EP0537163A1 - Transformation de plantes a l'aide de sequences de genes de virus de plantes non structurelles - Google Patents

Transformation de plantes a l'aide de sequences de genes de virus de plantes non structurelles

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Publication number
EP0537163A1
EP0537163A1 EP91908562A EP91908562A EP0537163A1 EP 0537163 A1 EP0537163 A1 EP 0537163A1 EP 91908562 A EP91908562 A EP 91908562A EP 91908562 A EP91908562 A EP 91908562A EP 0537163 A1 EP0537163 A1 EP 0537163A1
Authority
EP
European Patent Office
Prior art keywords
plants
rna
tmv
sequence
virus
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Withdrawn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
EP91908562A
Other languages
German (de)
English (en)
Other versions
EP0537163A4 (en
Inventor
Milton Zaitlin
Daniel Golemboski
George Lomonossoff
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.)
Cornell Research Foundation Inc
Original Assignee
Cornell Research Foundation Inc
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Filing date
Publication date
Application filed by Cornell Research Foundation Inc filed Critical Cornell Research Foundation Inc
Publication of EP0537163A1 publication Critical patent/EP0537163A1/fr
Publication of EP0537163A4 publication Critical patent/EP0537163A4/en
Withdrawn legal-status Critical Current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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/8283—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 virus 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • N. D. Young et al reported [see J. Cell Science Supplement 7:277 (1987)] that the ⁇ '-proximal region of the genomic RNA which encodes two coinitiated proteins, the 126K and 183K proteins, is considered to be components of the replicase.
  • the 183K protein is generated by a read-through of the UAG stop codon of the 126K protein.
  • the other two proteins (with known functions), the 30K protein and the coat protein are each synthesized from separate subgenomic mRNAs on which each gene is 5' proximal.
  • Support for its function as a mRNA and as a subgenomic RNA is derived from the observation that it is found on polyribosomes and that there is a double-stranded RNA of a size corresponding to the double- stranded version of the h subgenomic RNA [see A. Zelcer et al, Virology 113:417 (1981) and P. Palukaitis et al, Virology 131 :533 (1983)].
  • the following sequence of the region of the TMV genome containing the readthrough portion of the 183K protein gene is:
  • the 54K protein has not been found in infected tissues.
  • antibodies to a ⁇ -galactosidase fusion protein for 432 amino acids specific to the read-through of the 126K protein expressed in Escherichia coli were prepared, the 54K protein in protoplast extracts could not be detected by either immunoprecipitation or Western blotting under conditions where the antibody would detect the 183K protein [see T. Saito et al, Mol. Gen. Genet. 205:82 (1986)].
  • the 54K protein has not been detectable in Western blots using antiserum made to the whole protein [see G. J.
  • a novel aspect of the present invention is the conveyance of viral resistance to a plant which has previously undergone transformation of its normal genome with a portion of the replicase region of a viral genome, in its "sense" orientation.
  • Figure 1 depicts plant expression vectors containing the TMV 54K coding sequence inserted between the CaMV 35S promoter and the nopaline synthase polyadenylation site.
  • this figure shows plasmids which were derived by insertion of the TMV cDNA into either the Xholl site or the Smal site in the polylinker region of pMON316.
  • the numbers in these vectors refer to nucleotides in the TMV genome.
  • the NPTII gene confers a selectable kanamycin resistance marker to transformed plants.
  • TMV strain Ui was purified from infected N. tabacum cv. Turkish Samsun plants as described by A. Asselin et al [see Virology 91 :173 (1978)]. Virus RNA was isolated by phenol extraction and ethanol precipitation. N. tabacum cv. Xanthi nn was used as a TMV-susceptible, systemic host, and N. tabacum cv. Xanthi nc as a local lesion host. Plants were maintained in a greenhouse or in a growth chamber with a 14 hour per 24 hour light cycle and at 24°C.
  • EXAMPLE II cloning of the 54K ⁇ ene A clone of the TMV 54K gene was obtained by using a 22 base oligonucleotide primer consisting of a Bam HI site linked to the 5' end of a sequence complementary to base residues 4906 to 4923 of the TMV RNA sequence. First strand DNA was synthesized by M-MLV reverse transcriptase and was rendered double stranded by sequential treatment with reverse transcriptase and Klenow relying on loop-back synthesis [see T. Maniatis et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY) (1982)].
  • the presence of an intact open reading frame was verified by insertion of the TMV sequence into a T7 transcription vector; the T7 transcript was synthesized and translated in a reticulocyte lysate system.
  • In vitro translation yielded the desired 54K product which confirmed that the AUG at position 3495 functions as an initiation codon and that the UAA codon at position 4919 functions as the stop codon.
