Fig 1 sequence, as described in relation to the cloning protocols above. This can be used in the identification and isolation of a promoter from a genomic library containing DNA derived from a plant source (i.e. an appropriate Brassica B genome species) .
  • the level of promoter activity in a putative sequence is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter when exposed to L . maculans or an elicitor associated therewith.
  • the amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
  • reporter gene facilitates determination of promoter activity by reference to protein production.
  • the reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product.
  • a detectable signal preferably a visually detectable signal, such as a coloured product.
  • Many examples are known, including ⁇ -galactosidase and luciferase.
  • a promoter which is a variant (e.g. truncated derivative, or homologue from another species) of the Lml promoter. This can be generated or identified as described above in relation to variants of the gene,- it will share homology with the Lml promoter and retain promoter activity. Also provided is a construct comprising this promoter.
  • Fig 2 Schematic presentation of homology to Arabidopsis thaliana BACs T19F6 and end sequence of T32A16.
  • the upper bar represents the full sequence of cloned insert 27:8
  • ORFs identified by MacVector 6.5 ORF marked red (orf1-1008) has amino acid sequence homology to T19F6_16.
  • Genomic DNA from leaves of Rapid Cycling (RC) Brassica nigra was isolated according to Bernatzky and Tanksley (1986) with further modifications by Landgren and Glimelius (1990) .
  • This DNA was used as template (lng) in the following PCR reaction: 96°C 1 in x 1 cycle,- (96°C 20 sec, 55°C 30 sec, 72°C 90 sec) x 30 cycles,- 74°C5 min x 1 cycle 4°C indefinite.
  • AmpTaq DNA polymerase Perkin Elmer
  • a total volume of 50 ml was used in the PCR reactions.
  • PCR products were cross-hybridized and one clone (designated no. 27) was selected which derived from the following PCR primer pair:
  • This clone was used to screen a cDNA library (Clontech) , prepared from L . maculans stressed RC B . nigra plantlets .
  • the cDNA was cloned into the EcoRI cloning site in the lambda ZAP II vector. All handling of the library followed the instructions provided by Clontech. Thirteen positive cDNA clones were picked up and excised into pBluescript SK- plasmids . After cross-hybridization and restriction mapping, two clones were selected 27:3 and 27:8 for further studies . The work described below is concerned only with clone 27:8.
  • pCD24 was formed by digesting a fragment carrying the aadA gene driven by the nos promoter and terminated by the ocs sequence digested from SLJ551 (Dr JJ Jones, Sainsbury lab. Norwich, UK) with Hpal/Hindlll . The fragment was treated with Kleenow and blunted into the Sinai site of the binary vector pPZPlOl (Hajdukiewicz et al . 1994). To enable further work, pCD24 was digested with EcoRI blunt ended with Kleenow, and re-ligated (pCD29) .
  • the 35S- 35S3 ' sequence from pCD26 was digested by HindiII and ligated into the HindiII site of pCD29 creating the pCD32 binary plasmid. Subsequently, the cDNA clone (27:8) from the B . nigra cDNA library was inserted in the EcoRI site located between the 35S promoter and its 3' terminating sequence of pCD32, forming the binary vector pCD39 (see Fig 7) . Transfer of the binary vector to Agrobacterium tumefaciens and Agrobacterium mediated transformation of the blackleg susceptible oilseed rape spring cultivar Hanna was performed as described by Schroder et al . (1994) .
  • Plant DNA from transformed and control plants was prepared from freeze dried leaves according to Sharpe et al . (1995) using the Kirby mix (Covey & Hull, 1981) as extraction buffer.
  • the DNA was digested with EcoRI and HindiII respectively, and 10 mg of restricted and DNA was separated on 0.8% agarose gel and transferred to a Hybond N+ filter (Amersham) using 0.4M NaOH.
  • the filters were hybridised with a 2.5 kb fragment corresponding to the 27:8 insert which was radiolabeled with 50mC ⁇ ( 32P) dCTP (Pharmacia oligolabelling kit) .
  • the plasmid 27:8 was used as a positive control on the filters. Hybridisation and washes of the filters were performed according to Sharp et al .
