WO2005098004A2 - Stimulation inductible de satellites integres - Google Patents

Stimulation inductible de satellites integres Download PDF

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Publication number
WO2005098004A2
WO2005098004A2 PCT/EP2005/003864 EP2005003864W WO2005098004A2 WO 2005098004 A2 WO2005098004 A2 WO 2005098004A2 EP 2005003864 W EP2005003864 W EP 2005003864W WO 2005098004 A2 WO2005098004 A2 WO 2005098004A2
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Prior art keywords
plant
gene
virus
rna
nucleotides
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PCT/EP2005/003864
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English (en)
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WO2005098004A3 (fr
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Michael Metzlaff
Veronique Gossele
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Bayer Bioscience N.V.
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Publication of WO2005098004A2 publication Critical patent/WO2005098004A2/fr
Publication of WO2005098004A3 publication Critical patent/WO2005098004A3/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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • Methods and means are provided to improve the silencing phenotype when using particular ?RNA vectors derived from satellite viruses and at least one corresponding helper virus to introduce inhibitory RNA into plant cells or cells of a plant.
  • Stable introduction of a chimeric gene which upon transcription yield the particular -RNA vectors, or their complementary sequences, into the cells of a plant enhances and prolongs the silencing phenotypes observed upon infection with a corresponding helper virus.
  • the timing of the silencing phenotype can now be manipulated at will, i.e. is inducible, as it becomes dependent upon the inoculation by the corresponding helper virus.
  • Satellite viruses have been observed in plants to accompany several autonomous viruses, i.e. tobacco mosaic virus, tobacco necrosis virus, panicum mosaic virus and maize white line mosaic virus. Satellite viruses encode their own coat protein needed for virus particle assembly but their replication depends on the supply of viral replicase in trans from an appropriate, corresponding helper virus. Co-inoculations of both the helper and the satellite virus, result in systemic accumulation of satellite virus particles or viral ?RNA in infected plants at levels, which significantly exceed the level of accumulating helper virus particles! (10- 100 fold).
  • WO00/63397 and WO2003/052108 (herein incorporated by reference) and Gosssele et al. (2002) describe the use of satellite viruses as vectors for the transient delivery of gene silencing-inducing inhibitory -RNA. It was demonstrated that the satellite virus coat protein coding region is dispensable for replication and systemic spread, when in- vitro transcribed satellite virus ?RNA is co-infected with a corresponding helper virus, such as e.g. TMN-U2 particles. Those infected plants were symptom-less unless viral R ⁇ A vectors derived from these satellite viruses carrying insertions of short fragments of endogenous plant genes were used.
  • the virus infection and consequently also the induction of post-transcriptional gene silencing, attenuates in the recovery phase, which continuously develops post infection by activation of the plant defense system.
  • the viral -RNA vectors comprising a gene-silencing construct, as described in WO2003/052108, as a cDNA copy, operably linked to a plant expressible promoter and optionally transcription termination signals; stabl into the genome of plant cells.
  • the mRNA transcribed from such a chimeric gene does not encode a viral replicase, and hence cannot be amplified.
  • the viral -RNA vector replicates and forms double-stranded RNA structures that trigger the gene silencing.
  • RNA using the transgenic method herein described will also compensate any potential negative effect of RNA replication and accumulation which may be caused by large inserts of foreign DNA in the viral RNA vector.
  • Gleba et al., 2004 describe engineering viral expression vectors for plants using a "deconstructed virus” strategy.
  • nucleotide sequence encoding the N-terminal amino acids of the coat protein may comprises nucleotides 162 to 328 of SEQ ID No 2 and the ten contiguous nucleotides may be the nucleotides from position 1365 to position 1374 of SEQ ID No 1.
  • the ?RNA vector may comprise nucleotides 1365 to 1385 of SEQ ID No 1 or 1365 to 1386 of SEQ ID No 1 or 1365 to 1394 of SEQ ID No 1.
