EP1078089A2 - Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes - Google Patents

Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes

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Publication number
EP1078089A2
EP1078089A2 EP99950352A EP99950352A EP1078089A2 EP 1078089 A2 EP1078089 A2 EP 1078089A2 EP 99950352 A EP99950352 A EP 99950352A EP 99950352 A EP99950352 A EP 99950352A EP 1078089 A2 EP1078089 A2 EP 1078089A2
Authority
EP
European Patent Office
Prior art keywords
plant
plants
increased
tolerance
use according
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99950352A
Other languages
German (de)
English (en)
Inventor
Wolfgang Rohde
Dirk PRÜFER
Eckhard Tacke
Peter Pasemann
Francesco Salamini
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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 Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority to EP99950352A priority Critical patent/EP1078089A2/fr
Publication of EP1078089A2 publication Critical patent/EP1078089A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/8273—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 drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the invention relates to the use of nucleic acids encoding a (poly) peptide with an intrinsic affinity for plasmodesms, for the production of plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperatures (cold , Heat) and corresponding procedures.
  • a plant, a plant tissue or a plant cell is advantageously transfected with the nucleic acid.
  • the nucleic acid preferably encodes a virus-encoded transport protein, which in a particularly preferred embodiment is a derivative of the pr17 protein with a hydrophilic N-terminal extension.
  • a goal of classic plant breeding is the creation of high-yielding varieties that have an increased tolerance to environmental factors or are resistant to stress factors.
  • stress factors can be both biotic (insects, viruses, fungi, etc.) and abiotic (extreme temperatures, salt, drought).
  • biotic insects, viruses, fungi, etc.
  • abiotic extreme temperatures, salt, drought
  • wild plants in stress-dominated locations have adapted to the extreme living conditions, drought, heat or salinity of the soil limit the possibilities for cultivating crops in such areas.
  • agriculture in other locations also suffers major losses due to abiotic stress, as the example of the drought in 1983 in the USA has shown: Almost half of the total corn harvest and one third of the expected soybean yield were destroyed by the continuing drought. All abiotic stress conditions mentioned have in common that they are intracellular
  • Proteins and metabolites of plant metabolism such as sugar alcohols,
  • Proline or glycine betaine as accumulating as a result of abiotic stress
  • glycine betaine Among the plant metabolites identified as anti-stress factors or osmoprotectors is glycine betaine, the effectiveness of which has been demonstrated, for example, in maize (Saneoka, (1995) Plant Physiol. 107: 631-638). Betaine aldehyde dehydrogenase (BADH) is involved in the synthesis of glycine betaine in plant chloroplasts (Rhodes, (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 357-384). Transgenic tobacco plants expressing a bacterial BADH (Holmstrom, (1994) Plant. J.
  • the invention thus relates to the use of a nucleic acid which encodes a (poly) peptide with an intrinsic affinity for plasmodesms for the production of plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperatures; i.e. Cold and / or heat.
  • a plant, a plant tissue or a plant cell is usually transfected with such a nucleic acid by conventional methods.
  • All higher plants are characterized by the fact that they are capable of photosynthesizing sugars and their derivatives, which, as mentioned above, can serve as osmoprotectors under stress conditions.
  • the intracellular Concentration of the sugar and sugar derivatives - especially in the leaves to protect the photosynthesis-active chloroplasts - was solved according to the invention in a surprisingly simple manner by the measure identified above.
  • this measure is technically simple for the person skilled in the art, it is particularly advantageous that, according to the invention, the stress resistance can be increased with a mechanism which is valid in many and possibly even all plants.
  • the plant tolerance to both abiotic and biotic factors could be increased with the method according to the invention.
  • the term "increased salt concentration” refers to salt concentrations in the soil, which lead to an increased ion concentration in the plant, which then leads to reduced growth.
  • the absolute salt concentrations in the soil, which are to be regarded as increased, are different for different plants, but can be determined by a person skilled in the art using conventional methods, e.g. based on the disclosure content of Greenway and Munns (1980) Ann. Rev. Plant Physiol. 31: 149-190.
  • nucleic acid encoding a (poly) peptide with an intrinsic affinity for plasmodesms in plant cells this is linked to regulatory sequences which ensure transcription in plant cells.
