EP0673423A4 - Virus resistenz hervorgerufen durch eine pflanzentransformation mit einem fragment einer replikase von einem pflanzen virusgenom. - Google Patents

Virus resistenz hervorgerufen durch eine pflanzentransformation mit einem fragment einer replikase von einem pflanzen virusgenom.

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Publication number
EP0673423A4
EP0673423A4 EP19930914378 EP93914378A EP0673423A4 EP 0673423 A4 EP0673423 A4 EP 0673423A4 EP 19930914378 EP19930914378 EP 19930914378 EP 93914378 A EP93914378 A EP 93914378A EP 0673423 A4 EP0673423 A4 EP 0673423A4
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plants
tmv
rna
kda
virus
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EP0673423A1 (de
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Milton Zaitlin
Daniel Golemboski
George Lomonossoff
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Cornell Research Foundation Inc
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Cornell Research Foundation Inc
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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
    • 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

  • Plant virus sequences other than those coding for the viral coat protein have been tested to determine if transformed plants can be made to exhibit resistance to post-transformation viral infection.
  • RNAs from one of three regions tested (5' sequences of RNA 1) of the CMV genome gave a low level of resistance in one transformant line.
  • Other forms of resistance using plant transformations with DNAs prepared from satellite RNAs of plant viruses have been reported, such as the use of the satellite of CMV [see Nature 328:799 (1987)] and the concept of the ribozyme based on sequences from satellite RNAs which possess the capacity to self cleave [see Nature 334:585 (1988)].
  • the invention described herein represents an entirely new type of virus-induced resistance which may be transferred from one plant generation to another.
  • the present invention discloses that transgenic plants containing a coding sequence, taken from all or part of the replicase portion of the viral genome, are resistant to subsequent disease by the virus; although there may be a very benign degree of virus synthesis in the inoculated leaf, the virus has been found not to spread and hence no disease develops.
  • the use of the 54 kDa coding sequence from TMV and a modified cDNA of RNA-2 which encodes one component of the polymerase of cucumber mosaic virus are described as two specific examples of the broader technology according to the present invention.
  • the present invention defines a means for bringing about viral resistance in plants which have been transformed with DNA copies of fragments or segments taken from the replicase portion of the pathogenic virus genome.
  • the present invention defines transformed plants and their seeds which carry a portion of the viral genome which codes for a portion of the replicase genome of the pathogenic virus.
  • transformed plants that contain a portion of the viral replicase gene within their genome are resistant to subsequent viral disease from the virus from which the portion was de ⁇ ved, and these plants may also be resistant to subsequent disease from other closely related viruses.
  • the presence of the 54 kDa sequence prevents the development of local chlorosis or necrosis and any systemic development of symptoms or virus replication associated with TMV infection.
  • TMV genome The organization of the TMV genome is fairly well understood and accepted by the scientific community. Reading from the 5' towards the 3' end of TMV RNA, open reading frames code for 126- and 183 kDa proteins, a 30 kDa movement protein, and the 17.5 kDa coat protein.
  • one aspect of the genome strategy that has not been fully elucidated is the exact nature of the replicase enzyme responsible for the synthesis of the genomic and subgenomic RNAs. While it is generally accepted that the virus codes for four proteins, two of which are coded for by the genomic RNA, and two of which are coded for by individual subgenomic RNAs, it is not generally accepted that the virus codes for at least one other additional and separate protein. N. D. Young et al reported [see J. Cell Science Supplement 7:277
  • the 5'-proximal region of the genomic RNA which encodes two coinitiated proteins, the 126 kDa and 183 kDa proteins, are components of the replicase.
  • the 183 kDa protein is generated by a read-through of the UAG stop codon of the 126 kDa protein.
  • the other two proteins (with known functions), the 30 kDa protein and the coat protein are each synthesized from separate subgenomic mRNAs on which each gene is 5' proximal.