  • the viral ?RNA vector may be derived from satellite tobacco mosaic virus (STMV).
  • the viral RNA vector may not comprise a nucleotide sequence essentially similar to the nucleotide sequence normally encoding the complete coat protein, if the essentially similar sequence does not encode a functional coat protein.
  • this essentially similar nucleotide sequence is non-functional, for example due to the presence of the short stretch of nucleotides 1365 to 1394 of SEQ ID No 1 between the nucleotides normally encoding the N-terminal amino acids and the nucleotides normally encoding the central and C- terminal amino acids of the coat protein.
  • the nucleotide sequence comprising the N-terminal amino acids of a coat protein may be either derived from a wild type satellite virus, particularly from STMV, or natural variants or strains thereof, or it may be synthetic. It may also comprise modifications, such as nucleotide changes, deletions or insertions.
  • the gene-silencing constructs may comprise at the same time sense and anti-sense ?RNA targeted towards the same nucleotide sequence whose expression is to be reduced.
  • the sense and antisense ?RNA may be at least partly complementary to each other and capable of forming a stem- loop structure.
  • Such a configuration has been shown to increase the efficiency of gene-silencing, both in occurrence and level of gene-silencing (Waterhouse et al. 1998).
  • a vector comprising a gene- silencing construct may comprise more than one gene-silencing construct, and that several target gene(s) or gene-families may be silenced as a result of carrying out the method of the invention with such vector.
  • plants are inoculated with a solution containing a corresponding heiper virus.
  • the solution may further contain additional compounds to improve inoculation and infection of the plants, such as, but not limited to abrasives, adherents, tensio-active products and the like.
  • Plants may be infected during different developmental stages, in order to maximize the gene- silencing phenotype under investigation.
  • different parts of plants may be inoculated to optimize observation of the gene-silencing phenotype.
  • a “gene silencing phenotype” refers to the phenotype of a plant, into which an inhibitory RNA for one or more target genes has been introduced, and where the inhibitory ?RNA has caused a quantitative or qualitative change in the phenotype compared to the phenotype of plants into which said inhibitory ?RNA has not been introduced.
  • Transgenic plants according to the invention may develop a gene-silencing phenotype after inoculating tissue with a corresponding helper virus.
  • the gene-silencing phenotype is not observed in plants which are not inoculated or in plants which are mock-inoculated.
  • the gene-silencing phenotype may be morphological, such as chlorosis, necrosis, photobleaching, tissue distortion, or it may be developmental, such as a change in the time of flowering, it may be lethal to, or have severe effects on the normal development of the plant tissue if essential genes are targeted, or it may be molecular, such as changes in concentrations of molecules or metabolites in the tissue.
  • the methods to assay and/or quantify the gene-silencing phenotype may be diverse and may have to be adapted according to each gene silencing phenotype.
  • a phenotype which manifests in a macroscopically visible phenotype visual assessment may be made. If the phenotype does not manifest itself in a way that is macroscopically visible alternative methods of assessment may need to be employed. Such methods may comprise analysis of molecule or metabolite presence and/or concentrations, microscopic assays or enzyme assays. This is not to mean that in the case that the phenotype manifests itself in a macroscopically visible phenotype, alternative assays for assessment may not be used, such as but not limited to detection of presence and concentration of mRNA of the target gene(s) in the plant tissue. ' • '
  • the chimeric gene comprising the cDNA copy of the viral RNA vector is operably linked to a plant-expressible promoter and optionally to a 3' end transcription termination signal.
  • plant-expressible promoter means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Hapster et al., 1988), the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue- specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), orgari-primordia specific promoters (An et al., 1996), stem-specific promoters (Keller et al., 1988), leaf specific promoters (Hudspeth et al., 1989), mesophyl-specific promoters (such as the light-inducible Rub
  • the promoter may be a constitutive promoter, such as the Cauliflower Mosaic Virus 35S promoter described in US5352605 and 5530196; enhanced 35S promoter as described in US5164316; the Cassava Vein Mosaic Virus promoter, as described in WO97/48819; the maize ubiquitin promoter, as described in EP342926; the Arabidopsis actin 2 promoter as described in An et al. (1996); or the rice actin promoter as described in US5641876. '
  • a promoter can be utilized which is specific for one or more tissues or organs (e.g. leaves and/or roots) of a plant.
