WO1992020800A1 - Proteines intervenant dans la pathogenese des plantes - Google Patents

Proteines intervenant dans la pathogenese des plantes Download PDF

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Publication number
WO1992020800A1
WO1992020800A1 PCT/EP1992/001063 EP9201063W WO9220800A1 WO 1992020800 A1 WO1992020800 A1 WO 1992020800A1 EP 9201063 W EP9201063 W EP 9201063W WO 9220800 A1 WO9220800 A1 WO 9220800A1
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Prior art keywords
protein
plant
proteins
amino acid
sequence
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PCT/EP1992/001063
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English (en)
Inventor
Yigal Cohen
Karl Guegler
Egon Moesinger
Thierry Niderman
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Sandoz Ltd.
Sandoz-Patent-Gmbh
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Publication of WO1992020800A1 publication Critical patent/WO1992020800A1/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
    • 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
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
    • 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

Definitions

  • the present invention concerns a family of pathogenesis-related (PR) proteins referred to as P14 proteins, functionally equivalent protein analogues thereof, fungicidal compositions containing the proteins and their use in combating fungal diseases in plants.
  • PR pathogenesis-related
  • the present invention is further directed to biotechnological processes for producing such proteins as well as transgenic plant cells and plants that demonstrate resistance to fungal diseases.
  • a stress e.g. an abiotic stress such as UV-irradiation, or a biotic stress caused by pathogenic organisms such as fungi, bacteria, viruses or by insects
  • the plant defends itself through several mechanisms, including thickening of cell walls by deposition of additional callose or lignin at the site of infection and production of substances that are toxic to the organism.
  • proteins are generally characterized as being acid soluble, protease resistant, relatively small in size, accumulating predominantly in the intercellular space of the plant, and as producing a characteristic pattern in gel electophoresis.
  • hydrolytic enzymes e.g. chitinases, ⁇ -1,3 glucanases and proteases
  • peroxidases e.g. peroxidases, peroxidases and proteinase inhibitors.
  • Stressed plants are also found to produce a group of proteins having a molecular weight between 10 and 20 k Daltons.
  • One such protein described in the literature is the PR-protein produced by a tomato plant upon infection by a pathogen, this protein being referred to as the P14 protein of tomato.
  • This protein has been described as having a molecular weight of about 15.5 kD and an amino acid sequence thought to correspond to P14 has been published (Lucas et al., The EHBO Journal, vol. 4, no. 11, pp.2745-2749, 1985).
  • P14 protein is actually a family of related proteins.
  • genes coding for five different P14 proteins in tomato hereinafter referred to as P14a, P14b, P14d, P14e and P14f.
  • the c-DNA coding sequences and corresponding amino acid sequences including leader peptide sequences, for these five tomato P14 proteins are given hereinafter in the SEQUENCE LISTING as SEQ.ID N0:1, 2, 3, 4 and 5.
  • the present invention includes P14a, P14b, P14c, P14d, P14e and P14f per se, e.g. in purified or isolated form.
  • amino acid sequence published by Lucas et al. appears to correspond to P14a but lacks an internal sequence of five amino acids, amino acid residues 99 to 103 of the mature protein of SEQ ID NO 1.
  • the five P14 proteins have been synthesised and found to possess potent fungicidal activity.
  • This invention is therefore directed to fungicidal compositions that are effective in protecting plants and plant loci against fungal attack comprising a fungicidally effective amount of a P14 protein.
  • the P14 protein amino acid sequences can be modified by adding, substituting or removing one or more amino acids to provide modified products which are fungicidally active.
  • this invention is further directed to fungicidally active modified P14 proteins hereinafter referred to as P14 analogues.
  • a DNA sequence coding for a P14 protein or analogue or portions of such a sequence can be incorporated into a vector by conventional techniques and used to transform or transfect host cells in order to produce the P14 protein product in useful quantities.
  • DNA portions thereof, including genomic DNA sequences derivable directly from plant material, can also be incorporated into the genome of a plant in order to give the plant resistance to fungal disease.