  • the product was verified as the desired 54K protein by immunoprecipitation using 54K antiserum.
  • EXAMPLE III lant ransformation Cut pieces of sterile, TMV susceptible, Nicotiana tabacum cv. Xanthi nn leaves were transformed by the modified Agrobacterium tumefaciens GV3111 containing the TMV 54K coding sequence as described by Horsch [see Science 227:1229 (1985)]. Transformed calli were selected on regeneration medium supplemented with kanamycin at a concentration of 300 ⁇ g/ml. Resistant calli were induced to regenerate shoots and roots, transferred to soil, and maintained in a greenhouse.
  • Genomic DNA was isolated from transformed and untransformed N. tabacum cv Xanthi nn. BamHI digests of the genomic DNA were hybridized to a 32 P- labeled TMV 54K sequence specific probe. Hybridization to a 3.0 kb fragment verified the presence of a full length 54K coding sequence.
  • the 54K sequence insert is 1.44 kb and another 1.59 kb is contributed by flanking vector DNA.
  • the TMV 54K transcripts extracted from transformed plants were also examined by Northern analysis for RNA.
  • the expected size for the chimeric MRNA of 1.6 kb was identified in total RNA from each transgenic plant. Plants containing the integrate plasmid that lacks the 3' nopaline synthase untranslated region and the ti homologous region also synthesize a 1.6 kb transcript. In addition, a larger transcript was synthesized which might result from the lack of the termination sequence usually contributed by the nos 3' sequence. In all plants, a number of smaller unidentified transcripts were also detected. Plants transformed with the vector alone did not produce any transcripts that hybridize with the the 54 sequence probe.
  • the transgenic plants were also analyzed for expression of the TMV 54K protein in accordance with Example IV.
  • a 54K protein could not be detected from the 54K transgenic plants or from protoplasts prepared from 54K transgenic plants or the controls.
  • An antiserum to the 54K protein was made by injecting rabbits with a synthetic polypeptide representing an internal region, specifically amino acid residues 164 to 179, of the 54K protein.
  • An in vitro translation product of the 54K T7 transcript was immunoprecipitable with the antiserum raised against the synthetic polypeptide.
  • total extracts of the transformed and untransformed plants were prepared by homogenizing leaf samples in 50 mM Tris-HCI, pH 7.5, 1% SDS, 10 mM 2-mercaptoethanol buffer; subjected to electrophoresis in a 12.5% SDS-polyacrylamide gel; and transferred to nitrocellulose filter paper. The filter was incubated first with specific antibodies followed by gold and conjugated anti-rabbit antibodies and silver enhancement.
  • EXAMPLE VI inoculation of transformed plants Rl seedlings from self-fertilized transgenic plants were routinely inoculated with either 100 ⁇ g TMV-Ui per ml of 50 mM phosphate buffer, pH 7.2, with CeliteTM added as an abrasive, or TMV-Ui RNA at a concentration of 300 ⁇ g/ml in pH 8.6, 50 mM Tris-phosphate buffer. Two leaves of each plant were inoculated. The volume of the inoculum was not standardized since inoculum concentration is the critical determinant as long as there is sufficient volume for adequate spread. In subsequent experiments, a closely related TMV mutant - mutant b6 as described by F.
  • Progeny seedlings from self-fertilized transgenic plants were also analyzed for inheritability of the resistance phenomenon.
  • R1 generation seeds were germinated on tissue culture medium containing 300 ⁇ g kanamycin per ml. Kanamycin- sensitive seedlings were considered to be those that were chlorotic and did not grow beyond the cotyledon stage.
  • the segregation ratio of the seedlings expressing kanamycin resistance to those susceptible to kanamycin indicates that in each of the original transformants the NPTII gene was integrated at multiple loci.
  • transgenic plants containing a coding sequence portion of a viral genome associated with the replicase region of the virus are resistant to infection with the virus from which the portion was initially obtained.
  • the resistance to viral infection utilizing a replicase related code sequence as described in the present invention is not as "fragile" as coat protein-induced resistance in which resistance breaks down when high concentrations of inoculum are used.
  • complete resistance is observed in plants challenged with high concentrations of virus or viral RNA.
  • the protection mediated by the coat proteins of TMV and A1 MV can be overcome by inoculating with viral RNA
  • the induced resistance according to the present invention utilizing the 54K code sequence remains uncompromised when challenged with viral RNA.
  • vectors which are within the range of substitutes or equivalents are those such as pBIN19, pBI101 , pRokl , pAGS135, pARC12, PGA470, pRAL3940, and pCT1T3, among others.
  • TMV plant virus
  • other plant viruses such as cucumber mosaic, alfalfa mosaic, members of the potexvirus, bromovirus, potyvirus and luteovirus groups which also contain viral replicase regions within their genomes are also encompassed by the present invention, as are the host plants transformed with genetic sequences related to the replicase portions of these viruses.