  • tissues or organs e.g. leaves and/or roots
  • the light inducible promoter of the gene encoding the small subunit of ribulose 1,5-bisphosphate as described in US5034322 is preferentially active in leaves, while WO00/29566 describes a promoter preferentially active in roots.
  • an inducible promoter may be used.
  • Such a promoter may be induced after application of a chemical, for example a dexamethasone inducible promoter as described in by Aoyama and Chua (1997) or by a change in temperature, for example a heat shock promoter as described in US5447858 or in Severin and Schoef-fl (1990), or a promoter induced by other external stimuli.
  • a chemical for example a dexamethasone inducible promoter as described in by Aoyama and Chua (1997) or by a change in temperature, for example a heat shock promoter as described in US5447858 or in Severin and Schoef-fl (1990), or a promoter induced by other external stimuli.
  • 3' non-translated sequences or 3' transcription termination signals are well -known in the art and a suitable 3' nontranslated sequence may be obtained from a nopaline synthase gene, from an octopine synthase gene (Gielen et al. 1984) or from the T- DNA gene7 (Velten et al. 1985; Dhaese et al. 1983).
  • RNA which is biologically active i.e., which is either capable of interaction with another nucleic acid or which is capable of being translated into a biologically active polypeptide or protein.
  • a gene is said to encode an RNA when the end product of the expression of the gene is biologically active RNA, such as e.g. an antisense ?RNA, a ribozyme or a replicative intermediate.
  • a gene is said to encode a protein when the end product of the expression of the gene is a biologically active protein or polypeptide.
  • a gene may further comprise elements for cap-independent translation such as an internal ribosome entry sequence or the first and second translation enhancing elements as defined in WO 97/49814.
  • the alignment of the two sequences is performed using the Wilbur and Lipmann algorithm (Wilbur and Lipmann ,1983) using a window-size of 20 nucleotides or amino acids, a word length of 2 amino acids, and a gap penalty of 4.
  • SEQ ID No 1 nucleotide sequence of the tomato phytoene desaturase (pds) encoding cDNA (Genbank Accession No. X59948)
  • SEQ ID No 2 nucleotide sequence of the genome of STMV (Genbank Accession No. M25782).
  • SEQ ID No 3 nucleotide sequence of T-DNA vector pTVE481 (Example 1)
  • Example 1 Construction of a T-DNA vector comprising a cDNA copy of an STMV-derived viral vector RNA with capable of reducing the expression of a phytoene desaturase gene.
  • a chimeric gene was constructed comprising a cDNA copy of an STMV derived viral vector RNA capable of reducing the expression of a phytoene desaturase gene. To this end, the following fragments were operably linked using conventional cloning techniques:
  • This chimeric gene was introduced into a T-DNA vector between the T-DNA border sequences together with a chimeric bar gene, to create T-DNA vector pTVE481.
  • the T-DNA vector was introduced into an Agrobacterium strain containing a helper Ti- plasmid (pGV4100).
  • the T-DNA was introduced via Agrobacterium mediated transformation, according to standard protocols, into cells of tobacco SRI, and transgenic tobacco plants comprising the T-DNA with the chimeric genes were regenerated. Full green primary transformants were grown in soil.
  • Example 2 Analysis of gene-silencing in transgenic Nicotiana tabacum plants upon infection with a helper virus.