  • proteins and compositions of the invention are typically intended for use in agriculture. However, the proteins and compositions may also be employed in pharmaceutical and veterinary uses to protect or treat humans or animals against fungal diseases.
  • Any fungicidally active P14 protein or analogue may be used in the present invention.
  • additional fungicidally active tomato P14 proteins may be used in the present invention.
  • a P14 protein is a protein, typically produced by a stressed plant, having a molecular weight of about 15 kD, e.g. about from 14 kD to about 16 kD, and having an amino acid sequence which is at least 70%, preferably at least about 80%, homologous to the mature protein amino acid sequence of any one of the tomato P14 proteins the sequences of which are disclosed herein (i.e. P14a, P14b, P14d, P14e and P14f), but especially P14a (i.e. the amino acid sequences of residues 1 to 135 inclusive of the amino acid sequence of SEQ ID NO 1).
  • the terms "homology” or “homologous” when applied to sequences, either amino acid or nucleotide, are intended to indicate the level of identity between the sequences concerned.
  • the amino acid sequences of two related proteins, or the DNA sequence coding therefor may be aligned with one another at regions of identical, i.e. homologous, sequence, and the differences between one sequence and the other, whether these be by way of substitution deletion or insertion of amino acid or nucleotide residues, identi t -ed.
  • the percentage homology between the two sequences is determined with respect to one of the sequences, the reference sequence, by calculating the percentage of changes between the other sequence or sequences and the reference sequence with respect to the reference sequence. The homology by percentage is then 100 minus the percentage of changes between the other sequence or sequences and the reference sequence.
  • the mature P14b protein is about 96% homologous to the mature P14a protein (5 amino acid substitutions, both 135 amino acids in length).
  • the mature P14d protein is about 88% homologous (16 substitutions, both 135 amino acids in length)
  • the mature P14e protein about 90% homologous (2 substitutions and 9 deletions in 135 amino acid residues)
  • the mature P14f protein about 78% homologous (29 substitutions, both 135 amino acids in length) to the mature P14a protein.
  • P14 proteins for use in the present invention may be obtained from plant material by extraction and purification procedures.
  • the P14 family of proteins of tomato can be obtained from tomato plants and separated into the individual protein components by the procedures described in detail in Example 1.
  • the P14 proteins can be altered, i.e. functionally equivalent analogues can be prepared by standard techniques.
  • This invention therefore also concerns itself with P14 protein analogues in which the amino acid sequence of a mature P14 protein has been modified by adding, substituting or removing one or more of the amino acids without substantially reducing the level of fungicidal activity, i.e. which retain substantially the same level of fungicidal activity as the P14a protein or possess an even higher level of fungicidal activity.
  • Such altered sequences preferably possess substantial sequence homology to a P14 protein, typically at least 70%, preferably at least 80% homology.
  • the fungicidally active P14 analogues have mature protein amino acid sequences which are at least 70% homologous, or more preferably a least 80% homologous to one ore more of the mature protein sequences of the specific tomato P14 proteins (P14a, P14b, P14d, P14e or P14f) disclosed herein.
  • altered sequences that demonstrate P14-like fungicidal activity are at least 70% homologous to the protein extending from amino acid position 1 to 135 of SEQ ID NO 1, more preferably at least 80% homologous.
  • a protein or protein analogue is fungicidally active if it has a fungicidal activity at least one tenth of that of P14a when tested in a standard fungicidal activity test.
  • the proteins and analogues have a level of fungicidal activity substantially the same as, or more preferably higher than, P14a.
  • the P14 proteins may be obtained from plant material, the P14 proteins and analogues are preferably prepared by recombinant DNA techniques.
  • the invention includes DNA sequences coding for the P14 proteins and analogues, vectors containing these DNA sequences and host cells transformed with these DNA sequences.
  • the DNA sequences of the invention may code for any fungicidally active P14 proteins or analogues as hereinbefore defined.
  • Preferred DNA sequences are sequences coding for P14a, P14b, P14d, P14e or P14f or analogues thereof i.e. proteins having at least 70%, preferably at least 80% homology with any one of these proteins.