  • regulatory sequences which ensure transcription in plant cells.
  • promoters include promoters in particular.
  • any promoter active in plant cells can be used for the expression.
  • the promoter can be selected so that the expression is constitutive or only in a certain tissue, at a certain time in plant development or at a time determined by external influences.
  • the promoter can be homologous or heterologous to the plant.
  • Useful promoters are, for example, the 35S RNA promoter from the Cauliflower Mosaic Virus (CaMV) and the ubiquitin promoter from maize for constitutive expression.
  • a transcription termination sequence is usually present which serves to correctly terminate the transcription and can also be used to add a poly-A tail to the transcript which has a function in the stabilization the transcripts are attached.
  • Octopin synthase genes from agrobacteria have been described in the literature (cf. Gielen, EMBO J. 8 (1989), 23-29) and can be exchanged as desired. In a preferred one
  • the promoter is the 35S CaMV promoter.
  • the plant which is used or produced in one of the methods or uses according to the invention can contain further recombinant DNA molecules which, for. B. can be used for crop protection or quality improvements of the plant or its harvest products.
  • plant protection measures are: (i) herbicide tolerance (DE-A-3701623; Stalker (1988) Science 242, 419), (ii) insect resistance (Vaek (1987) Plant Cell 5, 159-169), (iii) virus resistance (Powell (1986) Science 232, 738-743) and (vi) ozone resistance (Van Camp (1994) BioTech. 12, 165-168).
  • quality increases are: (i) increase in the shelf life of fruit (Oeller (1991) Science 254, 437-439), (ii) increase in starch production in potato tubers (Stark (1992) Science 242, 419), (iii) change in Starch (Visser (1991) Mol. Gen. Genet. 225, 289-296) and lipid composition (Voelker (1992) Science 257, 72-74) and (iv) production of non-plant polymers (Porier (1992) Science 256, 520- 523).
  • transgenic plants The basic requirement for the production of transgenic plants is the availability of suitable transformation systems.
  • Several methods are currently available for transformation.
  • the most frequently used method for the transformation of dicotyledonous plants is the Agrobacterium -mediated gene transfer.
  • the natural ability of the soil bacteria to exploit genetic material in the plant genome is used.
  • Other suitable methods are, for example, protoplast transformation by polyethylene glycol-induced DNA uptake, electroporation, sonication or microinjection, and the transformation of intact cells or tissue by micro- or macroinjection into tissue or embryos, tissue electroporation, incubation of dry embryos in DNA-containing solution, vacuum infiltration of seeds and organic Gene transfer (for an overview: see, for example, Potrykus, Physiol. Plant (1990), 269-273 and Christou (1996) Trends in Plant Science 1, 423-431).
  • transgenic plant cells can be regenerated into whole plants using techniques known to those skilled in the art.
  • Plants are transgenic or not.
  • the transgenic plants can act principally plants of any plant species, ie both monocot and dicot plants.
  • a selectable marker is integrated into the plant genome in a conventional manner, which enables the identification of transgenic cells. Currently, selections are being transformed
  • Plant cells primarily used genes that have a herbicide or
  • Suitable resistance genes are, for example, the bar-
  • Phosphinothricin mediates (De Block (1987) EMBO J. 6, 2513-2518) or the nptll-
  • Another method takes advantage of the ability of a deaminase
  • Selection markers do not necessarily have to be present in the vector which contains the recombinant DNA molecule, but can also be co-transformed with them
  • This option is useful, for example, if no physical coupling of the marker gene and the information to be transmitted is desired.
  • a plant is also regenerated from the transfected plant cell.
  • the plants can be regenerated by customary methods which are familiar to the person skilled in the art.
  • TP virus-coded transport proteins
  • the virus-encoded transport protein is the potato leaf roll virus (PLRV) transport protein pr17 or a derivative thereof.
  • PLRV potato leaf roll virus
  • the TP of the potato leaf roll virus (potato leafroll virus, PLRV) and the crop plant potato were chosen as the model system.
  • the PLRV-TP which is a feature of a particularly preferred embodiment of the method according to the invention, is a 17 kDa protein (pr17) which is responsible for the transfer of the genomic RNA from cell to cell. It is encoded by the open reading frame (ORF) ORF4; this gene is located within ORF3, the gene for the viral capsid protein CP, but in a different reading frame.
  • the protein has an amino-terminal domain for the formation of homopolymers (Tacke (1993), Virology 197: 274-282.) And a carboxy-terminal domain for binding single-stranded nucleic acids (Tacke (1991), J. Gen. Virol. 72: 2035-2038).
