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EP0558491A1 - Resistance des plantes aux virus - Google Patents

Resistance des plantes aux virus

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Publication number
EP0558491A1
EP0558491A1 EP19910914997 EP91914997A EP0558491A1 EP 0558491 A1 EP0558491 A1 EP 0558491A1 EP 19910914997 EP19910914997 EP 19910914997 EP 91914997 A EP91914997 A EP 91914997A EP 0558491 A1 EP0558491 A1 EP 0558491A1
Authority
EP
European Patent Office
Prior art keywords
rna
replicase
protein
virus
plant
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Withdrawn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
EP19910914997
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German (de)
English (en)
Inventor
Kenneth William Bryherhurst Wood Road Buck
Robert James 70 Holden Road Hayes
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.)
Syngenta Ltd
Original Assignee
Zeneca Ltd
Imperial Chemical Industries Ltd
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Filing date
Publication date
Application filed by Zeneca Ltd, Imperial Chemical Industries Ltd filed Critical Zeneca Ltd
Publication of EP0558491A1 publication Critical patent/EP0558491A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • 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
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds

Definitions

  • This invention relates to the prevention of disease in plants, more specifically, to the production of plants with recombinant genomes (transgenic plants) which are resistant to infection by viruses.
  • RNA positive-strand (messenger-sense) RNA
  • examples include poliovirus, foot and mouth disease virus, tobacco mosaic virus, cucumber mosaic virus and bacteriophages MS2 and Q fl .
  • RNA-dependent RNA polymerase RdRp
  • RdRp The best characterised RdRp is that of bacteriophage Q_ which has been purified to homogeneity and shown to consist of one virus-encoded polypeptide and three host polypeptides ( 3lumenthal & Carmichael, 1979). This enzyme has a specific template requirement for Q_ RNA and catalyses both stages of the replication process.
  • a number of template-dependent RdRp preparations have been obtained from cells infected by eukaryotic viruses, ie, poliovirus (Van Dyke and Flanegan, 1980, Baron and Baltimore, 1982, Hey e_t at * .. , 1987; Lubinski et al., 1987), black beetle virus (Saunders & Kaesber ⁇ , 1985), turnip vellow mosaic virus (Mouches e_t al_. , 1985), brome mosaic virus (Miller and Hall, 1983; Quadt et. at., 1988) , cowpea chlorotic mottle virus .Miller and Hall, 1984) and alfalfa mosaic virus (Houwing and Jaspers, 1986).
  • eukaryotic viruses ie, poliovirus (Van Dyke and Flanegan, 1980, Baron and Baltimore, 1982, Hey e_t at * .. , 1987; Lubinski et al., 1987), black beetle virus (Sa
  • RNA 1 (3.4 kb)
  • RNA 2 (3.0 kb)
  • RNA 3 2.1 kb
  • RNA 1 (3.4 kb)
  • RNA 2 (3.0 kb)
  • RNA 3 (2.1 kb)
  • RNA 1 (3.4 kb)
  • RNA 2 (3.0 kb)
  • RNA 3 (2.1 kb)
  • RNA 1 (3.4 kb)
  • RNA 2 3.0 kb)
  • RNA 3 2.1 kb
  • RNA 3 also encodes the virus coat protein which is translated from a subgenomic RNA designated RNA 4 (1.0 kb) .
  • RNA 1, 2 and 3 are required to infect plants systemically and the function of protein P3a is believed to be the potentiation of movement of virus particles from cell-to-cell .
  • attempts to produce a soluble, template-free CMV polymerase or to demonstrate the presence of proteins Pla and P2a in RNA containing membrane-bound polymerase preparations have hitherto been unsuccessful (Gordon e_t a_ . , 1982; Jaspers et a_l . , 1985) .
  • An object of the present invention is to provide a viral replicase capable of catalysing complete replication of its RNA template and the use of that replicase to impart virus resistance to plants .
  • HEET provided an RNA-dependant replicase (RdRp) comprising polypeptides which includes: proteins Pla and P2a of cucumber mosaic virus or homologous or analogous proteins from another virus; and polypeptide P50 of tobacco, or a homologue or analogue thereof from another plant species.
