WO1995004825A1 - Ameliorations concernant la resistance de plantes a des maladies - Google Patents

Ameliorations concernant la resistance de plantes a des maladies Download PDF

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Publication number
WO1995004825A1
WO1995004825A1 PCT/GB1994/001731 GB9401731W WO9504825A1 WO 1995004825 A1 WO1995004825 A1 WO 1995004825A1 GB 9401731 W GB9401731 W GB 9401731W WO 9504825 A1 WO9504825 A1 WO 9504825A1
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Prior art keywords
virus
mosaic virus
leu
plant
replicase
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PCT/GB1994/001731
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English (en)
Inventor
Rebecca Stratford
Robert Edwin Boulton
Elaine Susan Higgins
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Unilever Plc
Unilever N.V.
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Priority to AU13183/95A priority Critical patent/AU1318395A/en
Publication of WO1995004825A1 publication Critical patent/WO1995004825A1/fr

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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
    • 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

Definitions

  • This invention relates to a nucleotide sequence capable of increasing the resistance of a plant to viral disease, and to altered plants comprising the sequence of the invention.
  • TMV genomic RNA encodes two co-initiated proteins (126kD and 183kD) which are considered to be components of the replicase since they are both enriched in partially purified preparations of the viral replicase complex from TMV-infected plants.
  • the 183kD protein is generated by readthrough of the stop codon of the 126kD protein.
  • ORF open reading frame
  • the existence of the 54kDa protein could not be demonstrated in TMV-infected plants (although a 54KDa product was made in in vitro using rabbit reticulocyte lysate systems).
  • the putative 54kD protein would be in-frame with the 183kD and would share the gly-asp-asp (GDD) motif which is characteristic of many RNA-dependent RNA polymerases and is believed to be involved in catalytic function. This implies a role for the 54kD protein as a third viral encoded replicase component.
  • GDD gly-asp-asp
  • Transgenic tobacco plants were produced which contained all but the three 3' terminal nucleotides of the TMV 54kD gene which encodes the putative component of the replicase complex. Plants were resistant to infection with high doses of both TMV virus and TMV RNA but were susceptible to distantly related TMV strains and other viruses. There was no detectable virus in the inoculated or systemic leaves of resistant transgenic plants i.e. very high level resistance was demonstrated. This contrasts with coat protein mediated resistance which is often expressed as a delay in symptom development and breaks down under high inoculum pressure.
  • the 54kD is an authentic replicase component, perhaps functioning as a regulatory subunit. Overexpression or expression at inappropriate times might disrupt replicase activity.
  • the 54kD is a truncated, defective version of the 183kD and inhibits replication by competing with the 183kD during replicase complex assembly, possibly titrating out host components involved in replication.
  • T V 54kD gene Since it is not known whether the T V 54kD gene is an authentic replicase component or a deletion mutant of the 183kD protein, it is difficult to establish which of the models describes the mechanism. It is possible that the actual clone used in the transformation constituted a mutated 54kD gene since there were five polylinker amino acids added to the C terminus.
  • PEBV Pea early browning virus
  • Transgenic plants expressing the read-through portion of the 201kD replicase gene of PEBV were resistant to high levels of virus inoculum (MacFarlane and Davies, 1992 PNAS 89 / 5829-5833).
  • the 141kD and 201kD proteins of PEBV share considerable homology with the 126kD and 183kD proteins of TMV and the PEBV 54kD read-through protein is also homologous to that of TMV. Constructs with mutations that caused premature translation termination did not confer resistance indicating that, like TMV, transcription of the viral sequence is not sufficient to generate resistance, the protein translation product being required for protection.
  • the PEBV read-through protein is an authentic replicase component or a truncated mutant of the 201kD protein.
  • the clone used in the experiment was derived by PCR and contained base changes relative to the wild type sequence. Three base changes introduced amino acid substitutions and three were silent third base pair changes. Hence it is again possible that the clone employed was effective in conferring resistance because the sequence was altered from that of the wild type.
  • RNA 2 sequences from alfalfa mosaic virus does not render host plants resistant to that virus (van Dun & Bol, 1988 Virology 167, 649-652) and protoplasts from transgenic plants expressing full-length RNA 2 sequences from brome mosaic virus do not resist BMV replication (Mori et al, 1992 J. Gen. Virol. 73. 169-172). In fact, both of these genes acted in trans to support the replication of defective viral genomes.