  • This protein which is phosphorylated in planta (Tacke (1993), op. Cit; Sokolova (1997), FEBS Lett. 400: 201-205), is expressed seven times more strongly than the viral coat protein gene (Tacke (1990), J. Gen Virol. 71: 2265-2272).
  • the pr17 is predominantly due to the plasmodesms between the sieve element and the escort cell of the
  • Phloems localized (Schmitz (1997), Virology 235: 311-322), to which the virus is limited during its propagation in the plant.
  • Expression of a mutated pr17 protein in transgenic potato plants causes broad-spectrum resistance to the most important potato viruses (Tacke (1996), Nature Biotechnology 14: 1597-1601).
  • Glucose and sugar derivatives such as starch.
  • the derivative is a pr17 protein with a hydrophilic N-terminal extension.
  • the polylinker (multiple cloning site; MCS) of the bluescript vector was fused translationally to the ⁇ ′ end of the pr17-WT gene, and both the first two AUG translation codons of pr17 were mutated into ACG codons by targeted mutagenesis AUG translation start codon inserted into the polylinker sequence ( Figure 1).
  • this gene was under the transcriptional control of the 35S promoter and terminator of the cauliflower mosaic virus (CaMV) in the vector pRT103 (Töpfer (1987), Nucleic Acids Res. 15: 5890) and these
  • the hydrophilic extension comprises the amino acid sequence MAELGSGSELHRGGGRSRTS.
  • the plant, the plant tissue or the plant cells come from the potato, from tobacco, from cereals or vegetables or are potatoes, tobacco plants, cereal plants or vegetable plants.
  • increasing the tolerance of the plants to fungal infections is one of the tolerance to infections with Phytophtora infestans.
  • transgenic lines obtained with the method according to the invention are also distinguished by a statistically significant tolerance to Phytophthora infestans, the causative agent of late blight.
  • the invention further relates to methods for producing plants or parts thereof with increased tolerance to drought and / or fungal infections and / or increased salt concentrations and / or extreme temperature (heat, cold), wherein (a) a plant, plant tissue or plant cell with one
  • step (c) following step (b), further plants or plant cells from the plant obtained in (b).
  • the (poly) peptide is a virus-encoded transport protein.
  • the virus-encoded transport protein is the potato leaf roll virus (PLRV) transport protein pr17 or a derivative thereof.
  • PLRV potato leaf roll virus
  • the derivative is a pr17 protein with a hydrophilic N-terminal extension.
  • the hydrophilic extension comprises the amino acid sequence MAELGSGSELHRGGGRSRTS.
  • the plant, the plant tissue or the plant cells come from the potato, from tobacco, from cereals or vegetables or are potatoes, tobacco plants, cereal plants or vegetable plants.
  • increasing the tolerance of the plants to fungal infections is one of the tolerance to infections with Phytophtora infestans.
  • a modification at the 5 'end of the pr17 gene was achieved by translational fusion of the multiple cloning site of the Bluescript vector, introduction of an optimized translation initiation codon and mutation of the two pr17-WT AUG initiation codons to ACG (FIG. 1).
  • This change results in the expression of a derivative (pr17-N) of the pr17-WT protein with a hydrophilic extension by the sequence MAELGSGSELHRGGGRSRTS at the amino terminus (Tacke (1996), op. Cit; Figure 2).
  • the preparation of the plasmid p17N is described in Schmitz (1996), Nucleic Acids Res. 24: 257-263 (referred to there as p17 / NIII).
  • Kanamycin sulfate Shoots formed after 6-8 weeks and became
  • Necrosis and bacterial rot can be avoided.
  • the leaf disks are placed in irrigation boxes (Gieffers et al., Op. Cit.)
  • Filter paper laid out with the bottom up.
  • the constant water film on the filter paper provides the leaf disks with sufficient water.
  • a drop with a sporangia suspension 200 sporangia / 20 ⁇ l is placed on the
  • the inoculated leaf disks are kept under a permanent temperature of 10 ° C and the above. Incubated light conditions. The infectious zoospores hatch under these conditions. After about 6 days, the first sporangia are formed, the
  • Infestation is assessed after 8 to 10 days.
  • Leaf tissue decay is estimated as a percentage.
  • the level of infestation determines the quantitative level of resistance.
  • Example 5 Induced tolerance to drought as an example of increased tolerance to abiotic stress
  • transgenic plants of the transgenic lines L4, L6, L7 and L8 and the starting variety Linda were kept under dry stress in the 6-8-leaf stage for 8 weeks, the plants being watered once after 3 and after 6 weeks.
  • the transgenic plants were shown in two independent experiments a significantly increased tolerance to water stress, as in Table 1 and
  • Example 6 Induced tolerance to Phytophthora infestans as an example of increased tolerance to fungal attack
  • Example 7 Induced tolerance to salt as another example of increased tolerance to abiotic stress