  • the following sequence of the region of the TMV genome containing the readthrough portion of the 183 kDa protein gene is:
  • This sequence depicts a portion of the h subgenomic RNA beginning at nucleotide residue 3405 [the complete genome of TMV is 6,395 nucleotides long and may be found in Goelet et al, Proc. Natl. Acad. Sci USA 79:5818 (1982)].
  • the h RNA terminates at nucleotide 6395.
  • the 54 kDa open reading frame according to the present invention extends from nucleotide residues 3495 to 4919, and the underlined region designates the sequence used for the plant transformation more fully described in the following examples. More specifically, the gene portion for the 54 kDa protein within the h RNA sequence is:
  • AAGACCACCC UCAAGGAUUA UACCGCAGGU AUAAAAACUU GCAUCUGGUA 896 UCAAAGAAAG AGCGGGGACG UCACGACGUU CAUUGGAAAC ACUGUGAUCA 946
  • the 54 kDa 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 126 kDa protein expressed in Escherichia coli were prepared, the 54 kDa protein in protoplast extracts could not be detected by either immunoprecipitation or Western blotting under conditions where the antibody would detect the 183 kDa protein [see T. Saito et al, Mol. Gen. Genet. 205:82 (1986)].
  • the 54 kDa protein has not been detectable in Western blots using antiserum made to the whole protein [see G. J.
  • the resistance exhibited by the 54-kDA transgenic plants differed in several important respects from TMV coat protein- mediated resistance: resistance was exhibited against both TMV RNA and TMV virions; it did not appear to break down over time or with increasing concentrations of innoculum; and it was effective against the TMV strain from which the 54 kDa protein gene was derived and a closely related mutant, but not against other TMV strains or other viruses.
  • 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 according to the present invention containing the TMV 54 kDa coding sequence inserted between the CaMV 35S promoter and the nopaline synthase polyadenylation site;
  • Figure 2 depicts the plant expression vector containing the modified Fny-CMV RNA-2 gene sequence inserted between the CaMV 35S promoter and the nopaline synthase polyadenlyation site;
  • Figure 3 depicts the construction of a modified Fny-CMV RNA-2 chimeric gene for integration into the genome of a host plant
  • Figure 1 shows plasmids which were derived by insertion of the TMV cDNA into either the Xho I site or the Sma I 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.
  • FIG. 2 shows the plant expression vector containing the modified Fny-CMV RNA-2 gene sequence according to the present invention inserted between the CaMV 35S promoter (35S) and the nopaline synthase polyadenlyation site (NOS polyA).
  • This plasmid (pCMV N/B-23), as will be described in detail within the following specification, was derived by inserting the modified Fny-CMV RNA-2 gene according to the present invention into the SatnHI site of pROK2, a binary plant transformation vector [see Nature 321 :446 (1986)]. This was accomplished by digesting pFny N/B-4 with Sphl which cut this plasmid at a site 5' of the RNA-2 cDNA sequences.
  • a BamY ⁇ -Sph ⁇ adapter was ligated to this Sph ⁇ site and then digested with BamH I which cuts at a site 3' of the RNA-2 sequences, thereby liberating the entire modified cDNA molecule.
  • This 3 kb fragment was subcloned via standard techinques into the Bam ⁇ site of pROK2 to generate pCMV N/B-23.
  • this construct was transferred to Agrobacterium tumefaciens strain LBA-4404 by tri-patental mating [see Methods Enzymol. 118:627 (1986)] mediated by E. coli strain MM294-pRK2013.
  • the trans-conjugates were selected by resistance to kanamycin and streptomycin at 50 and 125 ⁇ g/ml, respectively.
  • Figure 3 shows the construction of pCMV N/B-23 being done in two stages. First a 94 basepair region was deleted from the full length cDNA clone of Fny-CMV RNA-2. Second, this deletion derivative was subcloned into pROK2, the plant transformation vector.