  • Sequencing determination was performed on an ABI 377 automatic sequencer (Perkin Elmer Cetus, USA) . Sequencing reactions were prepared according to the manufacturer's instructions using the Thermo Sequenase TM dye terminator cycle sequencing pre-mix kit (Amersham Life Science, UK) . For each 20 ml reaction, we used 10 pmol of the primers and 2 mg of template DNA. Sequences were evaluated using ABI Edit View and Technelysium Chromas software.
  • RNA from B . napus cv. Hanna, B . nigra and transformants were all included. Both in situ experiments and slot blots indicate a background expression in Hanna, and a slightly higher expression in B. nigra.
  • the pCD 32 binary plasmid carrying the cDNA insert of the 27:8 clone was used in plant transformation as described above and seven shoots were obtained. Plants were, after establishment under in vi tro conditions, taken to the greenhouse to set seed. The following generation (Tl) was analysed for expression and segregation of the aadA marker gene as described by Schroder et al . (1994) . From this study one plant was considered to be homozygous (3B) , the second segregated 3:1 (1A) and the third line (4A) tested segregated in a 1:1 fashion.
  • spectinomycin marker gene was also assayed by PCR using primers specific to the aadA gene (5 ' -TTTGCCGACTACCTTGGT GAT-3') and the nos-promoter (5' -GACAGAACCGCAACGATTGA-3' ) . This yielded a band of approximately 1 kb in the spectinomycin resistant plants and no band of this size in the susceptible plants.
  • Spectinomycin resistant plantlets deriving from three Tl plants (1A, 3B, 4A) were further tested for L . maculans resistance.
  • the plants deriving from the 3B line did all express resistance to blackleg whereas the other two lines segregated into resistant and susceptible plants.
  • the resistance was clearest scored on adult leaves whereas the cotyledons often showed some signs of bleaching after the spectinomycin selection and re-planting which in some cases affected the result of the fungal inoculation.
  • B . napus cv. Hanna, B . nigra and B . napus transformants carrying the Lml cDNA were inoculated (Sjodin C and Glimelius K 1988 J. Phtopath.123 : 322-332) with four different isolates of L. maculans (Leroy, Lindsay, PHW1245 and 478) (Koch et al 1991 Mol . Plant- Microbe Interaction 4: 341-349; Agriculture and Agri-Food Canada Research Centre, Saskatoon, Canada) . Resistance was scored according to the Delwiche scale 11 days post inoculation of cotyledons and 19 days post inculation of adult leaves. Each plant was inoculated at four sites.
  • Homology search High homology (>90%) was found to the Arabidopsis thaliana BAC clone T19F6 (Fig 2) .
  • the homologous part corresponds to an unknown Arabidopsis gene, assigned T19F6_16 (Genebank entry AC002343.1).
  • Homology to a neighbouring, overlapping BAC clone, T32A16 was found in the same region. This BAC has so far only been end-sequenced.
  • ORFs 18 ORFs were identified in the sequence using MacVector 6.5 software (Fig. 3) . One of these ranged from 1-1008 bp on the complementary strand (assigned orfl- 1008) .
  • the translated sequence is 336 aa (Fig 4) .
  • Protein alignment The amino acid sequence of the unknown Arabidopsis gene T19F6_16 aligned with high homology to the amino acid sequence of orfl-1008 (Fig 5) .
  • a homologous region on a BAC clone has been identified in Rice.
  • BLAST searches revealed sequences from Arabidopsis, Oryza sativa and Lotus japonicus sequences which shared homology with the 3' end of the Lml gene. Possible trans- membrane regions have also been identified.
  • the only sequence to which a known function has been assigned is the L. japonicus nodule inception protein (nin) , which takes part in the symbiosis interaction with Mesorhizibium loti .
  • a Southern blot including DNA of B . napus, B . nigra, B . j uncea, B . carinata and transformed B . napus was hybridised with an internal probe covering bp 390-1946 of the cDNA sequence. The results (not shown) indicated that multiple genes with homology to this sequence are present in B . napus as well as in B. j uncea and B . carinata . Similar results have been found using internal PCR primers on genomic DNA, where multiple bands appear.
  • Brassica B genome Theoretical and Applied Genetics 99: 368-372.

