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WO1998003668A1 - Vegetaux a resistance phytovirale - Google Patents

Vegetaux a resistance phytovirale Download PDF

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Publication number
WO1998003668A1
WO1998003668A1 PCT/GB1997/001960 GB9701960W WO9803668A1 WO 1998003668 A1 WO1998003668 A1 WO 1998003668A1 GB 9701960 W GB9701960 W GB 9701960W WO 9803668 A1 WO9803668 A1 WO 9803668A1
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WO
WIPO (PCT)
Prior art keywords
plant
virus
rna
pvx
rdrp
Prior art date
Application number
PCT/GB1997/001960
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English (en)
Inventor
Iossif Grigorievich ATABEKOV
Youri Leonidovich Dorokhov
Sergey Yurievich Morozov
Original Assignee
Zeneca Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Zeneca Limited filed Critical Zeneca Limited
Priority to AU36279/97A priority Critical patent/AU3627997A/en
Publication of WO1998003668A1 publication Critical patent/WO1998003668A1/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
    • 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 virus resistance in plants.
  • RNA-dependent RNA polymerase RDRP; sometimes referred to as the viral "replicase"
  • This enzyme is responsible for synthesising complementary (-)-strand RNA, then using this RNA as a template for synthesis of genomic (+)-strand RNA (which can be packaged into virions) and (for some viruses) sub-genomic (+)-strand RNA's which can encode other structural and non-structural viral proteins.
  • genomic (+)-strand RNA which can be packaged into virions
  • sub-genomic (+)-strand RNA's which can encode other structural and non-structural viral proteins.
  • This process is shown diagramatically in Fig 1. Although it appears that host (plant) factors are also involved in viral RNA replication, the RDRP initiates (-)-strand RNA synthesis by binding to a specific sequence at the 3' end of the viral genome, synthesising RNA in the 5'— >3' direction.
  • the present invention seeks to provide, inter alia, a method for the protection of plants by inhibiting the replication of viruses, post infection.
  • a DNA construct comprising sequentially a promoter which is operable in plants, a polynucleotide comprising a sequence encoding an RNA capable of binding to an RNA dependent RNA polymerase of a plant virus, said RNA being the promoter region of a gene which when present in the viral genome is sub-genomic, and a terminator sequence, characterised in that the said sequence is heterologous with respect to the plant operable promoter.
  • a DNA costruct in which the polynucleotide is selected from the group of sequences as depicted in SEQ ID Nos.
  • DNA construct 1 or 2 as provided in the sequence listing herein, or is a polynucleotide which is complementary to one which when incubated at a temperature of between 60 and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequences as depicted in SEQ ID Nos. lor 2.
  • DNA constructs according to the present invention may contain a plant operable promoter which is constitutive, developmentally regulated or inducible. In addition to this the said promoter may be tissue specific and/or inducible.
  • the present invention further provides a method of imparting to or improving the ability of a plant to inhibit the replication of an infecting virus comprising inserting into the genome of plant material, a construct as described herein, regenerating said plant material, and selecting from the progeny those regenerants which have an improved ability to inhibit the infecting virus replication.
  • Transformation according to the present invention may be achieved via the Agrobaterium, particle mediated, fibre mediated or direct insertion methods.
  • the invention further provides a plant resulting from any of the aforesaid methods, characterised in that the said plant exhibits an increased inhibition of viral replication in said plant.
  • plants according to the present invention are potato, tomato, letuce and tobacco.
  • the invention may be considered to operate by producing an abundance of the RDRP binding sequence, which upon infection by a specific virus, will actively compete for binding of the RDRP produced by the infecting virus. Binding of the RDRP's to the "transgenic sites"(which are abundant, in-situ and immediately available for binding upon production of the viral RDRP) will reduce the degree of binding to the endogenous infecting virus. This will have the effect of inhibiting the production of infecting viral genomic RNA and thereby interrupting viral replication which depends thereon.
  • FIGURE 1 - A simplified infection cycle for a prototype (-t-)-strand RNA virus (based on TMV). Although the virus shown here is rod-shaped, not all (+)-strand RNA viruses are rods.