  • the plasmid pFny206 containing the full length cDNA clone of Fny-CMV RNA-2 in plBI-76 was digested with the restriction enzymes Ncol and BstEII. This DNA was then treated with the Klenow fragment of E. coli DNA polymerase 1 which acts as a DNA modification enzyme to obtain a blunt ended molecule. This blunt ended DNA was then ligated and transformed by standard methods into E. coli JM 101. The plasmid which resulted from this was labelled pFnyN/B-4. This plasmid was analyzed by various restriction enzyme digestions and DNA sequencing and was shown to have a 94 basepair deletion. This deletion also resulted in a change in the open reading frame such that this gene now encoded a truncated protein of approximately 75 kDa. Cloning also resulted in the retention of an AUG as a potential translation initiator
  • this modified Fny- CMV RNA-2 gene contained in pN/B-4 was subcloned into plant transformation vector pROK2. To facilitate this subcloning, a BamH 1 site was added to the 5' end of this gene. pN/B-4 was digested with SpM and a SamH1 -Sp ⁇ 1 adaptor was ligated to this Sp ?1 site located at the 5' end of the gene. Following this ligation reaction, the pB/N-4 was digested with Bam ⁇ - ⁇ 1 which released a fragment approximately
  • the plants are named in a code indicating which construct of Fny-CMV RNA-2 was transformed into the plant, what culture was used in a particular transformation, and the particular regenerated plant.
  • "N/B” indicates which construct of Fny- CMV RNA-2 was transformed into a particular plant; in the case of N/B 1-8, the construct in this plant is from pCMV N/B-23 (see Figure 2).
  • the numbers "1" or "2" indicates which of two culture tubes were used to transform tobacco with the N/B construct.
  • the second number appearing in the designation simply indicates the particular plant number. For example, the numeral "8" in the above example simply indicates that this is the eighth regenerated plant obtained in this transformation experiment.
  • 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.
  • a clone of the TMV 54 kDa gene was obtained by using a 22 base oligonucleotide primer consisting of a Bam 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 double-stranded cDNA was digested with BamH1 and ligated into the SamH1 site of M13mp18.
  • the 54 kDa insert was removed by digestion with Haell, treated with Klenow to blunt end the 3' overhang, and finally digested with Pstl.
  • the insert was ligated into Pstl/Smal digested pBS(-) resulting in plasmid pRTT-1 which contains the TMV sequence from nucleotide residues 3472 to 4914 of the TMV RNA sequence.
  • the orientation of the insert was such that transcription from the T7 promoter gives (+) sense transcripts as depicted in Fig. 1.
  • pMON316 contains a unique Xhol site in a polylinker region located between the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase 3'-untranslated region. A Smal site is found in the polylinker region as well as within the Ti plasmid homologous region of pMON316.
  • CaMV cauliflower mosaic virus
  • Plasmid pTS541A was generated by insertion of the TMV sequence into the Smal site which resulted in the deletion of the nopaline synthase 3'-untranslated region and a portion of the Ti homology region. Insertion of the TMV sequence into the Xhol site resulted in the formation of pTS541. Clones containing the 54 kDa sequence in either sense or antisense orientation were characterized and isolated. Each construct was transferred to Agrobacterium tumefaciens GV3111 carrying pTiB6S3-SE by means of a triparental mating system [see R. T. Fraley et al, Bio/Technology 3:629 (1985)], and transconjugants were selected by resistance to kanamycin and streptomycin.
  • the 54 kDa coding sequence was subcloned into the plant expression vector pMON316 such that it is preceded by the CaMV 35S promoter and followed by the nopaline synthase 3' untranslated region as depicted in Figure 2.
  • This construct was ultimately transferred into tobacco plants by Agrobacterium tumefaciens-mediated leaf disk transformation. Transformants were selected on the basis of kanamycin resistance and the production of nopaline synthase.
  • Four transformed plants were generated with pTS541 and four other plants with pTS541A which lacks the 3' nopaline synthase untranslated region and a portion of the Ti homology region located immediately downstream from the 54 kDa open reading frame.