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Abstract

La présente invention concerne des molécules d'acides nucléiques isolées qui comprennent une séquence nucléotide LM1 codant pour un polypeptide pouvant altérer, dans une plante, un phénotype de résistance à une maladie, dont Leptosphaeria maculans assure la médiation, plante dans laquelle l'acide nucléique est introduit et exprimé. Des séquences préférées de nucléotide LM1 codent pour le polypeptide de résistance LM1, illustré Figure 4 (c'est à dire la séquence complément de bp 1-1008 sur la Figure 2), ou pour des polypeptides de résistance variants, tels que des variants dérivés, allélique ou autre homologue. L'invention concerne aussi des sondes et des amorces, de vecteurs, des cellules hôtes, des produits d'expression, des plantes transgéniques, des procédés et des méthodes d'utilisation d'un quelconque de ces produits, pour, par exemple, influencer ou affecter, dans une plante, le phénotype de résistance de la maladie causée par L. maculans.
PCT/GB2000/003438 1999-09-08 2000-09-07 Gene de resistance de plante WO2001018221A1 (fr)

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AU70240/00A AU7024000A (en) 1999-09-08 2000-09-07 Nucleic and amino acid sequences relating to resistance to blackleg in plants

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008101343A1 (fr) * 2007-02-21 2008-08-28 University Of Manitoba Identification d'un gène candidat de résistance à la jambe noire (leptosphaeria maculans) lepr3 chez le colza (brassica napus)
WO2009043182A1 (fr) * 2007-10-05 2009-04-09 University Of Manitoba Marqueurs de région amplifiée de séquence connue (scar) pour la résistance de plantes à des pathogènes fongiques
US7601753B2 (en) 2001-08-10 2009-10-13 Palatin Technologies, Inc. Pyrrolidine melanocortin-specific compounds
WO2011044694A1 (fr) * 2009-10-13 2011-04-21 University Of Manitoba Gène de résistance à la jambe noire

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL EBI; 1 January 1998 (1998-01-01), ROUNSLEY S.D. ET AL.: "UNKNOWN PROTEIN. T19F06.16 OR T19F6.10 OR AT4G24020 from A. thaliana", XP002157332 *
DATABASE EMBL EBI; 26 June 1995 (1995-06-26), NEWMAN T. ET AL.: "14098 Lambda-PRL2 Arabidopsis thaliana cDNA clone 172F16T7, mRNA sequence.", XP002157333 *
DATABASE EMBL ebi; BEVAN M. ET AL.: "Arabidopsis thaliana DNA chromosome 4, BAC clone T19F6, partial sequence", XP002157334 *
DIXELIUS C ET AL: "Resistance to Leptosphaeria maculans is conserved in a specific region of the Brassica B genome.", THEORETICAL AND APPLIED GENETICS, vol. 99, no. 1-2, July 1999 (1999-07-01), pages 368 - 372, XP002157330, ISSN: 0040-5752 *
DIXELIUS C: "Inheritance of the resistance to Leptosphaeria maculans of Brassica nigra and B. juncea in near-isogenic lines of B. napus.", PLANT BREEDING, vol. 118, no. 2, May 1999 (1999-05-01), pages 151 - 156, XP002157331, ISSN: 0179-9541 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7601753B2 (en) 2001-08-10 2009-10-13 Palatin Technologies, Inc. Pyrrolidine melanocortin-specific compounds
WO2008101343A1 (fr) * 2007-02-21 2008-08-28 University Of Manitoba Identification d'un gène candidat de résistance à la jambe noire (leptosphaeria maculans) lepr3 chez le colza (brassica napus)
WO2009043182A1 (fr) * 2007-10-05 2009-04-09 University Of Manitoba Marqueurs de région amplifiée de séquence connue (scar) pour la résistance de plantes à des pathogènes fongiques
WO2011044694A1 (fr) * 2009-10-13 2011-04-21 University Of Manitoba Gène de résistance à la jambe noire

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