  • RdRp RNA-dependant replicase
  • the invention also provides a recombinant plant genome having ' virus resistance comprising a gene expressing a product which inhibits expression of or inhibits the catalytic activity of viral RdRp.
  • the said product may be an antibody directed against the protein Pla or P2a of cucumber mosaic virus, or a homologue or analogue of either protein from another virus, against polypeptide P50 of tobacco, or a homologue or analogue thereof from another plant species or against any other component of the replicase.
  • the said product may be RNA in antisense orientation to the RNA encoding the protein Pla or P2a of cucumber mosaic virus, or a homologue or analogue of either protein from another virus, to polypeptide P50 of tobacco, or a homologue or analogue thereof from another plant species or to any other component of the replicase.
  • the said product may also be RNA coding for a truncated or otherwise modified form of a protein (for example, Pla, P2a or P50) which is an essential component of the replicase. Production by the plant of such modified proteins can disrupt the function of the replicase.
  • a protein for example, Pla, P2a or P50
  • the said product may also be a ribozyme adapted to cut RNA coding for an essential component of the replicase.
  • the invention further provides monoclonal or polycional antibodies which inhibit the replication of cucumber mosaic virus _ir ⁇ vitro. Such antibodies may also inhibit the replication of other plant viruses.
  • the said antibodies may be prepared by a method comprising injecting a mammal with a substance selected from the group consisting of: (i) a purified RNA replicase extracted from a plant infected with cucumber mosaic virus; (ii) the protein Pla or a peptide fragment thereof;
  • the protein P50 or a peptide fragment thereof (v) any other protein component of a purified RNA replicase (particularly one of the minor protein constituents of the replicase that binds specifically to the negative strand of the viral RNA), or a peptide fragment thereof; and thereafter recovering polyclonal antibodies from serum extracted from the injected mammal or producing hybridomas which produce monoclonal antibodies or cloning DNA sequences from the animal or from the hydridomas into a vector and expressing the antibody in an expression system.
  • the said proteins Pla, P2a, and P50 may be produced by isolation from the purified replicase or by cloning the respective gene in an expression vector and expression thereof in a bacterial expression system.
  • Peptide ⁇ corresponding to regions of target proteins may be produced by degradation of the proteins or by direct synthesis.
  • the invention also provides a DNA sequence which encodes an antibody which inhibits replication of the cucumber mosaic virus replicase; and, additionally, tran ⁇ genic plants, resistant to
  • SUBSTITUTESHEET virus infection which contain such a sequence stably incorporated within their genomes.
  • RNA encoding the target protein may be suppressed by expression of RNA in antisense orientation to that RNA.
  • the catalytic activity of the target protein may be inhibited by expression of an antibody to the protein, or by expression of modified or truncated forms of the protein or of RNA coding for such forms.
  • viral replication in a host plant may be inhibited by expression of antisense RNA directed against the RNA encoding protein Pla or protein P2a of the virus or against protein P50 of the plant.
  • antibodies directed against the RdRp may be expressed in plant cells.
  • modified or truncated forms of these proteins, or of RNA coding for such forms, may be expressed in plant cells.
  • CMV RdRp which is soluble, completely dependent on addition of CMV RNA as a template and contains proteins Pla and P2a, as well as a host polypeptide. Furthermore the enzyme catalyses the synthesis of both stages of the replication process: ie, th- * synthesis of positive-strand RNA, as welsee as negative-strand RNA.
  • T complete replication of a eukaryotic virus RNA by a purified template-dependent RNA polymerase.
  • Figure i is the results of non-denaturing gel electrophoresis of products of RdRp reactions: Lanes 1 to 5, products of RdRp fraction 4 reactions; lanes 6 to 8 , products of RdRp fraction 6 reactions. Reactions were programmed with: lane 1, CMV RNA; lane 2, no added RNA; lanes 3 and 6, RNA 3; lanes 4 and 7, RNA 2; lanes 5 and 8, RNA 1. Double arrowheads indicate dsRNA bands. Single arrowheads indicate ssRNA bands. Bands were detected by autoradiography.