  • EXAMPLE III plant transformation Cut pieces of sterile, TMV susceptible, Nicotiana tabacum cv.
  • Xanthi nn leaves were transformed by the modified Agrobacterium tumefaciens GV3111 containing the TMV 54 kDa 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. EXAMPLE IV nucleic acid analysis DNA was isolated from leaves of plants by a modified procedure of Murray and Thompson [see Nucleic Acids Research 8:4321 (1980)].
  • SamH1 digests of the genomic DNA were hybridized to a 32 P-labeled TMV 54 kDa sequence specific probe. Hybridization to a 3.0 kb fragment verified the presence of a full length 54 kDa coding sequence.
  • the 54 kDa sequence insert is 1.44 kb and another 1.59 kb is contributed by flanking vector DNA.
  • the TMV 54 kDa 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 TMV 54 kDa sequence probe.
  • the transgenic plants were also analyzed for expression of the TMV 54 kDa protein in accordance with Example IV.
  • a 54 kDa protein could not be detected from the 54 kDa transgenic plants or from protoplasts prepared from 54 kDa transgenic plants or the controls.
  • EXAMPLE V immunological analyses An antiserum to the 54 kDa protein was made by injecting rabbits with a synthetic polypeptide representing an internal region, specifically amino acid- residues 164 to 179, of the 54 kDa protein. An in vitro translation product of the 54 kDa 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% sodium dodecyl sulfate (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- conjugated anti-rabbit antibodies and silver enhancement.
  • Leaf strips were extracted in a mortar with a similar solution, but one which did not contain the inhibitor. The extracts were then clarified by microfuge centrifugation, and the supernatants examined for the 54 kDa protein. The presence of the 54 kDa protein was sought by incubating the extracts of the labeled leaves or protoplasts with antisera described above; an immunoprecipitation, polyacrylamide gel, and autoradiography assays were also conducted.
  • This antiserum was confirmed as being very active with in vitro translation products of the 54 kDa gene transcripts, and it could easily precipitate a 54 kDa protein from in vitro translation products of the RNA prepared from TMV virions containing the RNA necessary for manufacture of the 54 kDa protein. Protein could not be detected in leaves of either TMV infected plants or 54 kDa 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.
  • a closely related TMV mutant - mutant b6 as described by F. Garcia-Arenal et al, Virology 132:131 (1984) which is easier to score as a consequence of the bright yellow symptoms it elicits in the leaf. Plants were scored daily by visual observation of symptom development. In some instances, the presence of virus in inoculated plants was determined by probing leaf extracts with labeled cDNA to TMV.
  • 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.
  • the resistance to viral infection utilizing a replicase related coding 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 non-inoculated leaves of plants challenged with high concentrations of virus or viral RNA.
  • the protection mediated by the coat proteins of TMV and A1MV can be overcome by inoculating with viral RNA
  • the induced resistance according to the present invention utilizing the 54 kDa code sequence remains uncompromised when challenged with viral RNA.
  • the level of resistance in 54 kDa transgenic plants does not appear to be due to the level of expression: plants with only one copy of the gene sequence did not show a decrease in resistance to intact virons or viral RNA.
  • a single copy of the TMV coat protein is also sufficient to protect the plant whereas one copy of the AIMV coat protein is not.
  • EXAMPLE VII Tobacco plants ⁇ Nicotiana tabacum L), cv. Xanthi nn, as well as the 54 kDa transgenic Xanthi nn, and the TMV local lesion indicator host Xanthi NN, were maintained under greenhouse conditions. Plants used for protoplast preparation were transferred to a growth chamber on a 14 hr light/10 hr dark cycle at 24°C for at least one week prior to use. The light intensity was reduced to 125-150 ⁇ E « m" 2 « s " 1 by shading with cheese cloth. TMV strains U1 and U2 [see Phytopathology 44:277 (1954)] were purified [see Virology 91 :133 (1978)].