  • the DNA sequence encodes a fungicidally active protein having an amino acid sequence having at least 70% homology to the protein extending from amino acid position 1 to 135 of SEQ ID NO 1, more preferably at least 80% homology.
  • DNA sequences may be obtained by methods well known in the recombinant DNA art.
  • c-DNA or genomic DNA sequences may be derived or obtained from plant tissue.
  • Sequences coding for P14 analogues may be synthesised in whole or in part using oligonucleotide synthesis techniques well known in the art or adaptable therefrom.
  • the DNA sequences may include 5' sequences which code for signal sequences, which may be natural or analogue P14 signal sequences or alternate signal sequences including those derived from other proteins. If DNA coding for a signal sequence is not present it will normally be necessary to include a translation start cordon (ATG) at or adjacent to the 5' end of the protein coding sequence.
  • ATG translation start cordon
  • the DNA sequences are incorporated under the control of appropriate regulatory sequences (e.g. promoter/enhancer sequences) into expression vectors and such vectors are used to prepare transformed host cells which may be cultured to express the proteins.
  • appropriate regulatory sequences e.g. promoter/enhancer sequences
  • the presence of an N terminal signal sequence in the expressed protein advantageously facilitates the processing and secretion of the mature protein from the host cell.
  • the DNA sequences may be used to transform plant cells and thus provide transformed plant cells and transgenic plants which express the P14 protein products and which are thereby resistant to fungal disease.
  • production of the P14 and P14 analogue proteins involves transforming or transfecting a host cell with a recombinant DNA vector as previously described and subsequently culturing the transformed or transfected host cell to produce the protein. While a variety of transformed or transfected cell systems may be employed, it is generally preferred to transform or transfect bacterial cells of either the gram-negative or gram-positive type.
  • One preferred type of gram-negative bacteria is E. Coli with which considerable experience in biotechnology has already been achieved and for which a wide variety of suitable and operatively functional plasmid and transfer and expression vector systems are known and available.
  • Pseudomonas fluorescens represents another type of gram-negative bacteria into which plasmids carrying the protein sequences have been incorporated.
  • Further suitable host cells include Bacillus thuringiensis (B.t. ), B.cereus, and B.subtilis.
  • Typical promoters to be used in transformation of E. Coli include Ptaci F rp > Ftre- P > T 3 , and T , all of which are commercially available, e.g. from Pharmacia.
  • Figure 1 which is a plasmid diagram of plasmid pBTlOO, the plasmid constructed for expression of a methionyl P14a product and
  • FIG. 2 which is a plasmid diagram of plasmid pBT103", the plasmid constructed for expression of a P14a protein product including its signal sequence peptide.
  • the P14 family of proteins of tomato were obtained from tomato plants and separated into the individual protein as described below.
  • Fresh tomato leaf material from uv-irradiated or P. infestans infected tomato plants was mixed and homogenised in a 5% acetic acid solution containing 0.1% Beta mercaptoethanol pH2.8, then centrifuged at 15000 r.p.m. for 20 minutes. The supernatant was adjusted to pH 5.5 and centrifuged again at 15000 r.p.m. for 20 minutes. The supernatant was precipitated by adding solid ammonium nitrate to give a final saturation of 80%. After 1 hour at 4°C, the precipitate was centrifuged at 10000 r.p.m. for 30 minutes to obtain a pellet.
  • the pellet was combined with the minimum volume of 50 mM Tris/HCl, 1 mM EDTA, 1 M NaCl and pH 7.5 buffer required to effect dissolution. This so-formed acidic leaf homogenate preparation was then subjected to overnight dialysis.
  • the P14 proteins were separated from the non-bound proteins fraction obtained above using a Mono Q anion exchange column (Pharmacia) in a 50 mM 1,3 diaminopropane, pH 10 buffer. The P14 proteins were eluted directly in the flow-through. This fraction, having a basic pH, was then separated on a Mono S cation exchange column (Pharmacia) in a 50 mM acetate buffer pH 4.4. From this column, three peaks corresponding to the P14a, P14b and P14c proteins, were eluted.
