Classifications

• • 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
  • C12N2770/34011—Potyviridae
  • C12N2770/34022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to the determination of the complete nucleotide sequence of the DNA complementary to the genomic RNA of potyvirus, including that of the potato virus Y, and to the use of this sequence to create genes capable to confer on plants a resistance to potyviruses, by the creation of transgenic plants resistant to phytopathogenic viruses and in particular to potyvirus.
• Viral conditions are a problem for the pathologist; in fact, because of the dependence of the virus on host cells, it is difficult to destroy it without running the risk of damaging the organism which it parasites.
• the means of control are therefore essentially preventive; vaccines are used in animals with immune systems and in plants, seed selection and resistance varieties.
• Curative methods are based on the use of molecules that interfere with replication (analogs of bases such as azidothymidine) which are not without side effects for the host.
• thermotherapy and the cultivation of meristems allow healthy plants to be regenerated from infected plants (Morel and Martin, 1952).
• Potyviruses are the largest group of plant viruses, comprising more than 70 apparently distinct members. These viruses are defined by the filamentary structure of their particle, 600 to 900 nm long, containing approximately 5% of nucleic acid and 95% of proteins. Each particle contains a single molecule of simple RNA positive strand of molecular weight 3-3.5 ⁇ 10 6 , and the subunits of a single polypeptide of molecular weight 32-34 ⁇ 10 3 . They induce the formation of inclusions of characteristic forms which accumulate in the cytoplasm and sometimes in the nucleus. They are transmitted naturally by aphids in a non-persistent fashion and some are also transmitted by seed.
• PVY Potato virus Y
• the Y virus and the leaf curly virus are considered to be the two most important viruses affecting the potato in the world.
• Early infection of tobacco with PVY can lead to a yield reduction of around 30%, and necrosis of the nerves from the nerves, caused by the N strain, can cause complete loss of the crop.
• Viruses belonging to group O generally induce severe systemic symptoms of embossing, and leaf necrosis drooping in potatoes, systemic necrosis in Physalis floridana and systemic mottling in tobacco.
• Group N strains of necrosis of the ribs of the tobacco
• Group C viruses including the PVC virus which is not transmissible by aphids, produce hypersensitivity reactions in many potato cultivars.
• These groups although very serologically related, show great intra-group variability. We can say that there are no reproducible serological differences between Yo, Yn and Yc. The reported inter-group cross-protection results are contradictory (De Bokx and Huttinga, 1981).
• the capsid protein sequence of an Australian isolate revealed 92% identity with the capsid of the Pepper Mottle Virus (PeMV) and 62% with that of the Tobacco Etch Virus (TEV), the N- terminal of these proteins showing the greatest divergences (Shukla et al., 1986).
• potyviruses The molecular study of potyviruses is relatively recent. These viruses are difficult to purify because of their particle instability and their tendency to form aggregates.
• a protein (VPg) is covalently linked to the 5 'end of the viral RNA (Hari, 1981), which is polyadenylated at the 3' end (Hari, 1979).
• Initial "in vitro" translation studies suggested that genomic RNA could be translated into a high molecular weight polyprotein, which would then be matured into smaller proteins (Yeh and Gonsalves, 1985. Hellman et al., 1983).
• This hypothesis was confirmed by analysis of the nucleotide sequence of the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV) genomic RNAs (Allison et al., 1986. Domier et al., 1986), these RNAs would code for 345kD and 340kD polyproteins respectively. This mechanism is similar to that used by animal picornaviruses (Nicklin et al, 1986) and by
• the proteolytic cleavages in these viruses are mainly made at the Gln-Gly (Picornavirus) and Gln-Gly, Gln-Ser and Gln-Met (Comovirus) sites.
• Domier et al, 1986 proposed a proteolytic cleavage map of the TVMV polyprotein.
• the same group (Domier et al, 1987) subsequently published an analysis of the sequence homologies of the proteins thus defined, with those of the Picorna-, Como-, and Caulimoviruses.
• Carrington and Dougherty (1987) demonstrated that one of the two nuclear inclusions of TEV was a protease, which enabled these researchers to propose a partial genetic map of the Potyvirus genome by comparison with genomic RNAs of picornaviruses ( poliovirus), comovirus (CPMV), romancevirus (TBRV) and potyvirus (TVMV).
• nucleotide sequences derived from the pathogen can be used to modify the host genetically in order to that it becomes resistant to the pathogen. This is possible because the life cycle of a parasite, like that of any organism, results from a sum of molecular events regulated at various levels. Resistance therefore comes from an interruption in the development of the parasite by one of its genes expressing itself at the wrong time, in the wrong place, in too large a quantity or in a modified counter-functional form.
• the presence of the capsid protein in the cell prevents the entry of the virus or its decapidation: in fact the inoculation of viral RNA can overcome the protection observed in transgenic plants (Nelson et al., 1987 VIROLOGY, 158, 126-132).
• This protection results in a delay in the appearance of lesions on the inoculated leaves, suggests an inhibitory effect at later stages of the infection, such as a reduction in the rate of multiplication or a blockage of the transport of the virus. cell to cell.
