WO2005098004A2 - Inducible boost of integrated satellite rna viruses - Google Patents

Abstract

Methods and means are provided to improve gene silencing 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 characterized by the stable introduction into the cells of a plant of a chimeric gene which upon transcription yield the particular RNA vectors, or their complementary sequences.

Description

INDUCIBLE BOOST OF INTEGRATED SATELLITES.

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. As an additional advantage, 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.

?RNA 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 RΝA vectors derived from these satellite viruses carrying insertions of short fragments of endogenous plant genes were used. Pronounced "knockout phenotypes" due to induction of post-transcriptional gene silencing were described. Due to the transient character of helper virus infection, and thus also of the satellite virus replication cycle, the silencing phenotype occurs also transiently and disappears, when the plants recover from virus infection. As a consequence, the following limitations may be associated with the prior art methods of introducing inhibitory -RNA into plant cells:

1. Only on average 50 % of all inoculated plants show a gene silencing phenotype depending on plant vigor and the efficiency of helper virus infection combined with the efficiency of replication of the viral -RNA vector.

2. 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.

3. The efficiency of satellite virus replication can be significantly reduced, when larger plant target gene fragments are inserted.

4. Insertion of foreign sequences into satellite viruses can result in reduction or loss of systemic spread of satellite RNA and thereby in loss of induction of gene silencing in newly emerging tissue during plant development.

It has now been found that these problems can be solved by introducing 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. Upon infection of a corresponding helper virus, the viral -RNA vector replicates and forms double-stranded RNA structures that trigger the gene silencing. Using this method, an enhanced and prolonged silencing phenotype can be observed, when compared to the methods wherein similar helper virus and viral ?RNA vectors are provided transiently to the cells. Without intending to limit the invention to a particular mode of action, it is contemplated that since the satellite virus derived -RNA vector is already present in form of transgenic m-RNA before replication starts (using the in trans provided functions by the helper virus infection), an initial lag- phase in satellite virus derived vector ?RNA accumulation, which often causes problems in reaching sufficient levels of satellite virus -RNA at later time-points, can be overcome. Because of a much higher initial level of satellite virus derived vector ?RNA, compared with levels obtained via in-vitro-RNA inoculations, the level of satellite virus derived ?RNA vector is rapidly boosted to higher levels after helper virus inoculation.

The higher initial level of viral vector ?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.

Moreover, the method of introducing inhibitory -RNA can now also be applied more efficiently in plants w-hich support poorly the replication and/or systemic spread of the satellite-virus derived ?RNA vectors since the initial viral RNA vector may be already distributed equally throughout the plant as a consequence of transcription of the transgene.

In addition, the gene-silencing can be induced at any time, at will, during plant development, determined by the time of inoculation with the corresponding helper virus.

WO98/36083 discloses are DNA constructs ("amplicons"™ ) comprising a promoter operably linked to DNA which can be transcribed in a plant cell to an RNA transcript, which ?RNA transcript includes plant virus sequence (e.g. derived from potato virus X) which confers on the ?RNA transcript the ability to replicate in the cytoplasm of the plant cell. The constructs may lack portions of the viral genome not required for such replication (e.g. coat proteins) but preferably include targeting sequence which is capable of down-regulating (for instance gene silencing) expression of one or more target genes, which may be endogenous plant genes, transgenes, or pathogen genes. Also disclosed are corresponding vectors, host cells, plants (particularly having modified and/or silenced genes or being pathogen resistant) and plant products, plus also methods of producing and using these materials. However, this disclosure remains silent on the use of chimeric genes comprising a cDNA copy of satellite derived RNA vectors, or any of the beneficial effects associated with such methods.

WOOO/53780 features a multiple component RNA vector system, which consists of ?RNA virus-derived RNA replicons and helper viruses. The present invention further features a method for producing foreign RNAs, effector ?RNAs, proteins or peptides in plants using the multiple component ?RNA vector system. Moreover, the document discloses a method for stable and systemic production of foreign RNAs, effector ?RNAs, proteins and peptides using the multiple component ?RNA vector system.

WO 90/12107 provides a sequence of the RNA genome of satellite tobacco mosaic virus. Based on that sequence and the discovery that heterologous ?RNA can be accommodated in the genome without eliminating replicability, the invention provides compositions comprising modified genomes of the virus, or cDNAs of such genomes, to transform plant cells in vitro and in vivo to make desired ?RNAs or proteins. Further, the document provides methods and intermediates for making such compositions and methods of using such compositions.

Gleba et al., 2004 describe engineering viral expression vectors for plants using a "deconstructed virus" strategy.

The latter three of the above prior art documents hint at the possibility of providing viral RNA vectors derived from satellite viruses capable of silencing of a target gene, as a chimeric gene, at a hypothetical level. None of the documents, however, describe that a prolonged and enhanced silencing effect can be obtained as described in the current application for the specific viral -RNA vectors.