  • the P14 proteins were further purified on a double Superose 12 gel filtration column (Pharmacia) in a 100 mM tris/HCl, pH 7.5 buffer. Finally, the proteins are desalted by G25 gel filtration. The purity was monitored by a SDS-PAGE and silver staining.
  • This Example describes the identification of DNA sequences coding for five different tomato P14 proteins.
  • Conventional and well known molecular biology techniques were used, such as described by Maniatis et al. (Molecular Cloning, A Laboratory Manual, 2 nd Edition, Sambrook, Fritsch and Maniatis) and thus a detailed description of these techniques is not included.
  • This sequence comprises a cDNA sequence of 480 nucleoclides coding for a protein of 159 amino acids in length comprising a 24 amino acid signal peptide (residues - 24 to - 1) and a 135 amino acid mature protein sequence (residues 1 to 135).
  • This protein contains an additional stretch of 5 additional amino acids (residues 99-103 inclusive) as compared with the P14 protein sequence published by Lucas et al.. (ibid.).
  • Tr - next step was the cloning of a genomic library into the Lambda-GEM 4 vector from PROMEGA (10 6 clones) this library was screened with the P14a cDNA and 27 independent clones containing P14 genes were isolated and some of them purified to individual Lambda clones.
  • the P14b coding sequence comprises a DNA sequence 480 nucleotides in length coding for a 159 amino acid protein having 24 amino signal peptide and 135 amino acid mature protein portions.
  • P14b has 5 amino acid substitutions with respect to the P14a protein sequence all of these being present in the mature protein (residues 77, 90, 92, 130 and 132), representing a homology of about 96% between the P14a and P14b mature protein sequences.
  • the P14e protein is very similar to the P14a protein though has a stretch of 9 amino acids (residues 127 to 135) deleted from its c-terminus and the last two c-terminal amino acids (residues 125 and 126) substituted with respect to P14a (i.e. a homology of about 90% between the P14e and P14a mature proteins).
  • P14d mature protein has a homology of about 88% with P14a mature protein (16 amino acid substitutions in the mature protein sequence plus 2 amino acid substitutions and one insertion in the leader peptide sequence).
  • P14f similar to P14a, has a 24 amino acid leader peptide and 135 amino acid mature protein sequence, but has 29 amino acid substitutions in the mature peptide sequence, thus representing a homology of about 78% between the P14a and P14f mature protein sequences.
  • Example 3 Constructions of a Plasmid for Expression of Methionyl P14 Protein in E. Coli and Transformation of E. Coli cells
  • E. Coli expression plasmid pKK233.2 (Pharmacia) was cut with Ncol and Hindlll restriction enzymes and the resultant plasmid DNA gel purified. The large plasmid DNA fragment was then treated with calf intestinal phosphatase to remove phosphates.
  • Primer TB3 corresponds to SEQ ID NO 7 and TB5 to SEQ ID NO 8.
  • TB3 5' TCATAAGGATCCATAAGCTTAGTAAGG 3' TB5: 5' ACTCTTGTGCCATGGAAAATTCACCCC 3'
  • Primer TB5 is homologous to 5' terminus of the nucleotide sequence extending from nucleotide position 59 to position 480 in SEQ ID NO 1 and is designed to provide a Ncol site and ATG translation initiation codon adjacent to the 5' terminus of the DNA sequence coding for the mature P14 a protein.
  • Primer TB3 is homologous to the 3' terminus of the aforementioned nucleotide sequence and is designed to provide a Hindlll site at the 3' terminus.
  • primers were used to amplify through polymerase chain reaction (PCR) a nucleotide sequence containing the nucleotide sequence extending from nucleotide position 73 to position 480 in SEQ ID NO 1, and further containing the aforementioned additional restriction sites and ATG translation initiation codon. After amplification this sequence was purified using standard procedures and digested with Ncol and Hindlll. The product of this digestion was then mixed with the Ncol-Hindlll cut pKK233.2 vector fragment prepared above and the two fragments ligated. The resulting plasmid pBTlOO is shown in Figure 1. This plasmid was then used to transform competent JM105 E. Coli cells. Transformed colonies were subsequently screened for their plasmid content and their ability to express the P14 protein after induction according to standard techniques.
