WO1995030017A1 - Resistance aux maladies amelioree chez les plantes - Google Patents

Resistance aux maladies amelioree chez les plantes Download PDF

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Publication number
WO1995030017A1
WO1995030017A1 PCT/GB1995/000944 GB9500944W WO9530017A1 WO 1995030017 A1 WO1995030017 A1 WO 1995030017A1 GB 9500944 W GB9500944 W GB 9500944W WO 9530017 A1 WO9530017 A1 WO 9530017A1
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Prior art keywords
nucleic acid
acid molecule
plant
plants
sequence
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PCT/GB1995/000944
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English (en)
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Rebecca Stratford
Robert Shields
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Unilever Plc
Unilever N.V.
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Priority to JP7528054A priority Critical patent/JPH09512429A/ja
Priority to SK1392-96A priority patent/SK139296A3/sk
Priority to MX9604896A priority patent/MX9604896A/es
Priority to AU23138/95A priority patent/AU2313895A/en
Priority to EP95916767A priority patent/EP0758395A1/fr
Publication of WO1995030017A1 publication Critical patent/WO1995030017A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • 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

  • This invention relates to disease-resistance of plants, particularly resistance to nematodes.
  • Nematodes are small wormlike animals, many of which are plant, animal or human parasites which cause a variety of diseases.
  • All nematodes are bounded by a tough elastic cuticle synthesised by the underlying hypodermis.
  • the major structural component of the nematode cuticle is collagen, cross-linked by disulphide bonds and located primarily in the basal and inner cortical layers.
  • the external cortex, containing the epicuticle, is composed of a cross-linked protein called cuticulin.
  • nematode cuticular collagens differ markedly from vertebrate interstitial collagens in molecular weight, assembly and mode of cross-linking.
  • a significant difference between the derived amino acid sequence of cloned C. elegans and vertebrate collagen genes is that those of nematodes have relatively short (24-66) Gly-X-Y repeats (as opposed to 300 or more in vertebrates), and these repeats are interrupted by stretches where Gly is not present in every third position; cysteine residues provide the potential for intermolecular disulphide bridges.
  • Gly-X-Y repeats as opposed to 300 or more in vertebrates
  • Plant pathogenic nematodes are a major agricultural problem causing significant crop and yield losses. Plant tissue, particularly root tissue, is damaged by nematode feeding. Such feeding can cause mechanical tissue damage and the accompanying injection of nematode enzymes can cause further tissue disintegration. Nematode infections of roots result in root galls, and distortions in root growth. Similar symptoms accompany nematode infections of other parts of the plants.
  • Important plant pathogenic nematodes include: root knot nematodes, such as Meloidogyne (e.g. M. incognita); cyst nematodes, including Heterodera (e.g. H. glycines, H. schachti ⁇ ) and Globodera (e.g. G. rostochiensis, G. pallida) particularly on soyabean, wheat, sugar beet and potatoes; lesion nematodes such as Pratylenchus; burrowing nematodes exemplified by Radopholus similis; stem and bulb nematodes, including Ditylenchus, (e.g. D. dipsaci); citrus nematodes, including Tylenchulus; seed gall nematodes, such as Anguina; and foliar nematodes typified by Aphelenchoides.
  • root knot nematodes such as Meloidogyne (e.g. M
  • Nematode infection can be accompanied by bacterial or fungal infection.
  • damage caused by nematodes can lead to enhanced severity of bacterial or fungal infection.
  • Fusarium and Verticillium wilts, Pythium damping off, Rhizoctonia and Phytophthora root rots as well as Pseudomonas solanacearum and Corynebacterium insidiosum bacterial wilts are enhanced by nematode infection.
  • several nematodes Xiphinema, Longidorus, Trichodorus and Paratrichodorus
  • Xiphinema Xiphinema, Longidorus, Trichodorus and Paratrichodorus
  • Plant pathogenic nematodes have also been controlled by use of crop rotation or by the use of chemical nematicides.
  • Nematicides used commercially are generally either fumigants (e.g. halogenated aliphatic hydrocarbons and methyl isothiocyanate precursor compounds) or non-fumigants (e.g. organophosphates and oximecarbamates).
  • fumigants e.g. halogenated aliphatic hydrocarbons and methyl isothiocyanate precursor compounds
  • non-fumigants e.g. organophosphates and oximecarbamates.