  • Figure 2 relates to the analysis of ssRNA synthesised in an RdRp reaction:
  • Figure 2A The band corresponding to to ssRNA 1 (Figure 1) was gel extracted, heated to 100°C for 30 seconds, annealed to oligonucleotides, and treated with ribonuclease H. The products were then electrophoresed through 1.2% agarose- formaldehyde gels and detected by autoradiography.
  • Lane 1 ssRNAl reaction product; lane 2, ssRNA reaction products plus ribonuclease H; lane 3, ssRNA 1 reaction product + Pi + P2 + ribonuclease H; lane 4, ssRNA 1 reaction product + P3 + P4 + ribonuclease H; lane 5, ssRNA 1 reaction product + Pi + P2 + P3 + P4 + ribonuclease H.
  • the sizes, shown by the side of the gel photograph, are in kilobases.
  • the genomic RNA is indicated by a double arrowhead and ribonuclease H products by a single arrowhead.
  • Figure 2B is a diagram showing the expected products from ribonuclease H digestion of positive- strand RNA I annealed to PI and P2 or neqative-
  • EET strand RNA 1 annealed to P3 and P4.
  • the sequences of the oligonucleotides are :
  • FIG. 3 shows the results of gel electrophoretic analysis of proteins of RdRp fractions. Proteins were electrophoresed in
  • SDS-polyacrylamide gels and detected by silver staining (lanes 1 to 7), by Western blotting and probing with antisense to the protein Pla (lanes 8 to 11) or antiserum to protein P2a (lanes 12 to 15), or by blotting followed by antibody-linked polymerase assay (ALPA) with antiserum to protein Pla (lanes 16 to 19) or with antiserum to protein P2a (lanes 20 to 23).
  • APA antibody-linked polymerase assay
  • the M r of marker proteins are shown on the side of the gel: myosin (205 Kd) , 3-galactosidase (116 Kd) , phosphorylase B 7Kd), bovine serum albumin (66Kd), ovalbumin (45Kd).
  • FIG. 4 antibody-linked polymerase assays ALPA) of fraction 6.
  • Pla P/C and P2a P/C antibodies are polyclonal antibodies raised against CMV proteins Pla and P2a respectively.
  • mAB/lal and mAB/2a3 are monoclonal antibodies against protein Pla of RNA 1 and protein P2a of RNA 2 respectively.
  • Replicase P/C are polyclonal antibodies raised in rabbits injected with Fraction 6.
  • Figure 6 effect of polyclonal antibodies raised against peptides on replicase activity.
  • Figure 7 effect of mAB/HP on replicase assays .
  • Tt.s 32P-labelled 150 nucleotides from positive-sense (lanes 1 & 3) or negative-sense (lanes 2 & 4) CMV RNA 1 were incubated with Fraction 3 prepared from healthy plants 'lanes 1 & 2) or CMV infected plants (lanes 3 & 4) .
  • Figure 9 competition binding of 3' terminal 150 nucleotides of negative-sense RNA 1.
  • the 32P-labelled 3' terminal 150 nucleotides from negative-sense RNA 1 was incubated with Fraction 3 from healthy plants together with unlabelled 3' terminal 150 nucleotides of negative-sense RNA 1 (lanes 1, 2 & 3) or unlabelled 3' terminal nucleotides of positive sense RNA (lanes 4, 5 & 6) at molar ratios of 100:1 (lanes 1 & 4), 10:1 (lanes 2 & 5) or 1:1 (lanes 3 & 6).
  • Figure 10 effect of polyclonal antibodies on protein-RNA binding.
  • Figure 11 effect of mAB/HP on protein-RNA binding.
  • the 3' terminal 150 nucleotides of negative- sense RNA was incubated with Fraction 3 from healthy (lane 3) or infected plants (lanes 1 & 2). After 10 minutes incubation mAB/HP was added to the protein-RNA from infected plant complex (lane 1) .