  • PCR polymerase chain reaction
  • Plasmid PKK233.2 was cut with Ncol and Hindlll and purified as described in Example 2.
  • Primer TB4 corresponds to SEQ ID NO 9.
  • Primer TB4 is homologous to the 5' terminus of a nucleotide sequence including the nucleotide sequence extending from nucleotide position 1 to position 480 in SEQ ID NO 1 and is designed to provide a Ncol site adjacent to the 5' terminus of the cDNA sequence coding for P14a including the leader peptide.
  • Primers TB3 and TB4 were used to amplify via PCR a nucleotide sequence containing the nucleotide sequence extending from nucleotide position 1 to position 480 in SEQ ID No. 1, and further containing a Ncol restriction site at the 5' terminus and a Hindlll restriction site at the 3' terminus. After amplification this sequence was purified and digested by Ncol and Hindlll and a ligation mixture was formed with the Ncol-Hindlll cut pKK233.2 vector as described in Example 4. The resulting plasmid pBT103 is shown in Figure 2. Transformation of competent JM105 E. Coli cells and screening was carried out as in Example 3.
  • P14b, P14d P14e and P14f genes are constructed. Such vectors are used to transform competent E. Coli cells. The transformed E. Coli cells are cultured and the various P14 proteins (P14a, P14b, P14d, P14e and P14f) expressed. All the P14 proteins are tested for fungicidal activity and found to have a fungicidal activity similar to that of P14a.
  • the P14 and modified P14 proteins of this invention are formulated in fungicidal compositions together with agriculturally acceptable diluents.
  • diluents as used herein means a liquid or solid which is added to the protein to bring it into an easier or better applicable form.
  • diluents may include water, xylene, talc, kaolin, and diatomaceous earth, agents which stabilise the formulation, e.g. buffers, and surfactants for enhancing contact between the proteins and the plant to be treated.
  • Formulations used in spray form may contain surfactants such as wetting and dispersing agents.
  • surfactants include Tween-80 and Trixton-X-100.
  • the P14 proteins and modified P14 proteins are typically stable throughout a range of pH's and therefore water is often a suitable carrier.
  • the composition additionally contain a buffer to maintain the composition within a desirable pH range. Any buffer solution may be used which is compatible with the proteins and does not otherwise deleteriously affect the plant. Suitable buffers include, for example, a 50 mM tris/HCl, sodium succinate or sodium citrate.
  • the amount of P14 protein in the fungicidal composition is between about 1 and 1000 ⁇ g/ml, although amounts outside such range might also be acceptable, depending upon the particular protein being employed and the plant being treated.
  • composition additionally contains a surfactant
  • the amount required is readily ascertainable by persons skilled in the art and will typically range from between about 0.001 and about 20 % by weight.
  • the above-described formulations, containing the P14 protein or modified P14 proteins and optional diluents and additives are then applied to the plant or plant loci.
  • Application of the composition can be carried out in accordance with techniques well known to persons skilled in the art such as by preparing a spray for application to the plant. It will be appreciated that the amount of solution required for treatment of the plant or plant loci will depend on the subject of the treatment (plant, soil), the type of treatment (e.g. drenching, sprinkling, spraying), the purpose of the treatment (prophylactic or therapeutic), the type of fungi to be treated and the application time.
  • active ingredients e.g., fungicides with similar or complementary fungicidal activity or other beneficially acting materials such as insecticides may be applied together with the P14 or P14 analogue proteins in order to increase their spectrum of activity or agricultural utility.
  • P14a protein 20 ⁇ g/ml water Buffer 10 mM sodium phosphate pH 7.0
  • the fungicidal compositions of this application are desirably applied to plants that are attacked by pathogens at a rate ranging from 100 to 1000 1/ha. It may be desired with many plants to repeat the treatment after several weeks if necessary to provide the desired protection from the pathogens present on the plant. Moreover, as a preventive measure, plants that are susceptible to pathogenic attack may be sprayed with the fungicidal compositions of this application prior to the first sign of the presence of pathogen or of the infection of the plant by pathogen.