  • PVX Potato virus X
  • ORF 1 Expression of the first ORF (ORF 1) of PVX in transgenic plants conferred resistance to infection with PVX (Braun and Hemenway, 1992 Plant Cell 4 . , 735-744).
  • ORF 1 is considered to encode the putative replicase gene since it contains several conserved domains found in other RNA-dependent RNA polymerases.
  • this experiment was designed to express a full-length authentic replicase gene, it has been suggested that the clone used in the transformation was in some way defective. This is because PVX genomic length clones containing this gene sequence were only very poorly infectious suggesting that the cDNA was in some way mutated from the wild type sequence.
  • PVY Potato Virus Y
  • Potyviridae A family of plant viruses called Potyviridae has been proposed (Barnett, O.W. 1991 Arch. Virol 118, ppl39-141) and recently accepted (Ninth Plenary Meeting of the International Committee on Taxonomy of Viruses, held at the IXth International Congress of Plant Virology, Glasgow 1994) to include three genera: Potyvirus containing aphid borne viruses with Potato Virus Y (PVY) as the type species; the genus Baymovirus comprising fungal transmitted viruses with Barley Yellow Mosaic Virus (BaYMV) as the type species; and the genus Ryemovirus with ryegrass mosaic virus as the type species.
  • PVY Potato Virus Y
  • BaYMV Barley Yellow Mosaic Virus
  • viruses in the three genera have flexuous filamentous particles which contain a positive sense ssRNA genome and pin wheel inclusions accumulate in the cytoplasm of infected plants.
  • they have translation strategies based on polyprotein processing, the coat protein maps to the 3' end and there is a 3'-terminal poly(A) tract.
  • PVY geno ic RNA replicates through RNA intermediates in a DNA- independent fashion.
  • PVY RNA has one ORF encoding a single polyprotein of 352 kilodaltons (kDa) .
  • This polyprotein undergoes proteolytic processing to yield about eight viral proteins of 31 kDa, 62 kDa, 38 kDa, 71 kDa, 5.6 kDa, 50 kDa, 60 kDa and 30 kDa. These proteins can be found in the infected plant cell and form the necessary components for viral replication.
  • the 60 kDa viral encoded protein is known as the nuclear inclusion II or nuclear inclusion b (Nib) protein (or replicase protein) and is believed to function in the replication of the viral RNA.
  • a replicase gene derived from a particular potyvirus shares certain common features with replicase genes derived from other potyviruses.
  • One of these common features is the location of the gene within the potyviral genome.
  • the replicase gene is typically located directly upstream from the potyviral coat protein.
  • Another common feature is a defined type of cleavage site with a consensus sequence, which is processed by the potyviral protease responsible, at least in part, for cleavage of the polyprotein.
  • the replicase amino ac d sequences of the Potyviridae show many conserved features, which allows for aignment of the sequences (Koonin, 1991 J. Gen. Virol. 72, 2197-2206).
  • CyRSV Cymbidium Ringspot Virus
  • Transgenic tobacco plants expressing the wild type TMV 183Kda replicase gene were found not to be resistant to infection (Donson et al., 1993 MPMI 6., p635). A total of 16 lines were tested.
  • a Conference report describes mutation in the GDD motif of the replicase component on RNA 2. Mutation to VDD had no effect on resistance. Other mutations produced resistant lines at varying frequences: mutation to DDD (23% resistant lines generated), GGD (20%), GVD (40%).
  • Tobacco plants made transgenic to express the wild type TMV 183Kda replicase gene were not resistant to infection (Donson et al., 1993 MPMI 6., p635).
  • transgenic plants containing essentially the same sequences but with an additional insertion (bacterial transposable element) expected to terminate translation in the middle of the 183Kda gene were highly resistant to infection with TMV and other tobamoviruses.
  • Replicase-mediated resistance has been achieved for several different viruses via a number of different strategies.
  • TMV and PEBV which are in different virus groups but the same super ⁇ group
  • expression of the read-through protein (containing the GDD box) at the C-terminus of the replicase gene gave resistance.
  • the read-through protein may be a functional protein.
  • the majority of viruses do not have an equivalent of this read-through protein and resistance has been achieved by expression of defective replicase mutants or the full length sequence.
  • PVX a member of the same super-group as TMV and PEBV
  • expression of a C-terminal region (including the GDD box) of the single replicase gene did not confer resistance. Instead, either the full-length replicase or the N-terminal half gave resistance.