SUMMARY OF THE INVENTION

In one embodiment of the invention, a method is provided for introducing inhibitory RNA into cells of a plant comprising the steps of providing a transgenic plant with a chimeric gene comprising a DNA copy of a viral RNA vector stably integrated into the genome of the cells of the plant a chimeric gene, and infecting said transgenic plants with a corresponding helper virus, whereby the chimeric gene comprises, operably linked to a plant expressible promoter (such as, but not limited to a constitutive promoter or a tissue-specific promoter) and optionally a transcription termination signal, a DNA region which when transcribed yields a viral -RNA vector, the viral RNA vector being derived from a satellite RNA virus and comprising the N- terminal amino acids from the coat protein of the satellite virus; 10 contiguous nucleotides of nucleotide 1365 to 1394 of SEQ ID No 1 (i.e. selected from the nucleotide sequence of SEQ ID Nol from the nucleotide at position 1365 to the nucleotide at position 1394); and a nucleotide sequence capable of reducing the expression of a gene of interest in cells of the plant. The nucleotide sequence capable of reducing the expression of the gene of interest may comprise a nucleotide sequence of at least 27 nucleotides in length having at least 75% sequence identity to a nucleotide sequence within the target igene or the complement thereof. The target gene may be an endogenous gene or a transgene. Further, the 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).

In another embodiment, the invention relates to the use of a kit for introducing inhibitory RNA into a plant cell, comprising a cDNA copy of a viral vector as herein described, operably linked to a plant expressible promoter and a transcription termination signal, and a corresponding helper virus, characterized in that the cDNA copy of said viral ?RNA vector is intended for stable integration into the genome of said plant cells.

In yet another embodiment, the invention provides a plant cell comprising stably integrated into its genome a cDNA copy of a viral vector as herein described operably linked to a plant expressible promoter and a transcription termination signal or a plant consisting essentially of such plant cells.

^■ I DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In one embodiment of the invention a method is provided to introduce inhibitory ?RNA for specific target gene(s) into a plant cell leading to prolonged and very effective gene silencing. The method comprises providing a viral RNA vector which is derived from a satellite RNA virus and comprising a gene-silencing construct, as a cDNA copy operably linked to a plant-expressible promoter and optionally a transcription termination signal, stably integrated into the cells of the plant and infecting a plant with a corresponding helper virus.

Viral -RNA vectors derived from a satellite virus, suitable for the invention have been described in great detail in WO2003/052108. Corresponding helper viruses have also been described in great detail in WO2003/052108. The viral RNA vector is characterized in that it comprises the nucleotide sequence encoding the N-terminal amino acids of the satellite virus coat protein, a nucleotide sequence comprising at least 10 contiguous nucleotides of nucleotides 1365 to 1394 of SEQ ID No 1 and a gene-silencing construct. Preferably, the at least 10 contiguous nucleotides of nucleotides 1365 to 1394 of SEQ ID No 1 are downstream of the nucleotide sequence encoding the N-terminal amino acids of the satellite virus coat protein and the gene- silencing construct is preferably downstream of the at least 10 contiguous nucleotides of nucleotides 1365 to 1394 of SEQ ID No 1.

As used herein, "a satellite virus" indicates an RN A virus, preferably a single stranded ?RNA virus, the ?RNA genome of which is capable of replicating in a plant cell and being encapsidated by coat protein molecule^'s to form a virus particle or virion, only when provided externally with any number of required essential functions therefor. By "externally provided" is meant that such functions are either not encoded by the satellite viral genome or that the transcription/translation product is non-functional. Satellite viruses thus depend upon external provision of essential functions, and may lack the capacity to encode functional replicase, movement protein, or other essential functions required to complete their life cycle inside a plant cell. In a natural situation, such essential functions are usually provided by an autonomously replicating virus or so-called "helper virus".

The satellite virus genome normally comprises a nucleotide sequence encoding its coat protein and a "satellite RNA virus, wherein the virus comprises a nucleotide sequence encoding a coat protein" is used to mean a satellite RNA virus, the genome of which encodes the coat protein of this satellite RNA virus. Upon co-infection of a plant with the satellite ?RNA virus and a corresponding helper virus, the satellite virus coat protein would normally have the capability to encapsidate the satellite virus genome.

Satellite viruses useful for the present invention are well known in the art and include any satellite virus falling under the definition above, such as but not limited to satellite tobacco mosaic virus (STMV), satellite tobacco necrosis virus (STNV) or satellite maize white line mosaic virus. Satellite viruses useful for the present invention may include wild type isolates, but also encompassed by this definition are variants which result in reduced or minimal symptoms when infected on a host plant, particularly when co-inoculated with a corresponding helper virus. The definition also includes synthetic satellite viruses such as mutant viruses and chimeric satellite viruses.