  • the DNA sequences according to the present invention can also be inserted into the genome of a plant that is vulnerable to a fungal disease.
  • Any suitable method may be employed for such incorporation of the P14 and P14 analogue protein-coding DNA sequences into a host plant cell genome, such as for example, via Ti plasmid of Agrobacterium tumefaciens, electroporation, electrotransformation, ballistic gun, micro-injection, viral infection or the use of chemicals that induce or increase free DNA uptake, and the like.
  • Such procedures and the use of such in the transformation of plants are well known to one skilled in the art; cf. Genetically Engineering Plants for Crop Improvement, Gasser, C.S.
  • the DNA sequence encoding the P14 and P14 analogue proteins will be associated with appropriate regulatory sequences, such as for example, 5' promoter and 3' regulatory (e.g. terminator and poly A sequences) sequences which are functional in plants, and the whole will be incorporated into a vector.
  • the promoter may express the DNA constitutively or differentially.
  • promoters differentially regulating DNA expression are promoters inducible by disease vectors, e.g. so-called wound inducible promoters.
  • Suitable promoters include the known plant viral promoters such as CaMV 35S and the promoter associated with the P14 gene.
  • the fungicidal compositions of this application are suitably used to combat fungal diseases including rust fungi and powdery mildews in various plants.
  • fungal diseases including rust fungi and powdery mildews
  • the following is a list of fungi which may be treated using the protein products of the invention. The list further identifies crop plants which are effected by these fungi.
  • Fungicidal activity is also demonstrated against Phytophthora infestans and Phytophthora megasperma.
  • the proteins and compositions may also be used in pharmaceutical and veterinary applications.
  • pharmaceutically or veterinarily acceptable components are used in the fungicidal compositions.
  • FEATURES from 1 to 72 bp signal peptide from 73 to 477 bp mature peptide
  • MOLECULE TYPE genomic DNA
  • FEATURES from 1 to 72 bp signal peptide from 73 to 477 bp mature peptide
  • MOLECULE TYPE genomic DNA
  • FEATURES from 1 to 75 bp signal peptide from 76 to 480 bp mature peptide
  • MOLECULE TYPE genomic DNA
  • FEATURES from 1 to 72 bp signal peptide from 73 to 450 bp mature peptide
  • MOLECULE TYPE genomic DNA
  • FEATURES from 1 to 72 bp signal peptide from 73 to 477 bp mature peptide

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Abstract

On a identifié une famille de protéines P14 liées aux agents pathogènes de la tomate, et on a isolé des gènes codant pour cinq nouvelles protéines, P14a, P14b, P14d, P14e et P14f. On a constaté que les protéines codées par ces gènes ont une activité fongicide puissante. On décrit des méthodes de lutte contre les maladies fongiques, ainsi que des compositions fongicides dans lesquelles une protéine P14, de préférence P14e, P14b, P14c, P14d, P14e et P14f, ou l'un de leurs analogues actifs du point de vue fongicide, est utilisé à titre d'ingrédient actif. Les protéines P14 peuvent être obtenues à partir de matière végétale, mais les protéines et les analogues sont de préférence préparées par la technique de l'ADN de recombinaison. On décrit des séquences codant pour les protéines P14, des analogues et des vecteurs contenant les séquences d'ADN ainsi que des cellules hôtes transformées par les séquences d'ADN; on décrit également des procédés de production de la protéine par la culture des cellules hôtes transformées. L'invention concerne également des cellules de plantes et des plantes transformées auxquelles on a conféré une résistance aux maladies fongiques.