• the capsid protein of the alfalfa mosaic virus would play a role in the binding of the polymerase to the 3 'end of the RNA, and therefore an activator of the synthesis of the negative strand, and in the late stages it would repress it, making the overall synthesis of RNA not symmetrical, in favor of the production of the positive strand (Houwing and Jaspars, 1987).
• the bacteriophage QB capsid protein would have a double negative regulatory role, by suppressing viral replication, while promoting the translation of its own gene, and by suppressing the translation of the gene coding for the polymerase, by binding to its domain. initiation (Bernardi, 1972; Robertson, 1975). Using this latest data, Grumet, Sanford and Johnston, (1987, MOLECULAR STRATEGIES FOR CROP
• î R. LISS INC pp. 3-12 demonstrated the validity of the concept of resistance derived from the pathogen, by obtaining high levels of resistance to this bacteriophage in E. coli strains expressing its capsid protein, even at multiplicities of infection important.
• Tomato aspermy virus TMV
• the object of the present invention is to construct a gene coding for the capsid protein of potyvirus and in particular of PVY, under conditions such that its function cannot be altered, by adding an ATG sequence. at the 5 'end of the coding sequence for the potyvirus capsid.
• Potyviruses have the particularity of having as translation product, a single polyprotein which gives functional proteins only after proteolytic cleavage in precise places of the polyprotein, by a specific protease precisely coded by the viral genome; as a result, the present invention also aims to provide a system capable of blocking the action of the protease in order to prevent the appearance of viral proteins and, consequently, symptoms of l viral infection of the plants concerned.
• the subject of the present invention is transgenic plants resistant to infection by potyviruses, characterized in that they are obtained from plant cell lines, in particular from plant species liable to be infected by potyviruses, modified by introduction of a gene coding for the potyvirus capsid protein.
• said transgenic plants are characterized in that the gene introduced is a gene coding for the capsid protein of potyvirus, resulting from the addition of an ATG sequence to the 5 'end of the cDNA sequence encoding for said potyvirus capsid protein, provided with signals for regulating gene expression in a plant cell.
• the ATG initiation codon of the introduced gene is included in a consensus sequence, of the type AX 1 X 2 ATGG in which X or Y are identical or different and each represents one nucleotides A, T, G or C.
• X or Y are identical or different and each represents one nucleotides A, T, G or C.
• the seeds derived from these transgenic plants and used for the reproduction of these fall within the scope of the present invention.
• the present invention also relates to a gene coding for the capsid protein of potyvirus, characterized in that it results from the addition of an ATG sequence to the 5 'end of the cDNA sequence coding for said protein. of capsid of potyvirus.
• its ATG initiation codon is included in a sequence of type AX 1 X 2 ATGG, where X 1 and X 2 are as defined above.
• this gene comprises the DNA sequence complementary to the following nucleotide sequence (I):
• the present invention further relates to a DNA sequence constituted by a gene coding for the capsid protein of potyvirus as defined above, provided with signals for regulating genetic expression in a plant cell.
• the present invention further relates to a process for obtaining a gene coding for the capsid protein of potyvirus, characterized in that a fragment of a synthetic DNA comprising an ATG codon is inserted at a chosen point of the cDNA sequence coding for the capsid protein, the plasmids containing the desired junction then being isolated by hybridization with a synthetic oligonucleotide covering half the sequence of the synthetic DNA fragment and half the sequence of the 5 'end of the sequence of the gene coding for the potyvirus capsid protein.
• the present invention further relates to a process for obtaining cDNA from potyvirus genomic RNA, which is characterized in that the first strand of cDNA is synthesized by the action of reverse transcriptase on RNA genome of potyvirus using random hexamers as initiators of the first strand, the RNA / DNA hybrid obtained is converted into double-stranded DNA by the joint action of three enzymes, RnaseH. of E.
• the DNA fragments thus created are joined by the ligase of E. coli, the double-stranded cDNA is then treated with Sl-nuclease, the repair of its ends is carried out using T 4 -DNA-polymerase and the cDNA is separated according to its size on gel. low melting agarose, then ligated into a Smal cleavage site of an appropriate vector and the recombinant plasmids are introduced into E. coli, collected and sequenced by an appropriate sequencing method.
• Clones, obtained by insertion of double-stranded cDNA in an appropriate plasmid, are partially sequenced by the deletion method or by subcloning of restriction fragments in an appropriate vector, the selection of said clones being carried out by hybridization with a mixture synthetic oligonucleotides of 20 mothers deduced from the amino acid sequence of the N-terminus of the capsid protein.
• the present invention further relates to the complete nucleotide sequence of the PVY genomic RNA, which is characterized in that it comprises 9704 bases followed by a poly-A end, in that a non-coding region with the 5 'end, which comprises 185 bases, precedes the single and long open reading phase, in that said phase codes for a polyprotein of 3063 amino acids and in that it is followed by a non-region 331 base coding at the 3 'end.
• said complete sequence of potyvirus genomic RNA this is represented by the following nucleotide and amino acid (II) sequence:
• the position of the sequence of the capsid protein in the above-mentioned RNA sequence is such that its N-terminal end is located at position 2796 of the polyprotein.