  • nematicides are highly toxic to other animals, including man. They are therefore hazardous not only to the user but also to the environment.
  • several nematicides e.g., DBCP, EDB, D-D, aldicarb and carbofuran
  • proteases and collagenases have effects on a number of plant parasitic nematodes in vitro (Miller & Sands 1977, J. Nematol: 9, 192-197). However many collagenases have proteoiytic activity and several proteases can hydrolyse denatured collagen (gelatin) so it is by no means certain that the effect on nematodes is due solely to a collagenolytic activity.
  • Collagenolytic matrix metalloproteases have the following characteristics: (1) they are proteinases that degrade at least one component of the extracellular matrix of higher organisms; (2) they contain a zinc ion (hence the name metalloprotease) and are inhibited by chelating agents; (3) they are secreted in a latent pro-enzymic form and require activation for proteolytic activity; (4) they are inhibited by specific inhibitors of metalloproteases (TIMPs); and (5) they share sequence similarities.
  • TIMPs specific inhibitors of metalloproteases
  • Interstitial collagenases (commonly called “collagenasen”, “collagenase type 1 " or MMP1) degrade native collagens types I, II & III. These collagens (types I-III) in native form are not susceptible to stromelysin or to gelatinase. The enzyme cleaves a single bond between Gly-Ile or Gly-Leu three quarters of the way along the collagen triple helix. The collagen molecule unwinds and is then degraded by other proteases or metalloproteases.
  • Gelatinases (or MMP2) preferentially degrades denatured collagen types IV (this is basement membrane collagen), V & NIL
  • MMP3 Stromelysin has very broad activity but does not degrade interstitial collagen. MMP3 has approximately 54% sequence identity at amino acid level and 71 % nucleotide similarity to MMP 1. Gelatinase is evolutionarily more distant.
  • the collagenases were (i) collagenase (expressed by construct p Coll85.2, Goldberg et al. 1986), an MMP-1 type enzyme, (ii) a gelatinase, (expressed by construct pGel 186.2 Collier et al. 1988), an MMP-2 enzyme, and (iii) a stromelysin, expressed by construct p. Strom, (Wilhelm et al. 1987) an MMP-3 enzyme.
  • the enzymes encoded by these DNA constructs were either:
  • the invention provides a nucleic acid molecule capable of increasing the resistance to nematode infection of a plant, comprising a nucleotide sequence encoding a polypeptide with collagenase activity or a precursor thereof, the nucleotide sequence being in operable linkage to a promoter active in plants.
  • collagenase refers to any enzyme capable of degrading collagen.
  • the collagenase or precursor thereof is a metalloprotease, more preferably a matrix metalloprotease, and most preferably a type I matrix metalloprotease.
  • An example of a suitable starting material is the sequence of a type 1 matrix metalloprotease (MMP), shown in Figure 1.
  • MMP matrix metalloprotease
  • the particular sequence identified in Figure 1 encodes the active matrix metalloprotease (i.e. without pre-pro-sequence).
  • Such a sequence may be modified such that it directly encodes an active form of the enzyme expressible in plants. This is conveniently achieved by modifying the nucleotide sequence to include an ATG start codon (encoding methionine) substantially immediately adjacent 5' to, and in frame with, the sequence encoding the mature MMP enzyme.
  • the nucleotide sequence encodes a polypeptide which lacks the "pre -pro" portions of the collagenase enzyme, the polypeptide thus not requiring any processing before displaying collagenolytic activity.
  • nucleic acid molecule is adapted in various ways to improve expression in plants of the sequence encoding the collagenolytic polypeptide or precursor.
  • alterations to improve expression in host plants include changes to the sequence around the ATG codon, especially immediately adjacent 5' of the ATG codon, so as to render the sequence more closely consistent with the consensus sequence for a plant initiation codon.
  • the sequence will also comprise a polyadenylation signal derived from a plant source.
  • other alterations to the sequence are possible which can introduce optional desired characteristics into the protein.
  • the nucleotide sequence can be altered to more closely resemble plant codon usage, and leader (or signal) sequences can be inserted between the ATG codon and the sequence encoding the mature collagenase.
  • leader (or signal) sequences can be inserted between the ATG codon and the sequence encoding the mature collagenase.