  • the present invention relates to the finding that nucleotide sequences encoding portions of a potyvirus replicase, when introduced into susceptible plants, are capable of increasing the resistance of the plants to disease caused by potyviruses and related viruses.
  • potyvirus as used herein is intended to refer to any member of the family Potyviridae.
  • the invention provides a DNA molecule encoding a portion of a potyvirus replicase or a functional equivalent thereof, said molecule being capable, upon introduction into an appropriate plant, of increasing the resistance of said plant to viral disease.
  • the invention provides a method of increasing the resistance of a plant to viral disease, comprising introducing into the plant a DNA molecule encoding a portion of a potyvirus replicase or a functional equivalent thereof.
  • the invention provides a plant, having increased resistance to a viral disease, into which has been introduced a DNA molecule encoding a portion of a potyvirus replicase or a functional equivalent thereof.
  • DNA sequence of the invention need not be restricted to sequences encoding "authentic" portions of a potyvirus replicase.
  • functionally equivalent sequences would be of equal utility.
  • Such functional equivalents include, for example, potyvirus replicase consensus sequences, and sequences encoding portions of a substantially "authentic" potyvirus replicase to which have been made minor deletions, additions or substitutions which do not substantially affect the protective effects of the sequence in plants. It will generally be found that such functionally equivalent sequences will hybridise, under standard laboratory conditions (those described in the standard manual by e.g. Sambrook et al.), to homologous portions of a potyvirus replicase.
  • the portion of the potyvirus replicase may be the sequence of any portion of a replicase gene from any potyvirus. Preferably the portion is from the 3' end region of the replicase coding sequence (i.e. corresponds to a sequence in the 3' half of the coding sequence). Conveniently the portion of a potyvirus replicase is a portion of the Nib coding sequence of Potato Virus Y (PVY), desirably PVY strain O.
  • PVY Potato Virus Y
  • the portion of the potyvirus replicase comprises 1104 nucleotides or fewer, preferably between 1104 and 594 nucleotides, and more preferably 594 nucleotides or less.
  • portion of the potyvirus replicase encodes a polypeptide having the same amino acid sequence as the polypeptides encoded by the nucleotide sequence of Figure 7 or Figure 8.
  • portion of the potyvirus replicase comprises the nucleotide sequence of nucleotides 4-1104 Figure 7 or the nucleotide sequence of nucleotides of 36-632 of Figure 8.
  • the DNA molecule comprises signals necessary for the potyvirus replicase sequence to be translated, but such signals are preferably included.
  • Promoters operable in plants, for transcription of the potyvirus replicase sequence are well known to those skilled in the art (e.g. CaMV 35S, Rubisco gene promoter). Useful promoters might be constitutive, inducible or tissue-specific, depending on the circumstances. Examples of each type are well known.
  • the portion of the potyvirus replicase-coding sequence might be derived directly from a particular potyvirus, or may be modified in some way to increase levels of transcription and/or translation in a particular host plant.
  • the portion of a replicase-coding sequence could be a consensus sequence, comprising motifs or sequences characteristic of several different potyviruses.
  • Such a chimeric/consensus-type portion of a potyvirus replicase coding sequence might confer resistance to a wider range of viral diseases than a sequence derived from a single potyvirus.
  • the DNA molecule may comprise a portion of a plurality of potyvirus replicases, such that introducing the DNA molecule into the plant would increase the resistance of the plant to a plurality of virus diseases.
  • the DNA molecule preferably comprises a 3' non-translated region located downstream of the portion of a potyvirus replicase-coding sequence.
  • a 3' non-translated region comprises signals directing the addition of polyadenylated ribonucleotides to the 3' end of the transcribed mRNA.
  • Suitable 3' regions are known to those skilled in the art (e.g. that from the nopaline synthase gene).
  • Methods of introducing the DNA molecule into suitable host plants are well known (e.g. transformation with Agrobacterial strains, micro-injection, "biolistic” delivery methods).
  • the present invention should be of use in increasing the resistance of susceptible plants to onbe or more of the following viruses: pea seedborne mosaic virus, pea mosaic virus, wheat spindle streak mosaic virus, potato virus A, potato virus V, cocksfoot streak virus, ryegrass mosaic virus, carnation vein mottle virus, turnip mosaic virus and pokeweed mosaic virus.