  • the TMV strain U1 used in some of the following examples was derived from transcripts generated from a full length cDNA clone of the virus. Virus infection of whole plants was achieved by inoculation of both upper and lower surfaces of fully expanded Xanthi nn or 54 kDa transgenic tobacco leaves with 0.05, 0.5. or 1.0 mg/ml of TMV strain U1 in 0.05 M potassium phosphate, pH 7.0 buffer, with Celite as an abrasive. Viral RNA was prepared from TMV strains U1 and U2 by phenol extraction and ethanol precipitation. EXAMPLE VIII protoplast preparation and infection Protoplasts were obtained from leaves of 54 kDa transgenic plants and control, nontransgenic tobacco plants.
  • the protoplasts (0.5- 1.0 x 1 ⁇ 6 cells/ml) were infected by electroporation with viral RNA extracted from TMV strains U1 or U2. Electroporation was performed in a final volume of 2 ml of sterile 0.7 ml of sterile 0.7 M mannitol, using a single ring electrode (2.5 mm high, 1 cm gap) connected to a ProGenetor 1 electroporation apparatus by applying two 5 msec pulses of 300 V.
  • the viral RNA concentrations ranged from 10 to 100 ⁇ g/ml although routinely 10 ⁇ g/ml was used.
  • all experiments included a set of mock-inoculated protoplasts electroporated in buffer alone.
  • protoplasts were resuspended in incubation medium: 0.7 M mannitol containing 1 mM KNO3, 1 mM MgSO4, 0.1 mM CaCl2, 1 ⁇ M Kl, 0.01 ⁇ M CuSO4, 10 ⁇ g/ml rimocidin, and 100 ⁇ g/ml carbenicillin buffered with 50 mM citrate, pH 5.5 buffer.
  • the protoplasts (3 ml) were transferred to agar plates (1 % noble agar in incubation medium prepared in 60- x 15-mm petri dishes) and incubated in low light at 25°C [see Virology 161 :488 (1987)].
  • the released proteins were separated by SDS-PAGE, electoblotted to nitrocellulose, and probed using a rabbit poiyclonal antiserum (diluted 1 :1 ,000) to strain U1 TMV coat protein and [ 1 5 l] protein A.
  • L-[ 35 S] methionine was added to the incubation medium at a concentration of 10 ⁇ Ci/ml. After continuous labeling, protoplasts were washed in 0.7 ml mannitol and disrupted in buffer. [ 35 S]-labeled proteins were analyzed by SDS-PAGE [see Nature 227:680 (1970)] and autoradiography.
  • RNAs were separated on formaldehyde-containing, 1.2% agarose gels and were blotted to nitrocellulose, which was then probed with in vitro-synthesized, [ 32 P]-labeled, ssRNA transcripts.
  • Relative amounts of specifically hybridizing RNA bands were qualified by excising the appropriate areas of the nitrocellulose filter using an autoradiograph as a template and determining the amount of radioactive probe bound using a liquid scintillation spectrometer.
  • RNA probes were prepared from two DNA templates: 1) T3 polymerase transcription [see Molecular Cloning, Cold Spring Harbor Laboratories (1989)] of pBS126, a derivative of pBSM13- containing an insert corresponding to nucleotides 1-3,785 of strain U1 TMV, including the whole of the 126 kDa protein reading frame, yields a (+) sense transcript corresponding to this region of TMV genomic RNA and complementary to the 3' region of full length (-) sense TMV RNA; 2) SP6 polymerase transcription [see Molecular Cloning, Cold Spring Harbor Laboratories (1989)] of pSP64 derivative containing an insert corresponding to the coat protein gene of TMV (from nucleotide 5,663 to the 3' end).
  • T7 transcripts of pRTT-1 containing the sequence encoding the strain U1 TMV 54 kDa protein were prepared and used to program wheat-germ [see PNAS (USA)70:2330 (1973)] and reticulocyte lysate-derived [see Eur. J. Biochem. 67:247 (1976)], in vitro translation systems.