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

Abstract

Nouveau procédé consistant à transmettre une résistance virale à une cellule hôte de plantes par incorporation dans l'hôte d'un fragment du génome viral associé à la partie de réplicase du génome.
EP19910908562 1990-03-12 1991-03-11 Transformation of plants with non-structural plant virus gene sequences Withdrawn EP0537163A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US49147390A 1990-03-12 1990-03-12
US491473 1990-03-12

Publications (2)

Publication Number Publication Date
EP0537163A1 true EP0537163A1 (fr) 1993-04-21
EP0537163A4 EP0537163A4 (en) 1993-12-08

Family

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Family Applications (1)

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EP19910908562 Withdrawn EP0537163A4 (en) 1990-03-12 1991-03-11 Transformation of plants with non-structural plant virus gene sequences

Country Status (4)

Country Link
EP (1) EP0537163A4 (fr)
JP (2) JPH05508535A (fr)
CA (1) CA2078134A1 (fr)
WO (1) WO1991013542A1 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5596132A (en) 1990-03-12 1997-01-21 Cornell Research Foundation, Inc. Induction of resistance to virus diseases by transformation of plants with a portion of a plant virus genome involving a read-through replicase gene
US5633449A (en) * 1990-03-12 1997-05-27 Cornell Research Foundation, Inc. Induction of resistance to viral diseases in plants
EP0536106A1 (fr) * 1991-10-04 1993-04-07 Monsanto Company Plantes résistantes aux infections par PVX
GB9208545D0 (en) * 1992-04-21 1992-06-03 Gatsby Charitable Foundation T Virus resistant plants
JPH07507457A (ja) * 1992-06-08 1995-08-24 コーネル・リサーチ・ファウンデーション・インコーポレイテッド 植物ウイルスゲノムのレプリカーゼ部分との植物形質転換によるウイルス耐性
EP0578627A1 (fr) * 1992-07-09 1994-01-12 Monsanto Company Plantes résistantes aux virus
US5850023A (en) * 1992-11-30 1998-12-15 Monsanto Company Modified plant viral replicase genes
FR2700235B1 (fr) * 1993-01-14 1995-03-31 Agronomique Inst Nat Rech Plantes transgéniques résistantes aux virus végétaux et procédé d'obtention.
JPH08506489A (ja) * 1993-02-03 1996-07-16 モンサント・カンパニー Ｐｌｒｖ感染に耐性の植物
BR9406949A (pt) * 1993-07-09 1996-08-06 Asgrow Seed Co Genes do virus amarelo infeccioso da alface
CA2202761A1 (fr) 1994-10-18 1996-04-25 Sean Nicholas Chapman Procede de production de proteines chimeres
US6040496A (en) * 1995-06-30 2000-03-21 Novartis Finance Corporation Use of translationally altered RNA to confer resistance to maize dwarf mosaic virus and other monocotyledonous plant viruses
US6284947B1 (en) * 1999-02-25 2001-09-04 Pioneer Hi-Bred International, Inc. Methods of using viral replicase polynucleotides and polypeptides