  • the presence of a leader sequence is not considered to prevent a polypeptide being thought of as "mature", if removal of the leader sequence alone is sufficient to leave a fully active collagenase.
  • such a leader or signal sequence comprises the patatin signal sequence, but other signal sequences which target the protein out of the plant cell are known to those skilled in the art and should also be suitable, such as that from a pathogenesis related ("PR") plant gene.
  • PR pathogenesis related
  • the nucleic acid molecule of the invention may conveniently be a vector.
  • a vector will comprise a nucleotide sequence encoding a polypeptide with collagenase activity or precursor thereof (preferably a collagenolytic metalloprotease), in operable linkage to a promoter active in plants.
  • the vector will further comprise an origin of replication effective in bacteria, and/or regulatory signals recognised in plants.
  • promoters active in plants, are available to those skilled in the art and are known from the literature. These include constitutive and, more preferably, inducible promoters. Desirably the promoter should be induced in response to nematode attack.
  • the molecule also comprises means for preventing the expression in bacteria of the polypeptide having collagenase activity.
  • this comprises a 'spacer' fragment located between the collagenase coding sequence and an adjacent bacterial promoter. This facilitates the manipulation in bacteria of nucleotide sequences encoding polypeptides having collagenolytic activity, which the inventors have typically found to be toxic for commonly used laboratory bacterial strains.
  • a suitable spacer fragment may be derived from the barley beta-glucanase gene.
  • a molecule in accordance with the invention may be introduced into a plant or part thereof, using techniques well known to those skilled in the art (e.g. transformation or electroporation).
  • the invention provides a method of increasing the resistance of a plant to a nematode, comprising introducing into the plant a nucleic acid molecule in accordance with the invention, so as to cause the plant to express, or to increase the expression of, a polypeptide possessing collagenase activity, or a precursor thereof.
  • the invention provides a transgenic plant or the progeny thereof, having increased resistance to nematodes, comprising a nucleic acid molecule in accordance with the invention.
  • the invention provides a method of reducing or eliminating plant-infecting nematodes from an area of land, comprising planting the area with nematode-resistant plants comprising a nucleic acid molecule in accordance with the invention.
  • Nematodes are unable to replicate in such plants, thus the number of nematodes in the area will be reduced. In principle, if there are very few nematode-susceptible plants in the area of land, it should be possible to eliminate all plant- infecting nematodes from the area.
  • Figure 1 shows the nucleotide sequence encoding a mature MMP type I enzyme, together with the amino acid sequence of the mature polypeptide
  • Figure 2 shows the sequence of a spacer fragment used in constructing a vector which did not express in bacteria
  • Figure 3 summarises the method for construction of the MET COL and PAT COL plasmids;
  • Figure 4 shows in vitro processing of the polypeptide expressed by the PAT COL construct;
  • Figures 5 to 7 show bar charts of the number of nematode cysts in resistant transgenic plants comprising a nucleotide sequence in accordance with the invention, compared with control plants.
  • DNA encoding the pre-pro-form of an MMP type I enzyme was isolated, the mature form of which had the sequence shown in Figure 1 (nucleotide sequence Seq. ID No. 1, amino acid sequence Seq. ID No. 2).
  • the DNA sequence was adapted for expression in plants as described below.
  • the DNA fragment coding for the active MMP described above lacks a protein synthesis initiation codon (as the protein is synthesised as a pre-pro-protein in vivo), so an ATG (Met) codon was placed immediately upstream of the mature enzyme sequence with the aim of expressing an active MMP polypeptide in the plant.
  • This clone was designated MET COL.
  • the procedure was designed to minimise the number of extra amino acids that are placed in front of the mature enzyme, so as to minimise the extraneous amino acid content of the enzyme.
  • the polymerase chain reaction was performed on a nearly full-length clone of the MMP type I gene.
  • the forward oligonucleotide (RS-5, Seq. ID No. 3) was designed to contain a if ⁇ mHI site, plant initiation codon context (Lutcke et al., 1987 EMBO J. 6, 43-48), ATG codon and sequences homologous to the 5' end of the mature MMP sequence:
  • the reverse primer (MOH-32, Seq. ID No. 4) was designed to contain a Sail site, a termination codon and sequences homologous to the 3' end of the MMP gene:
  • MET COL was then transferred as a Bam ⁇ l-Kpnl fragment into the 35S promoter - Nos terminator cassette PBI 220.5 and subsequently transferred into Bin 19 (Bevan, M.W. 1984 Nucleic Acid Res. 12, 8711-8721) as a H./ ⁇ dIII-£c ⁇ RI fragment.
  • the sequence encoding MMP was further adapted to expression in plants by including a stretch of nucleotides which encoded a plant signal sequence (to make a construct termed "PAT COL").
  • the purpose of making these constructs is to direct the MMP enzyme to the extracellular compartment of the plant cell to attack the nematode cuticle.
  • patatin leader sequence was cloned by PCR from lambda pat 18 (Bevan et al., 1986 Nucl. Acids Res. 14, 4625-4638 and Goldsbrough, 1989 Ph.D. thesis "A molecular analysis of Patatin gene expression", Council for National Academic awards, London) which contains 5kb of the 5' end of the patatin gene in pUC.
  • the forward ohgonucleotide (RS-1, Seq. ID No. 6) contained a BamHI site, plant context and sequences homologous to the 5' end of the patatin coding region:
  • the reverse oligo (RS-3) was used in conjunction with RSI to clone the signal sequence plus the first amino acid of the mature patatin respectively (Seq. ID No. 7):
  • the ohgonucleotide was designed to contain a Dral site. Cleavage with Dral leaves a TTT codon which encodes phenylalanine (i.e. the first residue of the mature MMP).
  • V L T P G N P The sequence of RS-4 differs slightly from the cDNA sequence in that the valine residue is encoded by the codon GTA in the primer rather than by GTT. This alteration was made in order to introduce a Sn ⁇ Bl site, to facilitate cloning. Cleavage with SnaBl leaves a GTA codon which encodes valine (ie the second amino acid of the mature MMP), so the encoded amino acid sequence is not changed by the alteration in nucleotide sequence.
  • the insert was removed by cleavage with SnaBl and Sail and ligated with the patatin signal sequence PCR products from (b) (see Figure 3) and inserted into BS cut with BamHI and Sail. Fusion products were sequenced to ensure that the constructs were correct.
  • Figure 3 summarises the construction of MET COL and PAT COL.
  • the signal sequence and 'pro' sequence were removed from the sequence encoding a pre-pro-collagenase, to leave a sequence encoding the mature polypeptide with an ATG start codon added at the 5' end to make MET COL.
  • the patatin leader sequence was fused to the 5' end of MET COL.
  • the fusion construct was, therefore, cloned into a modified PBI 220.5 vector.
  • the vector was modified by insertion of a 540bp Sphl fragment into the Sphl polylinker site between the lacZ promoter and the CaMV 35S promoter.
  • the 540bp Sphl "spacer" fragment was derived from the upstream promoter region of a barley (1-3, 1-4) Beta-glucanase gene and includes 12 bp of PUC vector DNA at the 5' end.
  • the sequence (Seq. ID No. 9) is presented in Figure 2.
  • the pBI 220.5 vector modified in this way would prevent transcriptional readthrough of the fusion protein from the lacZ promoter, which might have been causing accumulation of a potentially deleterious fusion polypeptide in the bacterial cells.
  • Transformants containing the fusion construct were obtained and the construct was cloned into Bin 19 as a H dlll-EcoRI fragment. This vector was then used to transform Agrobacteria and transformants were obtained having the expected plasmid structure.
  • agrobacteria strains were grown on YEP with appropriate antibiotics.
  • the MMP constructs in the Bin 19 vector were introduced into agrobacterium strain GV 2260, by electroporation and transformants selected on kanamycin.
  • MET COL and PAT COL fusion constructs in the BS vector were used as templates for in vitro transcription of RNA using standard procedures.
  • In vitro transcripts were translated in a rabbit reticulocyte in vitro translation system in the presence (4b, 4c) or absence (4a) of dog pancreatic microsomes.
  • the products were separated on standard 10% acrylamide gels (SDS PAGE) and detected by autoradiography ( Figure 4).
  • MET-COL products ("Met) are shown on the right of each gel, PAT-COL products ("Pat”) on the left.
  • Figure 4a-c shows that the predominant translation product from the MET COL construct has an apparent molecular weight (Mwt) of 41kD (large arrow) which corresponds closely to the expected size. Minor bands are detected at 38kD and 32kD (small arrows) and may correspond to degradation products.