A "viral -RNA vector derived from a satellite RNA virus" should at least include cis elements from a satellite virus which are recognized by an externally provided replicase. Further, RNA vectors according to the invention comprise the nucleotide sequence encoding the N-terminal amino acids of the satellite virus coat protein or a sequence essentially similar thereto. As used herein the "nucleotide sequence encoding the N-terminal amino acids of the coat protein" is to be interpreted as the nucleotide sequence of a satellite virus ' yhich, when transcribed and translated yields the N-terminal amino acids of the mature coat protein. In particular, if the amino acids making up the complete coat protein are equally divided into N-terminal, central- and C-terminal amino acids, the N-terminal amino acids comprise the N-terminal third of all amino acids. Due to the redundancy of the genetic code, this definition comprises various nucleotide sequences which yield the same amino acid sequence if they were to be translated.

This is not to be interpreted to mean that 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. In the viral RNA vector, 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 viral RNA vector of the method of the invention further comprises at least 10 contiguous nucleotides of the nucleotides 1365 to 1394 of SEQ ID No 1. This sequence may either be a synthetic sequence, or derived from nucleotide sequences encoding a phytoene desaturase, such as but not limited to tobacco or tomato phytoene desaturase. It is preferred that these at least 10 contiguous nucleotides are comprised downstream of (i.e. they are 3' to) the nucleotide sequence encoding the N- terminal amino acids of the coat protein. Particularly suited for the invention are vectors comprising at least the 10 nucleotides 1365 to 1374 of SEQ ID No 1, the 21 nucleotides 1365 to 1385 of SEQ ID No 1, the 22 nucleotides 1365 to 1386 of SEQ ID No 1, or the 27 nucleotides 1365 to 1391 of SEQ ID NO 1.

Conveniently, the viral RNA vector comprises a number of unique or low-occurrence restriction recognition sites, such as provided by a polylinker having a nucleotide sequence as described in SEQ ID No 10 of WO2003/052108. One embodiment of the invention comprises so-called 'basic' viral ?RNA vectors into which a gene-silencing construct may be inserted, comprising a polylinker sequence downstream of the contiguous stretch of at least 10 contiguous nucleotides of nucleotides 1365 to 1391 of SEQ ID No 1. Conveniently a gene-silencing construct may be inserted into the polylinker sequence. Particularly suited for the invention are viral RNA vectors derived from ST?MV.

The viral RNA vector used in the methods of the invention may further comprise a gene-silencing construct.

"Gene-silencing constructs" as used herein is a nucleic acid, which by itself or when replicated or transcribed represent or yields "inhibitory RNA" comprising or consisting of sense RNA or antisense RNA, or a combination of both, comprising a nucleotide sequence which has at least 75%, preferably at least 80%, particularly at least 85%, more particularly at least 90%, especially at least 95% sequence identity with or is identical to the nucleotide sequence whose expression is to be suppressed, or its complement. Further, the nucleotide sequence of the sense or antisense region should preferably be at least about 19, 20 or 21 nucleotides, more preferably at least 27 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides in length, more preferably at least about 250 nucleotides, particularly at least about 500 nucleotides but may extend to the full length of the transcribed region of the gene whose expression is to be reduced. The gene-silencing construct may be identical in sequence and length to the target nucleic acids, or they may be exactly complementary in sequence and identical in length to the target nucleic acids.

It is clear for the person skilled in the art that 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). It is also within the realm of the invention that "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. In certain situations this may be desirable. For example, the function of two nucleotide sequences may be -known, but not their combined function and the phenotype (such as but not limited to, for example, the accumulation of certain metabolic intermediates) of the plant resulting from silencing of both concomitantly. This may for example be the situation for biosynthetic enzymes of a certain pathway. When several gene-silencing constructs are combined in one vector, the gene-silencing constructs may be arranged in tandem or may be inserted into one another.

It is equal whether the sequence identity of the gene-silencing construct with the sequence in the plant whose expression is to be suppressed is within a coding or non- coding region of the sequence in the plant whose expression is to be suppressed. It is also equal, whether the gene-silencing construct is inserted "in-frame" or not in frame into the viral ?RNA vector. "In-frame" as used herein means that, if the gene-silencing construct were to be translated, the resulting amino acid sequence of the gene- silencing construct would be identical to the corresponding sequence of the target gene.

As used herein the terms "gene-silencing" or "inhibitory" are not to be interpreted as meaning completely abolishing the expression of the target gene(s). Instead, these terms are intended to include any reduction in expression, measured either as a reduction in transcription and/or translation, as a reduction in the accumulation of transcripts or translation products such as proteins, or as a modification of the plant phenotype.