• the present invention further relates to the DNA sequence complementary to the sequence (II) defined above.
• the present invention also encompasses any nucleotide sequence specific for the PVY genome, included in the sequence (II) or in the DNA sequence complementary to said sequence II.
• EXAMPLE 1 Cloning and sequencing of the DNA complementary to the potyvirus Y genomic RNA of the potato (PVY): 1. Development of the conditions for obtaining the cDNA of the PVY RNA.
• the cDNA was synthesized by the method of
• the first strand is synthesized in a conventional manner by the action of reverse transcriptase, and the RNA / DNA hybrid obtained is converted into double-stranded DNA by the joint action of 3 enzymes: RNaseH from E. coli partially degrades the RNA part of the hybrid, creating primers for DNA polymerase I which displaces the cleavage by synthesizing DNA by its 3 '->5' activity while degrading the RNA by its 5 'activity -> 3 ', the DNA fragments thus created are joined by the ligase of E. coli chosen because of its inability to join DNA with free ends.
• the optimal concentration of KCl for obtaining long retrotranscripts of viral RNA is 140 mM.
• RNase A treatment was included at the end of second strand synthesis; in order to obtain perfectly blunt ends, the repair step has takes place for a very short time in the absence of nucleotides allowing the DNA polymerase of phage T4 to resect the 3 'ends of the DNA; the nucleotides are added and the incubation then continues normally. 2. Cloning and sequencing of the cDNA.
• plasmids pY139 (1kb), pY118 (1.8kb) and pY117 (3.6kb) were chosen to be sequenced.
• the partial sequence of plasmids pY139 and pY118 was carried out by a sequential deletion method based on the cleavage properties of DNAse I in the presence of Mn 2+ ions (LIN et al., 1985) and that of plasmid pY117 by sub -cloning of restriction fragments generated by the enzymes Rsal, Ddel, Hinfl and Taql in the vector
• Bluescript KS + (Stratagene).
• the insertion contained in the plasmid pY139 was found to originate from a cDNA initiated on a sequence of 5 adenines corresponding to two lysines at positions 176 and 177 of the protein sequence previously published (SHUKLA et al. 1986).
• the cDNA can be cloned by adding homopolymeric extensions ("tailing").
• the two successive precipitations eliminate 99% of the non-incorporated radioactivity, check the presence of a cDNA pellet with moni tor at this stage (and at the others).
• the percentage of conversion of RNA into cDNA can range from 15 to 50% depending on the quality of the RNA, the percentage of synthesis of the second strand is 80 to 100%.
• the optimal size of the extensions is 15 to 25 nucleotides, the reaction being approximately linear, it is therefore possible to reincubate at 25 ° C. for a time defined by the first incubation.
• 5M ammonium acetate take up to 20ng / ⁇ l in TE.
• A50 which has the advantage of separating DNA according to size.
• the columns are poured into a 1 ml pipette, with the smallest possible internal diameter.
• EXAMPLE 4 Obtaining clones by priming the cDNA on the viral RNA using random hexamers.
• the method described below makes it possible to obtain, in a single step, all the clones necessary for obtaining the gene coding for the capsid protein, then to sequence them and to order the data obtained by computer processing.
• This plasmid contains the origin of replication of phage M13 and makes it possible to obtain single-stranded DNA after superinfection of the strain with a phage such as M13K07.
• a phage such as M13K07.
• the method chosen is derived from that described by KALYAN et Al (GENE-1986, 42, 331-337) which makes it possible to add a fragment of synthetic DNA to a chosen point of a sequence, the splice being able to be checked at the base, thanks to the use of a very sensitive screen based on the characteristics of the molecular hybridization of an oligonucleotide probe.
• KALYAN et Al GENE-1986, 42, 331-337
• deletions were created in the plasmid, from a unique HindIII site located approximately 400 base pairs 5 'from the sequence GCAAATGACACA, corresponding to the first acids capsid protein amines.
• the deleted fragments were then ligated with a synthetic sequence adapter (VA) (VB):
• Hybridization temperature nb of bases, ie 55oC for this oligonucleotide probe, the washings being carried out in 0.9M Na at room temperature, then for 5 minutes at hybridization temperature - 3 ° C.
• Hybridization temperature 4 (G + C) +2 (A + T) -5 at 10 ° C 0.9M Na but seem to be more discriminating. A first positive clone was obtained, its insertion could not be released by digestion with the enzymes BamHI (the site created by addition of the adapters) and Hpall (sine placed at 16 base pairs downstream of the coder.
• pMARCEL 70 and pMRKE70 vectors were created from the pMARCEL19 and pMRKE35 vectors respectively, and contain a promoter constructed from that of 35 S RNA by duplicating a transcriptional activation region located from -343 to -90 relative to at the transcription initiation site (KAY et al., 1987), which promoter has the advantage of producing 10 fcis more transcripts than the one from which it originates.
• the cell colonies obtained are resistant to kanamycin.

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• 1988
  • 1988-05-30 FR FR8807150A patent/FR2631973B1/fr not_active Expired - Lifetime
• 1989
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