  • the predominant translation product from the PAT COL construct has an apparent Mwt of 42kD (large arrow 4a), corresponding to a patatin signal sequence-MMP I fusion.
  • the band size is decreased to that of the collagenase (mature) peptide indicating that correct processing of the patatin signal sequence has occurred.
  • the MMP expression vectors described above were introduced into plants.
  • the plants were originally derived from tuber sprouts and are the widely available varieties Desiree and Maris Piper (or 83 N28-47 control plants).
  • the plants were grown as in vitro shoot cultures at 24 °C under continuous light (70 ⁇ E m sec ) in 150 x 25mm glass tubes loosely capped with Magenta lids (Magenta Corp., Chicago, 111. USA).
  • Nodal stem cuttings with one leaf were taken from 5-6 week old plants and planted in MS (Marashige and Skoog) medium containing 1 % sucrose solidified with 0.9% agar (Sigma) and supplemented (after autoclaving) with STS which promoted plant growth and produced larger leaves.
  • the STS (silver thiosulphate) stock solution (6mM or 1.5 mg/ml.) was prepared by adding 12mM AgN0 3 to an equal volume of 96mM Na 2 S 2 O 3 and used at a concentration of l ⁇ g/ml.
  • Leaf explants were taken from plantlets at 5-6 weeks after subculturing; cut across the base (discarding the petiole and the lower l-2mm of leaf base) and the remaining leaf used in transformation experiments. These were carried out essentially as published by Visser et al. (1989 Plant Mol. Biol. 12, 329-337) but slightly altered from the protocol published. Briefly, explants were floated overnight on liquid M387. The medium (M387) contained MS salts and vitamins supplemented with: lOg/1 sucrose; 80mg/l NH 4 NO 3 ; 147mg/l CaCl 2 ; lOmg/1 NAA; and lOmg/1 BAP. The next day the explants were soaked in an overnight culture of A.
  • M379 callus induction media M379 callus induction media
  • vitamins supplemented with: lg/1 sucrose; 4g/l mannitol; 0.0175mg/l IAA; 2.25mg/l BA and solidified with 0.8% agar).
  • the explants were placed on plates with 500 ⁇ g/ml cefotaxime (Claforan, Roussel) and five to seven days later the explants were transferred to M384 (shoot induction medium: MS supplemented with 15g/l sucrose; 2.25mg/l BAP; 5mg/l giberellin GA3 and 200mg/l cefotaxime and 50 ⁇ g/ml kanamycin). Every three to four weeks the explants were transferred to fresh M384 medium with 200 ⁇ g/ml cefotaxime and 50 ⁇ g/l kanamycin. Transformed shoots were removed from the explants and grown in vitro as described above before transfer to soil for further growth. Further details of the method are described by Higgins et al. (1992 Plant Sci. 82, 109-118) and Hulme et al. (1992 Plant Cell, Tiss. and Org. Cult. 31, 161-167).
  • Two types of test for nematode resistance were performed using Potato cyst nematode G. pallida Pa2/3: pot tests on whole plants in the greenhouse; and canister tests on roots growing on tuber explants. Plants that showed resistance in the greenhouse pot test were re-tested in canister tests.
  • Plants were grown in infested soil, obtained from a nematode bed maintained especially for the purpose. As the concentration of cysts in the bed was high, the soil was mixed with peat to give a concentration of approximately 20 eggs and larvae per gram of soil. Approximately 2.5cm of the soil/compost mixture was put in the base of a 9cm pot and the pot then filled with sterilised potting compost. Tissue culture plants were planted in the pots and grown under lights (14h photoperiod) at about 20 C C for 8-10 weeks. The plants were then tipped out of the pots and the rootballs assessed for the presence of cysts. Only clones with a mean score of less than one cyst per rootball were retained.
  • PAT COL constructs a total of 61 Maris Piper transformants were tested, and eight clones were found resistant in canister tests. Seventy-seven Desiree transformants were tested and eighteen showed resistance.
  • MET COL constructs a total of 86 Maris Piper transformants were tested for resistance in glass house tests. Of these, seven were selected for re-test in canisters, of which four (#2, #25, #31 & #89) displayed moderate levels of resistance.
  • Figure 5 is a bar chart showing the number of G. pallida cysts found in the resistant Desiree PAT COL transgenic lines compared with control varieties of Desiree, Maris piper and the partially resistant 83N plants.
  • Figure 6 shows the number of cysts in resistant transgenic PAT COL Maris piper plants
  • Figure 7 shows the number of cysts in resistant transgenic MET COL Maris piper plants.
  • GTC AGG GGA GAT CAT CGT GAC AAT TCT CCC ⁇ T GAT GGA CCT GGA GGA 240
  • AAACCAAGCA AATCAGGGAG GGTAAAAGAT TCACTGCACT GGAAGCTAGT CAAAG ⁇ CAT 300