  • Figure 12 determination of the size of RNA binding proteins. Fraction 3 from healthy plants was incubated with 32P-labelled RNA corresponding to the 3 r terminal 150 nucleotides of positive-sense 'lanes
  • mAB/lal Replicase Very effective replicase inhibitor ( ⁇ 60%). Maps in helicase region around nucleotide 2880 of RNA 1.
  • Fraction 1 A membrane-bound polymerase (Fraction 1) was obtained by differential centrifugation of extracts of CMV-infected tobacco leaves. Treatment with a non-ionic detergent produced a soluble polymerase (Fraction 2) from which endogenous RNA was removed by nuclease digestion to give Fraction 3. More high purified preparations were obtained by chromatography on an anion-exchange column (Fraction 4), followed by separation on the basis of molecular size (Fraction 5) and finally fractionation on a high resolution anion-exchange column (Fraction 6). The specific activity of Fraction 6 was 760,000 times that of Fraction 1 (Table 2) .
  • RNA polymerase activity was assayed as described
  • SUBSTITUTESHEET leaves to the volume of each fraction used for the assay.
  • RNA polymerase activities of Fractions 4,5 and 6 were completely dependent on addition of CMV RNA. No activity was observed in the absence of added RNA or on addition of equivalent amounts of RNA from viruses in different taxonomic groups (Matthews, 1982 " ) namely tobacco mosaic virus, tomato bushy stunt virus or red clover necrotic mosaic virus. Hence the polymerase had a specific template requirement.
  • RNA 3 was used as a template, both RNa 3 and the subgenomic RNA 4 were synthesised.
  • the ssRNA 1 product was extracted from a gel and hybridised with two oligonucleotides Pi and P2 with sequences complementary to internal sequences in RNA 1 (Figure 2b). Digestion with ribonuclease H hydrolysed the RNA specifically at the sites of hybridisation with Pi and P2 to produce fragments of the calculated size (1.0 kb, 1.5 kb and 0.8 kb) ' Figure P2a, lane 3) with nucleotide incorporation in proportion to their length.
  • the ssRNA 1 reaction product consisted mainly of positive- strand RNA uniformly labelled along its length.
  • RNA undigested by ribonuclease H ( Figure P2a, lane 3) was shown to be the RNA 1 negative-strand.
  • Figure 2b oligodeoxynucleotides P3 and P4 with sequences complementary to internal sequences of the RNA 1 negative-strand
  • Figure 2b ribonuclease H digestion
  • the apparent Mr of the proteins that reacted with the protein Pla or P2a antibodies were 98 K and 110K respectively, values similar to those obtained for the in vit o translation products of RNA 1 and RNA 2 respectively (Gordon et al . , 1982). Similar results were obtained with RdRp Fraction 6. hence the purified RNA polymerase contained proteins Pla and P2a.
  • Fractions 4 and 5 contained a number of host proteins in addition to proteins Pla and P2a.
  • Fraction 6 contained one major host protein of apparent M 50 K, although a number of other minor proteins were also present.
  • Fraction 5 lost the ability to synthesise ssRNA but was still able to synthesise dsRNA with no apparent change in its protein composition.
  • the final stages of RdRp purification led to loss of the 50 K host protein ( Figure 3, lane 7); such loss was invariably accompanied by complete loss of RdRp activity.
  • Soluble, template-dependent RdRp preparations have previously been obtained for very few viruses of eukaryotes, the best studied being those of poliovirus 'Van Dyke & Flanegan, 1980; Baron & Baltimore, 1982; Hey et al . , 1986; Lubinski et al . , 1987; Plotch et al . , 1989) and brome mosaic virus (Miller & Hall, 1983; Quadt et al., 1988).
  • the inability of RdRp preparations to catalyse complete replication of RNA could be due to absence of a helicase subunit or inhibition of helicase activity in partially purified preparations.
  • sequence motifs characteristic of helicases and polymerases lie in proteins 2C and 3D respectively (Gorbalenya et al., 1989).