  • the 126 kDa protein is the more abundant of the two known viral- coded TMV replicase components and its synthesis, directed by the 5' proximal open reading frame of TMV genomic RNA is probably the first step in replication after (or during) virus uncoating.
  • the 126 kDa protein was not apparent among [ 3 5S]-labeied proteins extracted from 54 kDa transgenic protoplasts infected with strain U1 TMV. However, under the same conditions the 126 kDa protein was present in extracts of [ 5S]-labeled protein from nontransgenic tobacco protoplasts infected with strain U1 TMV.
  • strain U2 TMV The equivalent, faster moving protein encoded by strain U2 TMV was synthesized in both transgenic and nontransgenic protoplasts infected with that strain of TMV. Similarly, synthesis of strain U2 TMV coat protein was observed in both cell types. Synthesis of strain U1 TMV coat protein could not be observed in this way because it lacks methionine. Attempts to improve the sensitivity of detection of the 126- and 183 kDa proteins by immunoprecipitation with appropriate antisera were unsuccessful.
  • the 54 kDa protein or its RNA might directly inhibit replicase activity, or second, the 54 kDa protein or its RNA may act indirectly, for instance by inhibiting synthesis of the virus-coded replicase components, the 126- and 183 kDa proteins.
  • the second possibility was addressed by translating TMV
  • RNA in rabbit reticulocyte or wheat-germ in vitro translation systems that had also been programmed or preprogrammed with an in vitro- synthesized TNA transcript encoding the 54 kDa protein.
  • synthesis of the 126- and 183-, as well as the 54 kDa proteins occurred with no suggestion of specific inhibition of 126- or 183 kDa protein synthesis.
  • the 54 kDa protein or its RNA according to the present invention affect the synthesis of virus-coded replicase components. The results are, therefore, consistent with the first possibility, namely that the 54 kDa protein or its corresponding RNA affect replicase activity directly.
  • Example XII and which contains the Ncol and BstEII sites used to generate a 94 basepair deletion is:
  • Example XII the nucleotide and amino acid sequence of the carboxy- terminus of the modified Fny-cDNA clone is:
  • the region surrounding and including this 94 bp region contains four domains which are highly conserved among putative replicase encoding sequences in many positive-sense RNA plant and animal viruses.
  • This deleted 94 bp region also contained the third domain, the
  • Gly-Asp-Asp domain which has been shown to be necessary for replication of the bacteriophage Q ⁇ . As noted in the sequence above, this deletion also caused a shift in the open reading frame resulting in a truncated translation product.
  • Ri generation plants from the original transformants were tested for resistance to virus infection. Individual plants from 2 out of 18 transformed lines were resistant to systemic disease when inoculated with either Fny-CMV virions or RNA at concentrations as high as 500 ⁇ g/ml.
  • Ciral mosaic virus strains Fny, O, Sny, Ve85, Y, and LS were purified from Nicotiana tabacum cv. Vietnamese Samsun as described by Palukaitis [see Methods For Plant Biology, Weissbach and Weissbach eds., Academic Press, New York (1988)].
  • Viral RNA was isolated from intact cucumber mosaic virus virions by phenol/chloroform extraction and ethanol precipitation. Plants were routinely inoculated with virus in 50 mM sodium phosphate buffer, pH 7.2, or RNA in 50 mM Tris phosphate buffer, pH 8.9, with the use of the abrasive Celite. Nicotiana tabacum cv.
  • Turkish Samsun NN was used for plant transformation and as a cucumber mosaic virus-susceptible systemjc host. Plants were maintained in a green house or in a growth chamber at 24°C with a 16 hr/8 hr light-dark cycle.