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

Abstract

L'invention concerne l'utilisation d'acides nucléiques, lesquels codent pour un (poly)peptide avec une affinité intrinsèque pour des plasmodesmes, en vue de la production de végétaux ou de parties de ceux-ci présentant une tolérance élevée à la sécheresse et/ou aux infections fongiques et/ou aux concentrations salines élevées et/ou aux températures extrêmes (chaleur, froid), ainsi que des procédés correspondants. Avantageusement, on transfecte à cet effet un végétal, un tissu végétal ou une cellule végétale avec l'acide nucléique. De préférence, l'acide nucléique code pour une protéine de transport codée par un virus, laquelle est, dans un mode de réalisation particulièrement préféré, un dérivé de la protéine pr17 avec un prolongement N-terminal hydrophile.
EP99950352A 1998-05-13 1999-05-12 Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes Withdrawn EP1078089A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99950352A EP1078089A2 (fr) 1998-05-13 1999-05-12 Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP98108726 1998-05-13
EP98108726 1998-05-13
EP99950352A EP1078089A2 (fr) 1998-05-13 1999-05-12 Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes
PCT/EP1999/003291 WO1999058653A2 (fr) 1998-05-13 1999-05-12 Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes

Publications (1)

Publication Number Publication Date
EP1078089A2 true EP1078089A2 (fr) 2001-02-28

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EP99950352A Withdrawn EP1078089A2 (fr) 1998-05-13 1999-05-12 Procede pour la production de vegetaux a tolerance elevee a la secheresse et/ou aux attaques fongiques et/ou a des concentrations salines elevees et/ou a des temperatures extremes par l'expression de proteines localisees dans des plasmodesmes

Country Status (4)

Country Link
EP (1) EP1078089A2 (fr)
AU (1) AU4260999A (fr)
CA (1) CA2328131A1 (fr)
WO (1) WO1999058653A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090222949A1 (en) * 2005-06-21 2009-09-03 Ramot At Tel Aviv University Ltd. Constructs and Methods for Generating Plants Exhibiting Altered Plasmodesmatal Conductance
GB0613100D0 (en) * 2006-06-30 2006-08-09 Plant Bioscience Ltd Compositions and methods relating to cellular targeting

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU6953496A (en) * 1995-08-15 1997-03-12 Regents Of The University Of California, The Regulation of plant development and physiology through plasmodesmatal macromolecular transport of proteins and oligonucleotides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9958653A3 *

Also Published As

Publication number Publication date
AU4260999A (en) 1999-11-29
WO1999058653A3 (fr) 2000-03-09
CA2328131A1 (fr) 1999-11-18
WO1999058653A2 (fr) 1999-11-18

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