US6770800B2 (en) 1999-03-12 2004-08-03 Pioneer Hi-Bred International, Inc. Methods of using viral replicase polynucleotides and polypeptides
IL147920A0 (en) * 1999-08-02 2002-08-14 Keygene Nv Method for generating cgmmv resistant plants, genetic constructs, and the obtained cgmmv-resistant plants
ZA200607843B (en) * 2004-02-23 2008-06-25 Israel State Engrafted plants resistant to viral diseases and methods of producing same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989008145A1 (fr) * 1988-02-26 1989-09-08 Biosource Genetics Corporation Transformation chromosomique non nucleaire
WO1990013654A1 (fr) * 1989-05-05 1990-11-15 Biosource Genetics Corporation Sterilite male chez les plantes
EP0421376A1 (fr) * 1989-10-06 1991-04-10 Hoechst Schering AgrEvo GmbH L'ARN multifonctionnelle avec une activité d'automaturation, sa production et utilisation
EP0426195A1 (fr) * 1989-11-03 1991-05-08 S&G Seeds B.V. Améliorations concernant des compositions organiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989008145A1 (fr) * 1988-02-26 1989-09-08 Biosource Genetics Corporation Transformation chromosomique non nucleaire
WO1990013654A1 (fr) * 1989-05-05 1990-11-15 Biosource Genetics Corporation Sterilite male chez les plantes
EP0421376A1 (fr) * 1989-10-06 1991-04-10 Hoechst Schering AgrEvo GmbH L'ARN multifonctionnelle avec une activité d'automaturation, sa production et utilisation
EP0426195A1 (fr) * 1989-11-03 1991-05-08 S&G Seeds B.V. Améliorations concernant des compositions organiques

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
J. CELL. BIOCHEM. SUPPL. vol. 13D, 1989, page 346 YOUNG, M.J., ET AL. 'Barley yellow dwarf virus expression in wheat protoplasts and construction of synthetic genes to interfere with viral replication' *
J. VIROLOGY vol. 61, no. 12, December 1987, pages 3946 - 3949 INOKUCHI, Y., ET AL. 'Interference with viral infection by defective RNA replicase' *
PLANT MOLECULAR BIOLOGY. vol. 11, 1988, DORDRECHT, THE NETHERLANDS. pages 463 - 471 REZAIAN, M.A., ET AL. 'Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 87, August 1990, WASHINGTON US pages 6311 - 6315 GOLEMBOSKI, D.B., ET AL. 'Plants transformed wtih a tobacco mosaic virus nonstructural gene sequence are resistant to the virus' *
See also references of WO9113542A1 *

Also Published As

Publication number Publication date
CA2078134A1 (fr) 1991-09-13
EP0537163A4 (en) 1993-12-08
JPH05508535A (ja) 1993-12-02
WO1991013542A1 (fr) 1991-09-19
JP2002204694A (ja) 2002-07-23

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