  • Transgenic plants comprising the STMV-derived viral RNA vector with the pds silencing RNA region cloned as a cDNA copy under control of a CaMV35S promoter, were infected at the three leaf-stage with Tobacco Mosaic Virus U2 (see WO2003/052108). 8-10 days post inoculation a "pds knock-out phenotype" (white photo-bleached leaves, stems and flower bud carpels) developed in all seven inoculated plants. 14 days post inoculation all newly emerging leaves showed a very strong pds knock-out phenotype. None of the non-inoculated transformants used as control showed a similar silencing phenotype of the pds gene.

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Abstract

L'invention concerne des procédés et des moyens permettant d'améliorer le silençage génique lors de l'utilisation de vecteurs d'ARN spécifiques dérivés de virus à satellite et d'au moins un virus assistant correspondant, de manière à introduire un ARN inhibiteur dans des cellules végétales ou des cellules d'un végétal, caractérisés en qu'ils consistent en l'introduction stable dans les cellules d'un végétal d'un gène chimère produisant, au moment de la transcription, des vecteurs d'ARN spécifiques ou des séquences complémentaires de ceux-ci.
PCT/EP2005/003864 2004-04-07 2005-04-06 Stimulation inductible de satellites integres WO2005098004A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP04076007.6 2004-04-07
EP04076007 2004-04-07
US62885004P 2004-11-17 2004-11-17
US60/628,850 2004-11-17

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WO2005098004A3 WO2005098004A3 (fr) 2005-12-01

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011089021A1 (fr) 2010-01-25 2011-07-28 Bayer Bioscience N.V. Procédés de fabrication de parois de cellules végétales comprenant de la chitine
WO2012004013A2 (fr) 2010-07-08 2012-01-12 Bayer Bioscience N.V. Protéine transportrice de glucosinolate et ses utilisations
WO2012093032A1 (fr) 2011-01-04 2012-07-12 Bayer Cropscience N.V. Promoteurs dans certaines fibres
WO2012101118A1 (fr) 2011-01-24 2012-08-02 Bayer Cropscience N.V. Utilisation du promoteur rd29 ou de fragments de celui-ci pour l'expression de transgènes inductible par le stress chez le coton
WO2012136788A1 (fr) 2011-04-07 2012-10-11 Bayer Cropscience Nv Promoteur spécifique de graine dans le coton
WO2013023992A1 (fr) 2011-08-12 2013-02-21 Bayer Cropscience Nv Expression spécifique des cellules de garde de transgènes dans le coton
WO2013026740A2 (fr) 2011-08-22 2013-02-28 Bayer Cropscience Nv Procédés et moyens pour modifier un génome de plante
WO2014118123A1 (fr) 2013-01-29 2014-08-07 The University Court Of The University Of Glasgow Procédés et moyens pour augmenter la tolérance aux contraintes et la biomasse dans des plantes
WO2015000914A1 (fr) 2013-07-01 2015-01-08 Bayer Cropscience Nv Procédés et moyens pour moduler la durée de floraison de plantes monocotylédones
WO2016050512A1 (fr) 2014-10-03 2016-04-07 Bayer Cropscience Nv Procédés et moyens pour augmenter la tolérance au stress et la biomasse chez des plantes
WO2016113333A1 (fr) 2015-01-16 2016-07-21 Bayer Cropscience Nv Promoteurs préférentiels de gousses et leurs utilisations
WO2016128519A1 (fr) 2015-02-12 2016-08-18 Bayer Cropscience Nv Promoteurs préférentiels de l'apex de pousse et utilisations de ces promoteurs
WO2017060232A1 (fr) 2015-10-08 2017-04-13 Bayer Cropscience Nv Promoteurs préférentiels de graines et leurs utilisations