  • Soluble, template-dependent preparations of poliovirus RdRp either isolated from infected cells or produced in E. coli from cDNA clones, contain 3D, but not 2C, a protein known to be required for RNA replication in vivo (Li & Baltimore, 1988).
  • the absence of a helicase subunit could be a factor in the inability of poliovirus RdRp preparations to catalyse complete RNA replication.
  • the CMV RdRp produced an excess of positive-strands over negative-strands.
  • the ratio of positive-to negative-strands in the ssRNA product was 7:1. We have not measured this ratio in the dsRNA product, but even if nucleotide incorporation into dsRNA were completely in the negative-strand, the overall ratio of positive-to negative-strands synthesised would be about 2.5 to 1. Since an excess of positive-strand templates was always present, the polymerase must utilise the negative-strand template preferentially and clearly each negative-strand template was copied more than once .
  • RNA 4 is transcribed from a subgenomic promoter on the negative-strand of RNA 3 upstream of the RNA 4 start site (Miller et al., 1985 Marsh et al., 1988).
  • the purified CMV RdRp contained a host polypeptide, apparent M 50 K, in addition to the two virus-encoded polypeptides . This protein is apparently bound to the RdRp, because it could not be detected in material corresponding to Fraction 6 from healthy plants.
  • Viruses with small genomes often utilise host components for their replication.
  • the RdRp of bacteriophage Q fi consists of one virus-encoded and three host-encoded subunits and an additional host protein is needed for negative-strand synthesis on a positive-strand template (reviewed by Blumenthal & Carmichael, 1979).
  • a host protein has been shown to enable initiation of poliovirus negative-strand synthesis in vitro (Dasgupta, 1983, Hey et al . , 1987).
  • a highly purified preparation of an RdRp from plants infected by turnip yellow mosaic virus was shown to contain one virus-encoded polypeptide and one host polypeptide, although it was not established whether the host polypeptide was needed for activity and the reaction products were not characterised (Candresse et al., 1986).
  • a host RdRp is induced when tobacco is infected by CMV or other viruses ( Fraenkel-Conrat, 1986). This enzyme consists of a single polypeptide, about 130 K, has no template specificity and synthesises only short chains. It is clearly distinct from the RdRp described here which contains two virus-encoded polypeptides and a host polypeptide, M about 50 K, is specific for CMV RNA and catalyses the complete replication of CMV RNA.
  • Fraction 1 was obtained by isolation of tonoplast vesicles by the method of Bremberger et al (1988). After addition of NP40 (final concentration, 0.75%), the mixture was stirred at 4°C for 1 hour and then centrifuged at 35 000 g for 30 minutes. The supernatant was recentrifuged at 100 000 g for 1 hour. The final supernatant (Fraction 2) was treated with micrococcal nuclease (Miller & Hall, 1983) to yield Fraction 3.
  • Fraction 3 was applied to a DEAE-Biogel column (1 x 10cm) at a flow rate of 1 ml/min. Unbound proteins were washed through with 30 ml of TMDPGN buffer (TMDPG containing 0.75% NP40) and the RdRp activity was eluted with TMDPGN buffer containing 0.5 M KC1. Fractions of 1 ml were collected and assayed for RdRp activity. The pooled active fractions were termed Fraction 4. Q-Sepharose could be used in place of DEAE-Biogel.
  • Fraction 4 was passed through a Pharmacia FPLC Superose 6 column (30 x 1 cm) at a rate of 0.25 ml/min with TMDPGN buffer containing 0.5 M KCl and fractions of 1 ml were collected.
  • Fractions containing RdRp activity were pooled (Fraction 5), dialyzed against TMDPGN buffer and then applied to a Pharmacia FPLC Mono Q column (5 x 0.5 cm) at a flow rate of 1 ml/min. A linear gradient of 0 to 0.5 M KCl in TMPDGN buffer was then applied.
  • Fractions of 1 ml were collected and those with RdRp activity were pooled (Fraction 6).
  • Fraction 3 could be stored at -70°C for at least one month and thawed and re-frozen several times without significant loss of RdRp activity.
  • Fraction 4 could also be stored at -70°C, but after thawing could not be re-frozen without considerable loss of activity.
