WO2001064927B1 - Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria - Google Patents

Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria

Info

Publication number
WO2001064927B1
WO2001064927B1 PCT/US2001/006287 US0106287W WO0164927B1 WO 2001064927 B1 WO2001064927 B1 WO 2001064927B1 US 0106287 W US0106287 W US 0106287W WO 0164927 B1 WO0164927 B1 WO 0164927B1
Authority
WO
WIPO (PCT)
Prior art keywords
vector
plant
antimicrobial peptide
plastid
target
Prior art date
Application number
PCT/US2001/006287
Other languages
French (fr)
Other versions
WO2001064927A1 (en
Inventor
Henry Daniell
Original Assignee
Univ Auburn
Univ Central Florida
Henry Daniell
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Univ Auburn, Univ Central Florida, Henry Daniell filed Critical Univ Auburn
Priority to AU2001241813A priority Critical patent/AU2001241813A1/en
Priority to NZ521660A priority patent/NZ521660A/en
Priority to CA002401957A priority patent/CA2401957A1/en
Priority to EP01913116A priority patent/EP1263976A4/en
Publication of WO2001064927A1 publication Critical patent/WO2001064927A1/en
Publication of WO2001064927B1 publication Critical patent/WO2001064927B1/en

Links

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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation
    • 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
    • C12N15/8281—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 bacterial 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
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

This invention provides a novel method to confer disease resistance to plants. Plant plastids are transformed using a plastid vector which contains heterologous DNA sequences coding for a cytotoxic antimicrobial peptide. Transgenic plants are capable of fighting off phytopathogenic bacterial infection.

Claims

AMENDED CLAIMS
[received by the International Bureau on 23 July 2001 (23.07.01); original claims 1-18 replaced by amended claims 1-20 (2 pages)]
1 . A stable plastid transformation and expression vector which comprises an expression cassette comprising, as operably linked components in the 5' to the 3' direction of translation, a promoter operative in said plastid, a selectable marker sequence, a heterologous
DNA sequence coding for cytotoxic antimicrobial peptide (AMP), transcription termination functional in said plastid, and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome, whereby stable integration of the heterologous coding sequence into the plastid genome of the target plant is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome.
2. A vector of claim 1, wherein the vector further comprises a ribosome binding site (RBS) and a 5' untranslated region (5'UTR).
3. A vector of claim 1 , wherein the plastid is selected from the group consisting of chloropiasts, chromoplasts, amyloplasts, proplastide, leucoplasts and etioplasts.
4. A vector of claim 1 , wherein the antimicrobial peptide is a cationic amphiphipathic alpha-helix molecule which has affinity for negatively charged phospholipides located in an outer membrane of a target microbe and which is functional to form aggregates that disrupt and lyse the microbe membrane of the target microbe, and in preventing spreading of infection by the microbe.
5. A vector of claim 1, wherein the antimicrobial peptide is selected from the groups of defensins, PGLA (frog skin), cecropins, apidaeciπs, melittin, bombinin and magainins.
6. A vector of claim I , wherein the antimicrobial peptide is againin 1 or an analogue thereof. 7. A vector of claim 1 , wherein the antimicrobial peptide is magainin TI or an analogue thereof.
8. The antimicrobial peptide of claim 7, wherein the analogue of magainin JI is MSI- 99.
9. A vector of claim 1, wherein the selectable marker sequence is an antibiotic-free selectable marker.
10. A vector of claim 1 which is a universal vector competent for stably transforming a plastid genome of different plant species wherein flanking DNA sequences are homologous to a spacer sequence of a target plastid genome and the sequences are conserved in the plastid genome of different plant species.
- 19 -
AMENDED SHEET Afr
1 1. A stably transformed plant which comprises plastid stably transformed with the vector of claim 1, and the progeny or subsequent generations thereof, including seeds.
12. A stably transformed plant of claim 11 which is a solanaceous plant.
13. A stably transformed plant of claim 1 1 which is a monocotyledonous or dicotyledonous plant.
14. A stably transformed plant of claim 13 which is maize, rice, grass, rye, barley, oat, wheat, soybean, peanut, grape, potato, sweet potato, pea, canola, tobacco, tomato or cotton.
15. A stably transformed plant of claim 1 1 which is edible for mammals and humans.
16. A stably transformed plant of claim 1 1 in which all the plastids are uniformly transformed.
17. The stably transformed plant and progeny of claim 1 1 , wherein subsequent generations are capable of enhanced levels of expression of the cytotoxic antimicrobial peptide.
18. A stably trans formed plant of claim 1 1 in which transgenic plants germinated in the absence of antibiotic selectable marker sequence, like spectinomycin. 1 . A method for stably transforming a target plant to control a phytopathogenic bacteria which comprises transforming plastids of said target plant with a transformation and expression vector of claim 1, selecting transformed plant cells, and allowing the transformed plant cells to grow.
20. A method of claim 19, wherein said vector further comprises a ribosome binding site (RBS) and a 5' untranslated region (5'UT ).
- 20 -
AMENDED SHEET (ARTICLE 19,
PCT/US2001/006287 2000-02-29 2001-02-28 Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria WO2001064927A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2001241813A AU2001241813A1 (en) 2000-02-29 2001-02-28 Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria
NZ521660A NZ521660A (en) 2000-02-29 2001-02-28 Plastid expression constructs useful for genetic engineering of plant cells and which provide for enhanced expression of foreign peptides
CA002401957A CA2401957A1 (en) 2000-02-29 2001-02-28 Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria
EP01913116A EP1263976A4 (en) 2000-02-29 2001-02-28 Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18566200P 2000-02-29 2000-02-29
US60/185,662 2000-02-29