PCT/EP1992/001063 1991-05-15 1992-05-14 Proteines intervenant dans la pathogenese des plantes WO1992020800A1 (fr)

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GB919110544A GB9110544D0 (en) 1991-05-15 1991-05-15 Improvements in or relating to organic compounds

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Cited By (9)

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US6121436A (en) * 1996-12-13 2000-09-19 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
DE102008014041A1 (de) 2008-03-13 2009-09-17 Leibniz-Institut für Pflanzengenetik Und Kulturpflanzenforschung (IPK) Verfahren zur Erzeugung einer Breitband-Resistenz gegenüber Pilzen in transgenen Pflanzen
WO2013050593A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes présentant une résistance accrue à des pathogènes
WO2013050318A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue à des pathogènes
WO2013050611A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes présentant une résistance accrue à des pathogènes
WO2013053711A1 (fr) 2011-10-10 2013-04-18 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue aux pathogènes
WO2013053686A1 (fr) 2011-10-10 2013-04-18 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue à des pathogènes
WO2016069623A1 (fr) * 2014-10-27 2016-05-06 Academia Sinica Peptides de signalisation de défense des plantes et leurs applications
WO2016130020A1 (fr) 2015-02-13 2016-08-18 Bioforsk - Norwegian Institute For Agricultural And Environmental Research Gènes de résistance d'une plante

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US6121436A (en) * 1996-12-13 2000-09-19 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
US6316407B1 (en) 1996-12-13 2001-11-13 Monsanto Company Antifungal polypeptide from alfalfa and methods for controlling plant pathogenic fungi
US6916970B2 (en) 1996-12-13 2005-07-12 Monsanto Technology, Llc Transgenic plants comprising antifungal polypeptides from alfalfa and methods for controlling plant pathogenic fungi
DE102008014041A1 (de) 2008-03-13 2009-09-17 Leibniz-Institut für Pflanzengenetik Und Kulturpflanzenforschung (IPK) Verfahren zur Erzeugung einer Breitband-Resistenz gegenüber Pilzen in transgenen Pflanzen
WO2013050593A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes présentant une résistance accrue à des pathogènes
WO2013050318A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue à des pathogènes
WO2013050611A1 (fr) 2011-10-07 2013-04-11 Basf Plant Science Company Gmbh Procédé de production de plantes présentant une résistance accrue à des pathogènes
WO2013053711A1 (fr) 2011-10-10 2013-04-18 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue aux pathogènes
WO2013053686A1 (fr) 2011-10-10 2013-04-18 Basf Plant Science Company Gmbh Procédé de production de plantes ayant une résistance accrue à des pathogènes
TWI640540B (zh) * 2014-10-27 2018-11-11 中央研究院 植物防禦訊息胜肽及其應用
CN107106631A (zh) * 2014-10-27 2017-08-29 中央研究院 植物防御讯息胜肽及其应用
US20170332645A1 (en) * 2014-10-27 2017-11-23 Academia Sinica Plant defense signaling peptides and applications thereof
TWI610942B (zh) * 2014-10-27 2018-01-11 中央研究院 植物防禦訊息胜肽及其應用
EP3212209A4 (fr) * 2014-10-27 2018-10-03 Academia Sinica Peptides de signalisation de défense des plantes et leurs applications
WO2016069623A1 (fr) * 2014-10-27 2016-05-06 Academia Sinica Peptides de signalisation de défense des plantes et leurs applications
US10306895B2 (en) 2014-10-27 2019-06-04 Academia Sinica Plant defense signaling peptides and applications thereof
TWI686408B (zh) * 2014-10-27 2020-03-01 中央研究院 植物防禦訊息胜肽及其應用
AU2015339463B2 (en) * 2014-10-27 2021-05-27 Academia Sinica Plant defense signaling peptides and applications thereof
CN107106631B (zh) * 2014-10-27 2022-04-22 台湾地区“中央研究院” 植物防御讯息胜肽及其应用
US11363822B2 (en) 2014-10-27 2022-06-21 Academia Sinica Plant defense signaling peptides and applications thereof
WO2016130020A1 (fr) 2015-02-13 2016-08-18 Bioforsk - Norwegian Institute For Agricultural And Environmental Research Gènes de résistance d'une plante

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