  • a typical infection cycle involves the following steps. Infectious virus ( 1 a) enters the cell (A) and begins to uncoat (B). Translation of this partially uncoated virion (2) ensues (C) and virus uncoating continues (D), resulting in synthesis of RDRP (3) and liberation of genomic RNA (4).
  • the RDRP (in concert with host factors) synthesises (-)-strand RNA (E;5) and subsequently both (+)-strand RNA (F;6) and, in many cases, (+)-strand sub-genomic RNA(s) (7).
  • RNA's are typically used as transcripts for proteins involved in processes such as virus spread as well as the CP (G;8).
  • the CP (8) and newly synthesised (+)-strand genomic RNA (6) can reassemble to form infectious virions (lb).
  • Virus movement throughout the plant may be potentiated by virion-like particles (perhaps involving other proteins such as the movement protein or MP) which could also be assembled at this stage (not shown).
  • FIGURE 2 A summary of the principle of virus protection through competition for RDRP binding by means of example involving; A transgenic plant produced containing a chimeric gene (A) that in turn produces a transcript containing (at least 1) "Virus A” RDRP binding sequence (B). This RNA serves no function in the cell unless "Virus A” enters the cell and initiates infection (C). Upon initiation of infection, "Virus A” will produce its RDRP (D). The transgenic RDRP binding sequence transcript will compete for RDRP binding. The result of successful binding to the "transgenic RDRP binding sites", illustrated at (E), will reduce the amount of RDRP available for binding to the endogenous sites thereby interrupting infecting viral replication.
  • a transgenic plant produced containing a chimeric gene (A) that in turn produces a transcript containing (at least 1) "Virus A” RDRP binding sequence (B). This RNA serves no function in the cell unless "Virus A” enters the cell and initiates infection (C). Upon initiation of infection, "
  • FIGURE 3 Diagram of "X2-TM53", or Construct 1 Construct lb is the plasmid pRTlOl containing "X2-TM53", capable of producing RNA's which are more stable when electroporated into Barley protoplasts
  • FIGURE 4 Diagram of "T2-X53", or Construct 2 Construct 2b is the plasmid pRT 101 containing "T2-X53", capable of producing RNA's which are more stable when electroporated into Barley protoplasts
  • FIGURE 5 Diagram of Construct 3. This is a negative control and comprises TMV 5' and 3' regions, i.e it contains no sub-genomic promoter binding sequences.
  • FIGURE 9 Diagram illustrating a multiple resistance model. This construct contains two copies (arranged in tandem) of PVX SgPr.RDRP binding sequence and TMV SgPr.RDRP binding sequence flanked by a promoter and terminator to enable their transcription.
  • FIGURE 10 Schematic representation of an inoculated tobacco leaf.
  • the diagram illustrates the leaf regions used for the ELIS A analysis and the distance between the regions.
  • constructs illustrated in these examples contain viral genomic promoter binding sequences (eg. Fig. 3. TMV 5' and 3' in the case of construct 1) flanking either side of the sub-genomic promoter (SgPr.) binding sequence
  • SgPr. sub-genomic promoter
  • the 5' and 3' sequences were used in construct preparation as a model for the protection of the transcript from degradation and as a means by which the viral RDRP specificity between genomic and sub-genomic promoters could be tested.
  • Early experiments provided results indicating that viral genomic promoters are not competitive inhibitors for viral RDRP. This inability of viral genomic promoters to inhibit virus replication has been reported elsewhere. For example Dawson 1991, Virology 184, 277-289 and White et al 1992. J. Virol. 66. 3069-3076
  • This invention relates to the use of Sub-genomic promoters which have demonstrated resistance to virus infection.
  • the invention requires the production of transgenic plants in which copies of a RDRP binding site accumulate in the plant. Constitutive accumulation may be sufficient but developmentally regulated and/or tissue-specific accumulation of RDRP binding sites may be preferred.
  • tobacco mosaic virus TMV is known to initiate plant infection in epidermal cells, and therefore it may be preferable to block infection specifically in those cells.
  • protection may be produced via accumulation of RDRP binding sites within phloem cells.
  • Another means by which the copy number of the transcript can be produced involves transformation of a plant with multiple copies of the sub-genomic promoter RDRP binding sequence. When the chimeric gene is transcribed, an increased number of binding sites may be produced.
  • Fig.1. parts 1-3. RDRP binding sequences already present in the cytoplasm of these cells (due to transcription of the nuclear transgene) compete with the virus genome for RDRP binding and if sufficient transgenic SgPr. RDRP binding sequences are present, infection should be reduced at this point, see Fig. 2. This would also be true for a multiple resistance model. Transgenic plants producing SgPr.