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/807,720 A-371-Of-International US20020162135A1 (en) 2000-02-29 2001-02-28 Expression of antimicrobial peptide via the plastid genome to control phytopathogenic bacteria
US10/671,822 Continuation US20040093636A1 (en) 2000-02-29 2004-01-20 Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria

Publications (2)

Publication Number Publication Date
WO2001064927A1 WO2001064927A1 (en) 2001-09-07
WO2001064927B1 true WO2001064927B1 (en) 2002-02-07

Family

ID=22681933

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/006287 WO2001064927A1 (en) 2000-02-29 2001-02-28 Expression of an antimicrobial peptide via the plastid genome to control phytopathogenic bacteria

Country Status (5)

Country Link
EP (1) EP1263976A4 (en)
AU (1) AU2001241813A1 (en)
CA (1) CA2401957A1 (en)
NZ (1) NZ521660A (en)
WO (1) WO2001064927A1 (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100251425A9 (en) 1998-05-15 2010-09-30 University Of Central Florida Expression of human interferon in transgenic chloroplasts
CA2491639A1 (en) 2002-07-03 2004-01-15 University Of Central Florida Expression of the human igf-1 in transgenic plastids
EP1885173A4 (en) 2005-05-27 2009-02-18 Univ Central Florida Chloroplasts engineered to express pharmaceutical proteins
AU2008232543B2 (en) 2007-03-30 2013-01-17 University Of Central Florida Research Foundation, Inc. Chloroplasts engineered to express pharmaceutical proteins in edible plants
US10689633B2 (en) 2008-02-29 2020-06-23 The Trustees Of The University Of Pennsylvania Expression of β-mannanase in chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis
US9724400B2 (en) 2009-11-09 2017-08-08 The Trustees Of The University Of Pennsylvania Administration of plant expressed oral tolerance agents
EP2770991B9 (en) 2011-10-24 2017-01-25 The Trustees Of The University Of Pennsylvania Orally-administered plastid expressed cholera toxin b subunit-exendin 4 for use in the treatment of type 2 diabetes

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2030779A1 (en) * 1989-04-11 1990-10-12 Huw M. Davies Use of animal-derived anti-microbial peptides for control of plant pathogens
US5877402A (en) * 1990-05-01 1999-03-02 Rutgers, The State University Of New Jersey DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein
US5451513A (en) * 1990-05-01 1995-09-19 The State University of New Jersey Rutgers Method for stably transforming plastids of multicellular plants
AU723005B2 (en) * 1996-08-14 2000-08-17 Novartis Ag Peptide with inhibitory activity towards plant pathogenic fungi
US6235973B1 (en) * 1997-07-31 2001-05-22 Sanford Scientific, Inc. Expression of magainin and PGL classes of antimicrobial peptide genes in plants, and their use in creating resistance to multiple plant pathogens
EP2319933B1 (en) * 1997-08-07 2014-10-15 Auburn University Universal chloroplast integration and expression vectors, transformed plants and products thereof

Also Published As

Publication number Publication date
CA2401957A1 (en) 2001-09-07
EP1263976A1 (en) 2002-12-11
WO2001064927A1 (en) 2001-09-07
AU2001241813A1 (en) 2001-09-12
NZ521660A (en) 2004-05-28
EP1263976A4 (en) 2004-12-01

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