  • Figure la shows the genomic structure of PVY and the relationship thereto of two partial cDNA clones (B4 and C12);
  • Figure lb is a schematic representation of the insertion of B4 and C12 into the vector pUC18;
  • Figure lc is a schematic representation of the use of PCR to sub- clone Nib fragments of B4 and C12;
  • Figure Id is a schematic representation of the insertion of the Nib fragments of B4 and C12 into the vector pBI220.5;
  • Figure 2 shows a bar chart illustrating the resistance of transgenic plants to infection with PVY compared to control plants
  • Figure 3 shows a bar chart illustrating the resistance of transgenic plants compared to controls, in particular line 4.12 (containing the "small replicase” transgene);
  • Figure 4 shows a bar chart illustrating the amount of virus (as measured by ELISA) " in control plants and transgenic plants;
  • Figures 5 and 6 are bar charts showing the resistance of line 4.12 to both primary and secondary (respectively) PVY infection under field trial conditions.
  • Figures 7 and 8 show the nucleotide sequence of "large” (Seq. ID No. 1) and "small” (Seq. ID No. 2) replicase fragments respectively and the amino acid sequence of the polypeptides encoded thereby (Seq. ID Nos. 3 & 4 respectively).
  • a Defective Replicase Gene Induces Resistance to Potato Virus Y in Transgenic Potato Plants
  • Potato virus Y (strain 0) was purified from infected potato plants using a method essentially as described by Robaglia et al., (1989 J. Gen Virol 70. / pp935-947). Viral RNA was extracted from the virus particles and used in a cDNA synthesis reaction following standard procedures (Sambrook et al. 1989; Cold Spring Harbour Laboratory Press). The cDNA synthesis reaction was primed by an oligo dT primer and double stranded cDNA was cloned into the S al site of pUC18. Sequence analysis was performed on two cDNA clones (B4 and C12).
  • Clones B4 and C12 were selected for the construction of deletion mutant forms of the Nib replicase gene.
  • the Nib portion of the cDNA clones was subcloned out of B4 and C12 using PCR techniques (Fig. lc).
  • PCR was performed on clone B4 using the universal reverse primer (Reverse: 5'-AACAGCTATGACCATG-3'; Seq. ID No. 5) which is homologous to part of the pUC18 sequence flanking the polylinker site, and a second primer (PVY-1, Seq. ID No. 6) homologous to the 3' end of the Nib gene: PVY-1 5'-CGC GGTACC TTA TTGATGGTGTACTTCATA-3' Kpnl Stop nt 8572 codon
  • PCR amplification Thirty cycles of PCR amplification were performed, each cycle consisting of 30 sec at 94°C, 1 min at 55°C, and 2 min at 72°C.
  • the PCR products were cloned into the CaMV 35S promoter vector pBI220.5 using the Kpnl site encoded at the 5' end of the PVY-1 primer and either the BamHI (clone C12) or Kpnl (clone B4) sites located in the polylinker sequence upstream of the 5' end of the Nib replicase gene insert.
  • the resulting clones were sequenced in full to verify the Nib sequence and the data are presented below (Fig. 7, 8).
  • the construct derived from cDNA clone B4 was designated 'large replicase' and that derived from cDNA clone C12 was designated 'small replicase'.
  • the constructs were then further subcloned into the plant transformation vector pBinl9 (Bevan, M W 1984 Nucleic Acid Res. .12, p8711-8721) as Hindlll - EcoRI fragments.
  • the resulting plasmids were transformed into Agrobacterium strain LB4404 and transformed into the potato variety Maris Piper as described below.
  • the plants were originally derived from tuber sprouts and are the widely available variety Maris Piper.
  • the plants were grown as in vitro shoot cultures at 24°C under continuous light (70 ⁇ E m ⁇ 2 sec _1 in 150 x 25mm glass tubes loosely capped with Magenta lids (Magenta Corp., Chicago, 111. USA).
  • Nodal stem cuttings with one leaf were taken from 5-6 week old plants and planted in MS (Marashige and Skoog) medium containing 1% sucrose solidified with 0.9% agar (Sigma) and supplemented (after autoclaving) with STS which promoted plant growth and produced larger leaves.
  • the STS (silver thiosulphate) stock solution (6mM or 1.5 mg/ l. ) was prepared by adding 12mM AgN0 3 to an equal volume of 96mM Na 2 S 2 0 3 and used at a concentration of lug/ml.
  • Leaf explants were taken from plantlets at 5-6 weeks after subculturing; cut across the base (discarding the petiole and the lower l-2mm of leaf base) and the remaining leaf used in transformation experiments. These were carried out essentially as published by Visser et al. (1989, Plant Mol. Biol. 12., 329- 337) but slightly altered from the protocol published. Briefly, explants were floated overnight on liquid M387.