  • RDRP binding sites from heterologous viruses would exhibit resistance to either a single virus, or a combination of viruses from which the transgenic SgPr. binding sites were derived.
  • EXAMPLE 1 These experiments used a protoplast transient assay system, as a preliminary model for transgenic plant work. The experiments involved the co-introduction of RNA's containing RDRP binding sequences (synthesised in vitro) and infectious virus RNA. It has previously been shown that, for at least one other virus protection system, inhibition of virus replication can be obtained following co-introduction of the "protecting molecule" with the infectious particle.
  • RNA from Construct 1 consists of (from 5'— >3'): (i) the 5' end of the TMV genome (containing a sequence complementary to the TMV RDRP binding site), (ii) a tandem repeat of the complement of the PVX sequence (i.e.
  • RNA transcript produced from Construct 1 contains two copies of the minus(-) sense sub-genomic RDRP binding sites. The ability of this transcript to protect against virus infection is then tested by co- introducing TMV RNA or PVX RNA with transcript from Construct 1 into Barley protoplasts and monitoring TMV or PVX infection using an ELISA to the relevant CP. The results are shown in Table 1.
  • RNA from Construct 2 consists of (from 5'->3'): (i) the 5' end of the PVX genome (containing a sequence complementary to the PVX RDRP binding site), (ii) a tandem repeat of the complement of the TMV sequence found immediately upstream of the TMV capsid protein (CP) coding region in the virus RNA (each repeat contains a RDRP binding site which is used for synthesis of a sub-genomic RNA that produces TMV CP during infection), and (iii) the 3' end of the PVX genome (containing the PVX RDRP binding sequence).
  • RNA transcript produced from Construct 2 contains two copies of the minus(-) sense sub- genomic RDRP binding sites and the ability of this construct to protect against virus infection is tested by co-introducing PVX RNA or TMV RNA with transcript from Construct 2 into tobacco protoplasts and monitoring PVX or TMV infection using an ELISA to the relevant CP. The results are shown in Table 2.
  • constructs 1 and 2 were unsuccessful, probably due to the instability of the chimeric RNA's when elecroporated into the protoplasts because the RNA's were lacking a "cap structure". In order to circumvent this problem the constructs 1 and 2 were cloned into the plasmid pRTlOl under the influence of the 35S promoter and a poly a signal (see Fig. 3b and 4b).
  • Construct 3 As for construct 1, except without any SgPr. RDRP binding sequences. This construct was produced as a negative control and to identify whether under these circumstances transgenic binding sites using viral genomic promoters will compete for viral RDRP. The construct also aimed to determine whether the binding sites are virus specific.
  • Construct 4 As for construct I, except contains 1 copy of the SgPr. RDRP binding sequence from PVX.
  • Construct 5 As for construct 1 , except contains 4 copies of the SgPr. RDRP binding sequence from PVX.
  • Construct 6 As for construct 1, except contains 8 copies of the SgPr. RDRP binding sequence from PVX
  • This construct comprises from 5' to 3', a 5' region containing a promoter capable of driving the transcription of the coding sequence. Tandem repeats of PVX sub-genomic promoter RDRP binding sites in positive sense (+). Tandem repeats of TMV SgPr. RDRP binding sites in positive sense (+). A 3' region containing a terminator capable of terminating the transcription of the coding sequence.
  • the initial experiments used tobacco mosaic virus (TMV) and potato virus X (PVX), two viruses which are capable of infecting protoplasts. Electroporation was used to introduce the
  • RNA's into the protoplasts Barley protoplasts were transformed with chime ⁇ c RNA transc ⁇ pts produced in vitro from construct 1 by electroporation and infected with TMV and PVX. See Table 1.
  • PVX 3 2 control, vi ⁇ on DNA only
  • TMV 3.2 control, virion DNA only
  • Tobacco plants (Nicotiana sp.) were transformed with construct lb (pRTlOl "X2-TM53") containing two copies of PVX SgPr. binding sites, arranged in tandem and construct 3 (pRTlOl
  • Plant Line Inoculated leaf regions Upper non inoculated leaf a b c d non 1.309+/-0.05 0.805+/-0.32 0.263+/-0.12 0.052+/-0.02 1.71747-0.49 transgenic
  • pi -2 Plants transformed with construct 3 see fig.5.
  • pi -2(1) Plant line 1, transformed with construct lb see fig.3.
  • p 1-2(11) Plant line 2, transformed with construct lb see f ⁇ g.3.
  • RDRP binding sites is a process that is virus specific. Clarification of which is illustrated in tables 7 and 8.
  • MOLECULE TYPE other nucleic acid