  • the activities of Fractions 5 and 6 were completely lost after freezing and thawing. These fractions were stored unfrozen at 0°C and generally used within an hour of preparation.
  • RdRp activity was assayed by mixing 25 ⁇ l of each fraction with 25 ⁇ l of 100 mM tris-HCl pH 8.2, containing 4% v/v glycerol, 20 mM MgCl-,, 2 mM ATP,
  • RNA 1, RNA 2 and RNA 3 were synthesised by transcription i_n vitro using T7 RNA polymerase and vectors containing full-length clones of CMV RNA 1, RNA 2 and RNA 3
  • Proteins were electrophoresed in SDS-polyacrylamide gels (Laemlli, 1970) and detected by silver-staining (Ochs, 1983), by Western blotting (Sherwood, 1987) or by blotting followed by antibody-linked polymerase assay (Van der Meer et al., 1984; Candresse et al . , 1986). Production of Proteins Pla and P2a
  • Ndel sites were introduced into pCMVl and pCMV2 ( Figure 1) by _in_ vitro mutagenesis (Kunkel et al., 1987) using oligonucleotides
  • Pla and P2a coding regions were then cut out with Ndel and BaroHl and cloned into expression vector pET3a in E Coli BL21 cells (Rosenberg et al., 1987) . Expression and purification of proteins Pla and P2a were essentially as described ( Plotch et al , 1989). Electrophoretically homogeneous proteins were used
  • Fraction 6 replicase Approximately 10/vg of Fraction 6 replicase was injected into Balb/c mouse. After two similar injections two and four weeks later the spleen cells were fused with myeloma cells to produce hybridoma ⁇ . A number of hybridomas were screened for the production of antibodies against the host-encoded polypeptide. Only one (mAB/HP) was detected using Western blot analysis (see Figure 12, lane 1) . This antibody was then used in an ALPA against Fraction 6 replicase. As can be seen in Figure 4, lane 2, a band is present in Fraction 6 which corresponds to the host-polypeptide .
  • Two monoconal antibodies against Pla and three monoclonal antibodies a ainst P2a were detected using the replicase as an antigen as described above.
  • the monoclonal antibodies against Pla were termed mAB/lal and mAB/la2 , and those against P2a as mAB/2al, ⁇ AB/2a2 and mAB/2a3.
  • Polyclonal antibodies against the viral proteins Pla and P2a, and the monoclonal antibodies mAB/la and mAB/2a3 have been ⁇ hown to inhibit the activity of the replicase ( Figure 5). Polyclonal antibodies raised against the replicase are more effective than any antibodies against the single viral proteins.
  • polyclonal antibodies were raised against peptides corresponding to conserved regions of the Pla or P2a proteins.
  • polyclonal antibodies to peptides 2 and 3 have inhibitory effects similar to those raised against whole proteins.
  • antibodies against peptide 4 are notable for their ability to inhibit the replicase.
  • the antibodies, raised against the GDD region of P2a, are capable of near total inhibitions of the polymerase at extremely low levels. Monoclonal antibodies against this peptide are being raised.
  • the monoclonal antibody mHP has a smaller concentration dependent inhibitory effect on the replicase as shown by Figure 7.
  • Genes encoding a polypeptide can be cloned if some of the amino acid sequence data is known.
  • Oligonucleotides can then be designed to screen a cDNA library or to amplify the gene via the polymerase chain reaction. Approximately 5 g of purified HP was subjected to M-terminal sequencing via ⁇ dman degradation. Fortunately the M-terminus was not blocked, and fourteen amino acids could be designed to screen a cDNA library or to amplify the gene via the polymerase chain reaction. Approximately 5 g of purified HP was subjected to M-terminal sequencing via ⁇ dman degradation. Fortunately the M-terminus was not blocked, and fourteen amino acids could
  • RNA from CMV-infected M. tabacum has been isolated. Oligo-dT was used to prime first strand cDNA synthe ⁇ is using standard techniques. The mRNA was then hydrolysed u ⁇ ing ⁇ odium hydroxide, and the single-stranded cDNA was purified using a column of Sephacryl S-400. The cDNA was then amplified in a Vent DNA polymerase chain reaction using a 5'-pr ⁇ mer designed from the above amino acid sequence:
  • ATAAGAATGCGGCCGCGCXCCXATXCCXGGXGTXATG (Notl site underlined) and the 3'-primer: ATAAGAATGGCCXXXXXGGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • reaction products were purified using a Stratagene PrimerErase column, digested overnight with Notl and Sfil and ligated into Motl-Sfil digested pGem-llZf( + ) . A number of colonies were obtained with inserts of the correct length.