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Abstract

On décrit une molécule d'acide nucléique, pouvant accroître la résistance d'une plante aux infections à nématodes, et comprenant une séquence nucléotidique qui code un polypeptide à activité collagénase, ou un de ses précurseurs, et qui est liée fonctionnellement à un promoteur actif chez les plantes.
PCT/GB1995/000944 1994-04-29 1995-04-26 Resistance aux maladies amelioree chez les plantes WO1995030017A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP7528054A JPH09512429A (ja) 1994-04-29 1995-04-26 植物の疾病抵抗性におけるまたはそれに関する改良
SK1392-96A SK139296A3 (en) 1994-04-29 1995-04-26 Nucleic acid molecule having ability to increase resistance against infection by nematodes, a vector containing this molecule and method of increasing resistance against this infection
MX9604896A MX9604896A (es) 1995-04-26 1995-04-26 Mejoramientos en o relacionados a la resistencia de las plantas contra enfermedades.
AU23138/95A AU2313895A (en) 1994-04-29 1995-04-26 Improvements in or relating to disease-resistance of plants
EP95916767A EP0758395A1 (fr) 1994-04-29 1995-04-26 Resistance aux maladies amelioree chez les plantes

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WO1997020057A1 (fr) * 1995-11-29 1997-06-05 University Of Leeds Promoteurs specifiques des racines

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WO1992021757A1 (fr) * 1991-05-30 1992-12-10 Plant Genetic Systems, N.V. Promoteurs destines aux plantes et reagissant aux nematodes

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WO1992021757A1 (fr) * 1991-05-30 1992-12-10 Plant Genetic Systems, N.V. Promoteurs destines aux plantes et reagissant aux nematodes

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GALPER, S., ET AL.: "Nematicidal effect of collagen-ameded soil and the influence of protease and collagenase", REV. NEMATOL, vol. 13, no. 1, ., pages 67 - 72 *
MILLER, P.M, ET AL.: "Effects of hydrolytic enzymes on plant-parasitic nematodes", J. NEMATOL., vol. 9, no. 3, pages 192 - 197 *
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Publication number Priority date Publication date Assignee Title
WO1997020057A1 (fr) * 1995-11-29 1997-06-05 University Of Leeds Promoteurs specifiques des racines

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EP0758395A1 (fr) 1997-02-19
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SK139296A3 (en) 1997-06-04
AU2313895A (en) 1995-11-29
PL316992A1 (en) 1997-03-03
JPH09512429A (ja) 1997-12-16
HUT75768A (en) 1997-05-28
CZ315496A3 (cs) 1998-03-18

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