WO2017064173A1 (fr) 2015-10-16 2017-04-20 Bayer Cropscience Nv Plantes brassica dotées de propriétés modifiées de production de semences
WO2017178318A1 (fr) 2016-04-11 2017-10-19 Bayer Cropscience Nv Promoteurs spécifiques des graines et préférentiels de l'endosperme et leurs utilisations
WO2017178322A1 (fr) 2016-04-11 2017-10-19 Bayer Cropscience Nv Promoteurs spécifiques des graines et préférentiels de l'endosperme et leurs utilisations
WO2017178367A1 (fr) 2016-04-13 2017-10-19 Bayer Cropscience Nv Promoteurs préférentiels des graines et du funicule et leurs utilisations
WO2017178368A1 (fr) 2016-04-13 2017-10-19 Bayer Cropscience Nv Promoteurs spécifiques des graines et préférentiels de l'embryon et leurs utilisations
WO2017205665A1 (fr) 2016-05-25 2017-11-30 Cargill, Incorporated Nucléases modifiées pour générer des mutants de délétion dans des plantes
US10093907B2 (en) 2013-09-24 2018-10-09 Basf Se Hetero-transglycosylase and uses thereof
WO2020142598A2 (fr) 2019-01-04 2020-07-09 Cargill, Incorporated Nucléases modifiées pour générer des mutations dans des plantes
WO2024099765A2 (fr) 2022-11-10 2024-05-16 BASF Agricultural Solutions Seed US LLC Séquences nucléotidiques régulant la transcription et procédés d'utilisation

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WO2000063397A2 (fr) * 1999-04-20 2000-10-26 Aventis Cropscience N.V. Methodes et moyens d'administration d'arn inhibiteur a des vegetaux et applications associees
WO2003052108A2 (fr) * 2001-12-18 2003-06-26 Bayer Bioscience N.V. Methodes ameliorees et dispositif d'administration d'arn inhibiteur a des vegetaux et applications associees

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Publication number Priority date Publication date Assignee Title
WO2000063397A2 (fr) * 1999-04-20 2000-10-26 Aventis Cropscience N.V. Methodes et moyens d'administration d'arn inhibiteur a des vegetaux et applications associees
WO2003052108A2 (fr) * 2001-12-18 2003-06-26 Bayer Bioscience N.V. Methodes ameliorees et dispositif d'administration d'arn inhibiteur a des vegetaux et applications associees

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Title
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METZLAFF M. ET AL: "Satellite RNA Virus-based transient and inducible gene silencing in tobacco" SYMPOSIUM "NEW DIMENSIONS OF RNA IN CELLULAR FUNCTIONS", [Online] 21 February 2005 (2005-02-21), - 22 February 2005 (2005-02-22) XP002344682 Hokkaido University Conference Hall, Sapporo , Japan Retrieved from the Internet: URL:http://arabi4.agr.hokudai.ac.jp/RNA/ab stract/metzlaff.html> [retrieved on 2005-09-12] *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011089021A1 (fr) 2010-01-25 2011-07-28 Bayer Bioscience N.V. Procédés de fabrication de parois de cellules végétales comprenant de la chitine
US9279130B2 (en) 2010-01-25 2016-03-08 Bayer Cropscience Nv Methods for manufacturing plant cell walls comprising chitin
WO2012004013A2 (fr) 2010-07-08 2012-01-12 Bayer Bioscience N.V. Protéine transportrice de glucosinolate et ses utilisations
WO2012093032A1 (fr) 2011-01-04 2012-07-12 Bayer Cropscience N.V. Promoteurs dans certaines fibres
US9243260B2 (en) 2011-01-04 2016-01-26 Bayer Cropscience Nv Fiber selective promoters
WO2012101118A1 (fr) 2011-01-24 2012-08-02 Bayer Cropscience N.V. Utilisation du promoteur rd29 ou de fragments de celui-ci pour l'expression de transgènes inductible par le stress chez le coton