  • RNA-2 clone was modified by digesting pFny206 with the restriction endonuclease Ncol, phenol/chloroform extracted to remove these enzymes, and ethanol precipitated. This DNA was treated with Klenow fragment in the absence of nucleotides in an attempt to obtain a blunt-ended molecule. This DNA was then digested with the restriction endonuclease BstEII, treated with Klenow fragment in the presence of nucleotides to obtain a blunt ended molecule, phenol/chloroform extracted, and ethanol precipitated.
  • This linear molecule was then recircuiarized by T4 DNA ligase to generate plasmid (pFnyN/B-4) which contained a cDNA clone of Fny-CMV RNA-2 in which 94 nucleotides from nucleotide 1857 to nucleotide 1950, inclusive, were deleted.
  • This 94 nucleotide region contained the Gly-Asp-Asp domain that is highly conserved among replicase proteins of many positive-sense viruses. This deletion also caused a shift in the open reading frame which resulted in an in-frame translational stop codon 15 codons downstream of the deletion site.
  • This deletion therefore, not only deleted a 94 bp region, but also resulted in a truncated open reading frame. Cloning also resulted in the retention of an AUG as a potential translation initiator 87 nucleotides upstream of the AUG in the RNA 2 gene resulting in potential translation of an additional 29 amino acids at the amino terminus of the protein.
  • the protein encoded by this modified gene is thus approximately 75 kDA in size compared with a wildtype protein of 96.7 kDa.
  • pN/B-4 was digested with Sph which cut this plasmid at a site 5' of the N-terminus of the RNA-2 cDNA deletion clone.
  • An adapter containing a 5' SamH1 overhang and a 3' SpM overhang was ligated to this site and then digested with SamH1 which cuts at a site of 3' of the C-terminus, thereby liberating the entire modified cDNA molecule.
  • This 3kb fragment was subcloned via standard techniques into the SamHI site of the binary plant transformation expression vector pRok2 [see Nature 321 :446 (1986)], to generate pCMV N/B-23.
  • This construct was transferred to Agrobacterium tumefaciens strain LBA-4404 by tri-parental mating [see Methods Enzymol. 118:627 (1986)] mediated by E. coli strain MM294-pRK2013.
  • the transconjugates were selected by kanamycin and streptomycin at 50 and 125 ug/ml respectively.
  • the nucleotide sequence of Fny-CMV RNA- 2 in pFny206 and pCMV N/B-23 are shown in figure 2.
  • EXAMPLE XIII plant transformation Transformation of Nicotiana tabacum cv. Turkish Samsun NN was accomplished via Agrobacterium fumefaciens- e ⁇ aXed leaf disk transformation described by Horsch as in Example III.
  • Leaf discs growing in this medium were transferred to fresh medium every 7 to 14 days.
  • shoots formed which were transferred to root- inducing medium containing 300 ⁇ g/ml of Kanamycin, 0.2 mg/l BAP, and 0.1 mg/l of NAA. These shoots formed roots in approximately 2-3 weeks at which time they were transferred to vermiculite for 2 weeks, repotted to soil, and transferred to a growth chamber. Eighteen independent kanamycin resistant transformants were chosen for further analysis.
  • Genomic DNA was isolated from leaf tissue by a modified procedure of Murray and Thompson (1980). High molecular weight DNA was digested with restriction enzymes, separated on a 0.8% agarose gel, transferred to GeneScreenPlus (Dupont) nylon membranes. This membrane was hybridized in 5x SSC, 5x Denhardts, 5% dextran sulfate and 2% SDS to a 32 P-labeled DNA probe specific for Fny-CMV RNA-2 gene sequence. All DNA probes were prepared by the random hexamer primer reaction [see Anal. Biochem 132:6 (1983)] to specific activities of at least 5 x 10 8 cpm/ ⁇ g of DNA.
  • the level of resistance appears to be affected by the number of copies of the defective CMV replicase gene inserted into the genome.
  • the copy number of the original transformed plants tested in the detached leaf assay ranged from 1-3 copies per haploid genome for the five lines which were not resistant, to 3-5 copies and 5-7 copies for the resistant N/B 1-2 and 1-8 lines, respectively.