  • the medium (M387) contained MS salts and vitamins supplenmented with: lOg/1 sucrose; 80mg/l NH A N0 3 ; 147mg/l CaCl 2 ; lOmg/1 NAA; and lOmg/1 BAP.
  • transgenic lines (4.66 and 4.12) which contain the small replicase construct have a reduced incidence of infection relative to conrol lines and other transgenic lines.
  • Two transgenic lines were selected for a further PVY resistance test. These were line 4.12 (small replicase) and 2.41 (large replicase). Three plants of each line were tested along with non-transformed susceptible (M. piper) and resistant (Desiree and M. bard) controls. Also included in the test were other transgenic lines expressing sequences of PVY. Tests were performed as described above. The percentage of infected plants is shown in Fig. 3 and the mean of the ELISA reading (proportional to the virus titre) is shown in Fig. 4. No plants of the transgenic line 4.12 became infected and the ELISA value was not significantly different to that of healthy transgenic lines and resistant control varieties.
  • Line 4.12 was included in a transgenic virus resistance field trial experiment. Twenty plants (primary transformants) of line 4.12, together with resistant and susceptible control varieties, were planted in a randomised block design. Infector plants (PVY infected plants of the susceptible variety Craigs Snow white) were planted adjacent to the transgenic plots. PVY was aphid- transmitted from the infector plants by a combination of natural aphid infection and an artificially reared aphid population. Leaf samples were taken at the end of the growing season (late August) and tested for the presence of PVY primary infection by ELISA. The data (Fig. 5) clearly show that line 4.12 is resistant to primary infection with PVY.
  • FIG. 6 shows that line 4.12 is also resistant to secondary infection with PVY and has a reduced incidence of PVY infection relative to control plants of the resistant varieties M. bard and Pentland Crown.
  • Figures 7 and 8 show the respective nucleotide sequences of the "large” and “small” mutant replicases.
  • the polypeptide encoded is shown (in single letter code) above the nucleotide sequence.
  • MOLECULE TYPE cDNA (111)
  • HYPOTHETICAL NO (111)
  • ANTI-SENSE NO
  • GGT AAA GTG TGT G ⁇ GAT GAC ⁇ C AAT AAT CAA TU TAT TCA AAG AAT 192 Gly Lys Val Cys Val Asp Asp Phe Asn Asn Gin Phe Tyr Ser Lys Asn 50 55 60
  • GCA GAC ⁇ G GCT GAA CAC AGG C ⁇ GAG GCG A ⁇ TGC GCA GCT ATG ATA 864 Ala Asp Leu Ala Glu His Arg Leu Glu Ala He Cys Ala Ala Met He 275 280 285
  • GCA ⁇ A GAC GAT GAG T ⁇ GAG ⁇ T GAT TCT TAT GAA
  • GTA CAC CAT CAA 1104 Ala Leu Asp Asp Glu Phe Glu Phe Asp Ser Tyr Glu Val His His Gin 355 360 365
  • MOLECULE TYPE cDNA (111) HYPOTHETICAL! NO (111) ANTI-SENSE: NO
  • GCA GAC ⁇ G GCT GAA CAC AGG C ⁇ GAG GCG A ⁇ TGC GCA GCT ATG ATA 389 Ala Asp Leu Ala Glu His Arg Leu Glu Ala He Cys Ala Ala Met He 105 110 115
  • GCA ⁇ A GAC GAT GAG TTT GAG T ⁇ GAT TCT TAT GAA GTA CAC CAT CAA 629 Ala Leu Asp Asp Glu Phe Glu Phe Asp Ser Tyr Glu Val His His Gin 185 190 195
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO (ill) ANTI-SENSE: NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

On décrit une molécule d'ADN qui code une portion d'une potyvirus réplicase, molécule pouvant, après introduction dans une plante appropriée, accroître la résistance de cette plante à une maladie virale. On décrit aussi un procédé permettant d'accroître la résistance d'une plante à une maladie virale, ainsi que des plantes transgéniques présentant une résistance accrue à une maladie virale.