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Plant Pathology (AREA)
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  • Biophysics (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

Cette invention a trait à une technique visant à améliorer la résistance d'un organisme à une infection due à un virus spécifique ou bien à lui conférer cette résistance. Cette technique consiste à produire un produit de transcription d'ARN usurpant l'identité du site de fixation de l'ARN polymérase - ARN-dépendante du virus spécifique (RDPR), cette polymérase étant produite par le virus infectieux. Ces sites de fixation transgéniques entrent en concurrence avec la RDPR et la fixation de cette RDPR à la séquence transgénique plutôt qu'au virus infectieux débouche sur une incapacité du virus infectieux à se répliquer avec succès. Cette technique donne de remarquables résultats, s'agissant de faire reculer l'infection virale.
PCT/GB1997/001960 1996-07-22 1997-07-21 Vegetaux a resistance phytovirale WO1998003668A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36279/97A AU3627997A (en) 1996-07-22 1997-07-21 Virus resistance in plants

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GBGB9615349.9A GB9615349D0 (en) 1996-07-22 1996-07-22 Virus resistance in plants
GB9615349.9 1996-07-22

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999049031A1 (fr) * 1998-03-26 1999-09-30 Advanced Research And Technology Institute, Inc. Utilisation de molecules d'acide nucleique comme agents antiviraux
WO2000004141A3 (fr) * 1998-07-20 2000-04-27 Ribozyme Pharm Inc Utilisation de molecules d'acide nucleique comme agents antiviraux
CN111513077A (zh) * 2020-05-25 2020-08-11 中国农业科学院烟草研究所 一种RNAi纳米制剂及其制备方法和在TMV防治中的应用

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EP0425004A2 (fr) * 1989-10-03 1991-05-02 Aveve N.V. Manipulations génétiques avec l'ADN recombinant, contenant des séquences dérivées des virus d'ARN
WO1991013994A1 (fr) * 1990-03-13 1991-09-19 Commonwealth Scientific And Industrial Research Organisation Expression de genes
EP0479180A2 (fr) * 1990-10-05 1992-04-08 Hoechst Aktiengesellschaft Plantes résistantes aux virus, procédé pour leur production
WO1994016089A1 (fr) * 1992-12-30 1994-07-21 Biosource Genetics Corporation Amplification virale de l'arn messager recombine dans les plantes transgeniques
WO1994029464A1 (fr) * 1993-06-04 1994-12-22 Sandoz Ltd. Plantes resistantes aux virus
US5466788A (en) * 1985-03-07 1995-11-14 Mycogen Plant Science, Inc. Subgenomic promoter
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US5466788A (en) * 1985-03-07 1995-11-14 Mycogen Plant Science, Inc. Subgenomic promoter
CN1033645A (zh) * 1988-10-22 1989-07-05 中国科学院上海植物生理研究所 控制植物病毒病的基因工程方法
EP0425004A2 (fr) * 1989-10-03 1991-05-02 Aveve N.V. Manipulations génétiques avec l'ADN recombinant, contenant des séquences dérivées des virus d'ARN
WO1991013994A1 (fr) * 1990-03-13 1991-09-19 Commonwealth Scientific And Industrial Research Organisation Expression de genes
EP0479180A2 (fr) * 1990-10-05 1992-04-08 Hoechst Aktiengesellschaft Plantes résistantes aux virus, procédé pour leur production
WO1994016089A1 (fr) * 1992-12-30 1994-07-21 Biosource Genetics Corporation Amplification virale de l'arn messager recombine dans les plantes transgeniques
WO1994029464A1 (fr) * 1993-06-04 1994-12-22 Sandoz Ltd. Plantes resistantes aux virus
EP0806481A2 (fr) * 1996-05-09 1997-11-12 Metapontum Agrobios s.c.r.l. Méthode de préparation de plantes transgéniques résistantes aux infections virales et plantes obtenues

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CHEMICAL ABSTRACTS, vol. 113, no. 15, 8 October 1990, Columbus, Ohio, US; abstract no. 127723, JUN, W.: "Preparation of transgenic plants for control of virosis" XP002027388 *
HEMENWAY, C., ET AL.: "Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA", THE EMBO JOURNAL, vol. 7, no. 5, 1988, pages 1273 - 1280, XP002047300 *
HUNTLEY, C.C., ET AL.: "Minus sense transcripts of brome mosaic virus RNA-3 intercistronic region interfere with viral replication", VIROLOGY, vol. 192, 1993, pages 290 - 297, XP002047298 *
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POWELL P A ET AL: "PROTECTION AGAINST TOBACCO MOSAIC VIRUS IN TRANSGENIC PLANTS THAT EXPRESS TOBACCO MOSAIC VIRUS ANTISENSE RNA", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 86, no. 18, 1 September 1989 (1989-09-01), pages 6949 - 6952, XP000068469 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999049031A1 (fr) * 1998-03-26 1999-09-30 Advanced Research And Technology Institute, Inc. Utilisation de molecules d'acide nucleique comme agents antiviraux
WO2000004141A3 (fr) * 1998-07-20 2000-04-27 Ribozyme Pharm Inc Utilisation de molecules d'acide nucleique comme agents antiviraux
CN111513077A (zh) * 2020-05-25 2020-08-11 中国农业科学院烟草研究所 一种RNAi纳米制剂及其制备方法和在TMV防治中的应用
CN111513077B (zh) * 2020-05-25 2021-05-25 中国农业科学院烟草研究所 一种RNAi纳米制剂及其制备方法和在TMV防治中的应用

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GB9615349D0 (en) 1996-09-04

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