  • These two resistant lines also showed a difference in the number of resistant R1 plants at various inoculum concentrations (Table 1), which again suggests that the level of resistance of any given plant, particularly at very high inoculum concentrations of 100 to 500 ⁇ g/ml, may be affected by the number of copies of the modified RNA-2 Fny-CMV RNA 2 sequence present in the genome of that plant.
  • Virus as depicted in Table 2, was shown to be present only in those plants showing systemic symptoms.
  • the copy number in the susceptible N/B 1 -2 plants were 0 or 1 per haploid genome, whereas the resistant N/B 1-2 plants each contained 2 copies per haploid genome. This indicated that a copy of the gene is necessary but does not assure resistance, and the presence of multiple copies of the gene does not guarantee that a particular plant will be resistant. This is substantiated by analysis of the N/B 1-8 resistant and susceptible plants which showed that they all have incorporated three to four copies per haploid genome (Table 2). Furthermore, most of these insertions, whether from resistant or susceptible plants, appear to be in similar locations within the genome.
  • transgenic tobacco plants containing a modified CMV-replicase gene are highly resistant to infection by CMV.
  • level of CMV replicase mediated resistance is maintained at an inoculum dose of 500 ⁇ g/ml which is 10-fold higher than that previously shown for CMV coat protein mediated protection [see Bio/Technology 6:549 (1988)].
  • the data also show that transgenic tobacco plants transformed with a 54- kDa gene encoded in the read through of a replicase protein also exhibit TMV resistance.
  • replicase sequences are used for the plant transformation. Although the two systems exemplified herein, both of which involve transformation with viral replicase sequences, may operate by different mechanisms, it appears that this replicase-mediated resistance is generic and applicable to other plant and animal RNA viruses.
  • 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 and CMV other plant viruses such as, 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.
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:1 : GCAGGA
  • MOLECULETYPE DNA
  • SEQUENCEDESCRIPTION SEQID NO:4:

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DE19508290A1 (de) * 1995-03-09 1996-09-12 Hoechst Schering Agrevo Gmbh Verfahren zur Kontrolle unerwünschter Virenvermehrung sowie zur Herstellung virusresistenter Organismen
WO1999058696A1 (en) * 1998-05-12 1999-11-18 Institute Of Molecular Agrobiology Disease resistant transgenic plants
ID27743A (id) * 1998-05-12 2001-04-26 Inst Of Moleculer Agrobiology Daya tahan terhadap penyakit pada tanaman transgenik
EP1029923A1 (de) 1999-01-27 2000-08-23 D.J. Van Der Have B.V. Verfahren zur Vermittlung von BNYVV-Resistenz gegenüber Zuckerrüben
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WO1991016420A1 (en) * 1990-04-20 1991-10-31 The General Hospital Corporation Methods of preventing viral replication
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WO1991013542A1 (en) * 1990-03-12 1991-09-19 Cornell Research Foundation, Inc. Transformation of plants with non-structural plant virus gene sequences
WO1991016420A1 (en) * 1990-04-20 1991-10-31 The General Hospital Corporation Methods of preventing viral replication
WO1992003539A1 (en) * 1990-08-24 1992-03-05 Imperial Chemical Industries Plc Virus resistance in plants
EP0536106A1 (de) * 1991-10-04 1993-04-07 Monsanto Company Gegen Infektionen durch PVX resistente Pflanzen
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BRAUN, C.J., ET AL.: "Expression of amino-terminal portions or full-length viral replicase genes in transgenic plants confers resistance to potato virus X infection", THE PLANT CELL, vol. 4, no. 6, pages 735 - 744 *
CARR, JOHN P. ET AL: "Resistance in transgenic tobacco plants expressing a nonstructural gene sequence of tobacco mosaic virus is a consequence of markedly reduced virus replication", MOL. PLANT-MICROBE INTERACT. (1991), 4(6), 579-85 *
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