PCT/GB1994/001731 1993-08-06 1994-08-08 Ameliorations concernant la resistance de plantes a des maladies WO1995004825A1 (fr)

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Cited By (9)

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WO1996028562A2 (fr) * 1995-03-09 1996-09-19 Hoechst Schering Agrevo Gmbh Procede de controle de la proliferation de virus indesirables et procede de preparation d'organismes resistants aux virus
WO1997002352A1 (fr) * 1995-06-30 1997-01-23 Novartis Ag Utilisation d'arn modifie par traduction pour conferer une resistance au virus de la mosaique du mais et du sorgho et autres virus de plantes monocotyledones
WO1999028485A2 (fr) * 1997-12-03 1999-06-10 Gene Shears Pty. Ltd. Ribozymes capables de conferer une resistance a l'infection par le potyvirus, et plantes exprimant ces ribozymes
WO2002063019A2 (fr) * 2001-02-08 2002-08-15 Keygene N.V. Techniques permettant de generer une resistance contre le virus cgmmv dans des vegetaux
WO2002077209A1 (fr) * 2001-03-26 2002-10-03 Plant Science Sweden Ab Plantes transgeniques possedant une resistance contre le virus de tobacco rattle et sequence nucleotidique correspondante
US7309813B2 (en) * 1999-02-25 2007-12-18 Pioneer Hi-Bred International, Inc. Methods of using viral replicase polynucleotides and polypeptides
WO2009037369A1 (fr) 2007-09-21 2009-03-26 Consejo Superior De Investigaciones Cientificas Manipulation de la fonction du gène atdbp1 afin de générer une résistance au potyvirus
CN104846005A (zh) * 2015-03-02 2015-08-19 福建农林大学 利用人工合成mv3序列培育抗花叶病甘蔗品种的方法
CN116675751A (zh) * 2023-06-08 2023-09-01 山东农业大学 SWEET1g蛋白及其编码基因在抗马铃薯病毒中的应用

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WO1992003539A1 (fr) * 1990-08-24 1992-03-05 Imperial Chemical Industries Plc Resistance des plantes aux virus
EP0578627A1 (fr) * 1992-07-09 1994-01-12 Monsanto Company Plantes résistantes aux virus
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WO1996028562A2 (fr) * 1995-03-09 1996-09-19 Hoechst Schering Agrevo Gmbh Procede de controle de la proliferation de virus indesirables et procede de preparation d'organismes resistants aux virus
WO1997002352A1 (fr) * 1995-06-30 1997-01-23 Novartis Ag Utilisation d'arn modifie par traduction pour conferer une resistance au virus de la mosaique du mais et du sorgho et autres virus de plantes monocotyledones
US6040496A (en) * 1995-06-30 2000-03-21 Novartis Finance Corporation Use of translationally altered RNA to confer resistance to maize dwarf mosaic virus and other monocotyledonous plant viruses
WO1999028485A2 (fr) * 1997-12-03 1999-06-10 Gene Shears Pty. Ltd. Ribozymes capables de conferer une resistance a l'infection par le potyvirus, et plantes exprimant ces ribozymes
EP0922767A1 (fr) * 1997-12-03 1999-06-16 Gene Shears Pty Limited Ribozymes capables de conférer une résistance à une infection par Potyvirus, et plantes exprimant ces ribozymes
WO1999028485A3 (fr) * 1997-12-03 1999-09-02 Gene Shears Pty Ltd Ribozymes capables de conferer une resistance a l'infection par le potyvirus, et plantes exprimant ces ribozymes
US7309813B2 (en) * 1999-02-25 2007-12-18 Pioneer Hi-Bred International, Inc. Methods of using viral replicase polynucleotides and polypeptides
US7459604B2 (en) * 2001-02-08 2008-12-02 Keygene N.V. Methods for generating resistance against CGMMV in plants
WO2002063019A2 (fr) * 2001-02-08 2002-08-15 Keygene N.V. Techniques permettant de generer une resistance contre le virus cgmmv dans des vegetaux
WO2002063019A3 (fr) * 2001-02-08 2002-11-28 Keygene Nv Techniques permettant de generer une resistance contre le virus cgmmv dans des vegetaux
WO2002077209A1 (fr) * 2001-03-26 2002-10-03 Plant Science Sweden Ab Plantes transgeniques possedant une resistance contre le virus de tobacco rattle et sequence nucleotidique correspondante
WO2009037369A1 (fr) 2007-09-21 2009-03-26 Consejo Superior De Investigaciones Cientificas Manipulation de la fonction du gène atdbp1 afin de générer une résistance au potyvirus
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CN116675751A (zh) * 2023-06-08 2023-09-01 山东农业大学 SWEET1g蛋白及其编码基因在抗马铃薯病毒中的应用
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