  • host-encoded protein ⁇ may also be present, but at a much lower concentration.
  • host-encoded protein ⁇ might be expected to be pre ⁇ ent when the polymera ⁇ e interacts with the 3'-ends of either the positive (genomic) sense RNA and/or with the 3 '-end of the negative-sense RNA.
  • Short RNA molecules corresponding to the terminal 150 nucleotides of the positive and negative-sense RNA were prepared. These were labelled and allowed to interact with Fraction 3 prepared from either healthy or CMV-infected plants. As expected, both fragments interacted with proteins present in Fraction 3 from infected plants ( Figure 8, lane ⁇ 3 and 4).
  • HEET complex may form part of the replicase. It may be required specifically for the synthesis of positive (genomic) sense RNA.
  • RNA replication function and structure of Q ⁇ -replicase . Ann. Rev. Biochem. 48 , 525-548.
  • HEET Lot H. , Harchoux, G., arrou, J. , Kaper, J.M., West, C.K., Van Vloten-Doting, L. and Hull, R. '1974) .
  • Antibody-linked polymerase assay on protein blots a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis. EMBO J. 2, 233-237.

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Abstract

Une réplicase dépendant de l'ARN (RdRp) comprend des polypeptides P1a et P2a d'un virus mosaïque de concombre et polypeptide P50 de tabac. Il peut aussi contenir d'autres constituants de polypeptides mineurs. Chaque polypeptide peut être substitué par un autre provenant d'une source analogue de fonction équivalente. On décrit aussi un génome de plante recombinant présentant une résistance aux virus et comprenant un gène exprimant un produit qui inhibe l'expression ou l'activité catalytique de la réplicase d'ARN viral. Ce produit peut être notamment (1) un anticorps dirigé contre un constituant protéique de la réplicase ou (2) de l'ARN en une orientation anti-sens par rapport à l'ARN codant un constituant protéique de la réplicase.
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US5633449A (en) * 1990-03-12 1997-05-27 Cornell Research Foundation, Inc. Induction of resistance to viral diseases in plants
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
WO1993020686A1 (fr) * 1992-04-17 1993-10-28 Kirin Beer Kabushiki Kaisha Vegetal resistant a au moins deux virus et sa preparation
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
US6160201A (en) * 1993-07-09 2000-12-12 Seminis Vegetable Seeds, Inc. Lettuce infectious yellows virus genes
GB9316384D0 (en) * 1993-08-06 1993-09-22 Unilever Plc Improvements in or relating to disease-resistance of plants
DE19508290A1 (de) * 1995-03-09 1996-09-12 Hoechst Schering Agrevo Gmbh Verfahren zur Kontrolle unerwünschter Virenvermehrung sowie zur Herstellung virusresistenter Organismen
EP1358341B1 (fr) * 2001-02-08 2011-05-25 Keygene N.V. Techniques permettant de generer une resistance contre le virus cgmmv dans des vegetaux

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EP0298918B1 (fr) * 1987-07-10 2001-09-05 Syngenta Participations AG Résistance inductible contre des virus dans des plantes
CA1340323C (fr) * 1988-09-20 1999-01-19 Arnold E. Hampel Catalyseur d'arn pour le clivage de sequences specifiques d'arn
DE3933384A1 (de) * 1989-10-06 1991-04-18 Hoechst Ag Multifunktionelle rna mit selbstprozessierungsaktivitaet, ihre herstellung und ihre verwendung
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