WO2012136788A1 (fr) 2011-04-07 2012-10-11 Bayer Cropscience Nv Promoteur spécifique de graine dans le coton
WO2013023992A1 (fr) 2011-08-12 2013-02-21 Bayer Cropscience Nv Expression spécifique des cellules de garde de transgènes dans le coton
US9670496B2 (en) 2011-08-22 2017-06-06 Bayer Cropscience N.V. Methods and means to modify a plant genome
WO2013026740A2 (fr) 2011-08-22 2013-02-28 Bayer Cropscience Nv Procédés et moyens pour modifier un génome de plante
US10538774B2 (en) 2011-08-22 2020-01-21 Basf Agricultural Solutions Seed, Us Llc Methods and means to modify a plant genome
WO2014118123A1 (fr) 2013-01-29 2014-08-07 The University Court Of The University Of Glasgow Procédés et moyens pour augmenter la tolérance aux contraintes et la biomasse dans des plantes
WO2015000914A1 (fr) 2013-07-01 2015-01-08 Bayer Cropscience Nv Procédés et moyens pour moduler la durée de floraison de plantes monocotylédones
EP3431606A1 (fr) 2013-07-01 2019-01-23 Bayer CropScience NV Procédés et moyens pour moduler le temps de floraison dans des plantes monocotylédones
US11447791B2 (en) 2013-07-01 2022-09-20 Basf Se Methods and means for modulating flowering time in monocot plants
US10472645B2 (en) 2013-07-01 2019-11-12 Basf Se Methods and means for modulating flowering time in monocot plants
US10647965B2 (en) 2013-09-24 2020-05-12 Basf Se Hetero-transglycosylase and uses thereof
US10093907B2 (en) 2013-09-24 2018-10-09 Basf Se Hetero-transglycosylase and uses thereof
WO2016050512A1 (fr) 2014-10-03 2016-04-07 Bayer Cropscience Nv Procédés et moyens pour augmenter la tolérance au stress et la biomasse chez des plantes
WO2016113333A1 (fr) 2015-01-16 2016-07-21 Bayer Cropscience Nv Promoteurs préférentiels de gousses et leurs utilisations
WO2016128519A1 (fr) 2015-02-12 2016-08-18 Bayer Cropscience Nv Promoteurs préférentiels de l'apex de pousse et utilisations de ces promoteurs
WO2017060232A1 (fr) 2015-10-08 2017-04-13 Bayer Cropscience Nv Promoteurs préférentiels de graines et leurs utilisations
WO2017064173A1 (fr) 2015-10-16 2017-04-20 Bayer Cropscience Nv Plantes brassica dotées de propriétés modifiées de production de semences
WO2017178318A1 (fr) 2016-04-11 2017-10-19 Bayer Cropscience Nv Promoteurs spécifiques des graines et préférentiels de l'endosperme et leurs utilisations
US10975380B2 (en) 2016-04-11 2021-04-13 Basf Agricultural Solutions Seed, Us Llc Seed-specific and endosperm-preferental promoters and uses thereof
WO2017178322A1 (fr) 2016-04-11 2017-10-19 Bayer Cropscience Nv Promoteurs spécifiques des graines et préférentiels de l'endosperme et leurs utilisations
WO2017178368A1 (fr) 2016-04-13 2017-10-19 Bayer Cropscience Nv Promoteurs spécifiques des graines et préférentiels de l'embryon et leurs utilisations
WO2017178367A1 (fr) 2016-04-13 2017-10-19 Bayer Cropscience Nv Promoteurs préférentiels des graines et du funicule et leurs utilisations
US10900046B2 (en) 2016-04-13 2021-01-26 BASF Agricultural Solutions Seed US LLC Seed- and funiculus-preferential promoters and uses thereof
WO2017205665A1 (fr) 2016-05-25 2017-11-30 Cargill, Incorporated Nucléases modifiées pour générer des mutants de délétion dans des plantes
WO2020142598A2 (fr) 2019-01-04 2020-07-09 Cargill, Incorporated Nucléases modifiées pour générer des mutations dans des plantes
WO2024099765A2 (fr) 2022-11-10 2024-05-16 BASF Agricultural Solutions Seed US LLC Séquences nucléotidiques régulant la transcription et procédés d'utilisation

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