Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • 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

• plant defensin is used to denote those proteins having antimicrobial activity, especially antifungal activity or antifungal and antibacterial activity and also having the following characteristic structural features: a cysteine residue at positions 4, 15, 21, 25, 36, 45, 47 and 51; disulphide bridge formation between the cysteines at positions 4 and 51, 15 and 36, 21 and 45 and 25 and 47; an aromatic amino acid residue 4 amino acids upstream from the cysteine at position 15, a glycine residue 2 amino acids upstream from the cysteine at position 15, a glutamic acid residue 7 amino acids upstream from the cysteine at position 36, and a glycine residue 2 amino acids upstream of the cysteine at position 36 wherein the positions of the cysteine residues are defined relative to the amino acid sequence of Rs-AFPl as shown in SEQ ID NO 1, and homologues, active variants and derivatives thereof.
• Amino acids which differ from the basic sequence may be conservatively or non- conservatively substituted.
• a conservative substitution is to be understood to mean that the amino acid is replaced with an amino acid with broadly similar chemical properties.
• conservative substitutions may be made between amino acids with the following groups:
• Suitable homologues may be determined by testing antimicrobial properties of the peptide using routine methods, for example as illustrated hereinafter.
• variants of the invention will comprise antimicrobial proteins with an amino acid sequence with a FASTA opt score (as defined in accordance with FASTA version 3.0t82 November 1, 1997) against any one of the sequences of the antimicrobial proteins of the invention described herein as follows.
• variants of the invention will comprise antimicrobial proteins with an amino acid sequence with a FASTA opt score (as defined in accordance with FASTA version 3.0t82 November 1, 1997) of greater than or equal to 300 against Rs-AFPl or 2.
• amino acid positions mentioned above correspond to the amino acid positions found in the full length amino acid sequence of Rs-AFP2 as shown in SEQ ID NO 3 or to the equivalent positions in defensins from different sources when optimally aligned with the Rs-AFP2 sequence.
• the replacement amino acid residue at position 34 is preferably selected from the group consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine, methionine, cysteine, lysine, histidine or tyrosine residue, more preferably from the group consisting of a valine, leucine, isoleucine, tryptophan, phenylalanine, methionine, lysine or histidine residue and is most preferably selected from the group consisting of a valine, leucine or isoleucine residue.
• the replacement residues at position 38 are preferably selected from the group consisting of a leucine, isoleucine, tryptophan, phenylalanine, cysteine or a valine residue; more preferably from the group consisting of a leucine, tryptophan or phenylalanine residue.
• the replacement residue at position 41 is preferably selected from the group consisting of an isoleucine, tryptophan, phenylalanine, serine, threonine, tyrosine, glutamine, asparagine, lysine, arginine or histidine residue, and is more preferably selected from the group consisting of an isoleucine, tryptophan, phenylalanine, tyrosine, asparagine, lysine, arginine or histidine residue and is most preferably selected from the group consisting of a tryptophan, phenylalanine, lysine, arginine or histidine residue.
• proteins and peptides may be replaced by an alpha-aminobutyric acid group.
• the antimicrobial peptides of the invention are preferably at least six amino acid residues long, more preferably greater than 10 amino acid residues long preferably 12 amino acids long, most preferably 19 amino acids or longer, especially 20 amino acids in length.
• Short peptides will contain at least one modified residue as described above. Where this includes modified residues 37, 39 or 42, the peptide suitably contains more than one such modification.
• Solid compositions may be in the form of granules, or dusting powders wherein the active ingredient is mixed with a finely divided solid diluent, e.g. kaolin, bentonite. kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia. Fuller's earth and gypsum. They may also be in the form of dispersible powders or grains, comprising a wetting agent to facilitate the dispersion of the powder or grains in liquid. Solid compositions in the form of a powder may be applied as foliar dusts.
• a finely divided solid diluent e.g. kaolin, bentonite. kieselguhr, dolomite, calcium carbonate, talc, powdered magnesia. Fuller's earth and gypsum.
• a finely divided solid diluent e.g. kaolin, bentonite. kieselguhr, dolomite,
• the invention further provides a plant having improved resistance to a microbial pathogen and containing recombinant DNA which expresses an antimicrobial peptide or protein according to the invention.
• a plant may be used as a parent in standard plant breeding crosses to develop hybrids and lines having improved microbial resistance.
• the invention further provides a plant having improved resistance to a fungal pathogen and containing recombinant DNA which expresses an antimicrobial peptide or protein according to the invention.
• Boc- ⁇ -alanine was coupled using DCC and DMAP as catalyst, the Boc group was removed with TFA and after careful washing Fmoc-2,4-dimethoxy-4' (carboxymethyloxy)-benzhydrylamine (Rink Linker, Bachem, Laufelfingen, Switzerland) was coupled using the DOC/HOBt method.
• Fmoc-2,4-dimethoxy-4' (carboxymethyloxy)-benzhydrylamine (Rink Linker, Bachem, Laufelfingen, Switzerland) was coupled using the DOC/HOBt method.
• 240 dodecapeptides from AFP2 were synthesized simultaneously using standard Fmoc-chemistry and overnight couplings with DCC/HOBt as coupling method. After coupling of the last amino acid the Fmoc group was removed with 30% piperidine/DMF and the peptides were acetylated with aceticanhydride.
• Coupling step Fmoc-amino acid in NMP (0.4M, 0.25ml), 0.22ml of 0,45M HBTU/HOBt in DMF, and 0.2ml of 2M DIEA in NMP were transferred to the reaction vessel and allowed to react for 30 min. Then the reaction mixture was drained and the coupling procedure was repeated once.

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• Molecular Biology (AREA)
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• Biotechnology (AREA)
• Plant Pathology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Biochemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Microbiology (AREA)
• Biomedical Technology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biophysics (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Public Health (AREA)
• Cell Biology (AREA)
• Physics & Mathematics (AREA)
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• Dentistry (AREA)
• Mycology (AREA)
• Agronomy & Crop Science (AREA)
• Botany (AREA)
• Gastroenterology & Hepatology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Peptides Or Proteins (AREA)
• Agricultural Chemicals And Associated Chemicals (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

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• 1999
  • 1999-08-02 GB GBGB9918155.4A patent/GB9918155D0/en not_active Ceased
• 2000
  • 2000-07-31 WO PCT/GB2000/002941 patent/WO2001009174A2/en not_active Application Discontinuation
  • 2000-07-31 EP EP00953273A patent/EP1206557A2/en not_active Withdrawn
  • 2000-07-31 CA CA002378432A patent/CA2378432A1/en not_active Abandoned
  • 2000-07-31 AU AU65791/00A patent/AU6579100A/en not_active Abandoned
  • 2000-07-31 JP JP2001513980A patent/JP2003507009A/ja active Pending

Non-Patent Citations (1)

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