As used herein "phenotype" refers to any quantitative or qualitative characteristic of a plant, be it morphological (including macroscopic and microscopic characteristics), biochemical (including the presence, absence, or concentration of particular metabolites or molecules, such as mRNA or protein), functional or other. The gene silencing phenotype obtained according to the methods of the invention is prolonged when compared with the methods described in WO 2003/052108. This provides the possibility of analyzing plants not only macroscopically, but also microscopically or at a molecular and biochemical level. For example changes in metabolites, proteins, DNA or RNA levels, etc can be analyzed. Also, the response to external stimuli, such as changes in pathogen resistance/sensitivity, light sensitivity, heat or cold stress sensitivity, and the like, can be analyzed.

"Target gene" as used herein is the gene(s) of the plant which is/are to be silenced. As gene families may be silenced, the definition includes one or more endogenous genes or one or more chimeric genes (transgenes) or a combination of both.

"A corresponding helper virus" as used herein, indicates those -RNA viruses, preferably single stranded RNA viruses, which can supply the satellite virus or the derived viral ?RNA vector with the functions required in trans by that satellite virus or the derived viral RNA vector, to allow it to replicate in the cytoplasm of plant cells, and spread throughout an infected plant. Typically, corresponding helper viruses will provide the satellite virus or the vector derived thereof with a replicase (RNA dependent RNA polymerase) which recognizes the cis sequences present on the satellite virus ?RNA, and will allow replication of the satellite virus genome or the derived vector. Other proteins which may typically be provided by the helper virus are movement proteins, allowing inter alia, the plasmodesmata-mediated spread of viral particles from cell to cell. For satellite viruses or viral RNA vectors derived thereof which lack a functional coat protein encoding gene, corresponding helper viruses may also provide a functional coat protein. Preferably, the corresponding helper virus will be capable of autonomous systemic spread in an infected plant. However, such a systemic spread seems not to be a prerequisite for gene silencing. Functions required in trans for one particular viral ?RNA vector may be supplied in trans by different corresponding helper viruses. It is clear that the corresponding helper viruses may be wild type isolates of RNA viruses, preferably single-stranded RNA viruses such as the tobamoviruses. Particularly preferred are rod-shaped RNA viruses such as tobamoviruses including tobacco mosaic virus (such as TMV strains Ul, U2 U5), tomato mosaic virus, green tomato atypical mosaic virus, pepper mild mottle virus and odontoglossum ringspot virus. A preferred helper virus is tobacco mild green mosaic virus, such as for example TMV-U2 (see WO 2003/ 052108, SEQ ID No 7).

Also encompassed by the methods and means of the invention are variants of such wild type isolates, preferably variants or mutants which develop minimal symptoms when inoculated on host plants or when co-infected with a corresponding satellite virus or RNA vector derived thereof. Further preferred helper viruses may be variants or mutants of wild type isolates which have an extended host range such as tobamoviruses which can replicate and spread in corn or Brassicae.

In one embodiment of the invention, the viral RNA vector derived from satellite tobacco mosaic virus, comprises nucleotides 162 to 328 of SEQ ID No 1 and at least 10 contiguous nucleotides of nucleotides 1365 to 1394 of SEQ ID No 1. The viral vector may further comprise nucleotides 1172 to 1504 of SEQ ID No 3 and/or nucleotides 1700 to 2431 of SEQ ID No 3.

While the sequences in the sequence listing refer to DNA molecules, it is clear that, when it is stated in the description or the claims that a vector or nucleotide sequence comprises a nucleotide sequence as in the sequence listing, while it is clear that reference is made to an ?RNA molecule, the actual base-sequence of the RNA molecule is identical to the base-sequence of the sequence listing, with the difference that the T (thymine) is replaced by the U (uracil).

Methods to infect or inoculate plants and plant cells with viral ?RNA vectors, and helper viruses are well within in the realm of the person skilled in the art and may be performed according to the methods described in Walkey (1985). In one embodiment of the methods of the invention, 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. In addition, 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. Depending on the function of the endogenous gene(s) of which the expression is modulated, reduced, or eliminated, 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. For 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.

As used herein, the term "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 Rubisco promoters), root-specific promoters (Keller et al.,1989), tuber-specific promoters (Keil et al., 1989), vascular tissue specific promoters (Peleman et al., 1989 ), stamen-selective promoters ( WO 89/10396, WO 92/13956), dehiscence zone specific promoters ( WO 97/13865) and the like.

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. '

Alternatively a promoter can be utilized which is specific for one or more tissues or organs (e.g. leaves and/or roots) of a plant. For example 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. Alternatively, 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.

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).

As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.

The term "gene" means any DNA or RNA fragment comprising a region (the "transcribed region") which is transcribed into an ?RNA molecule (e.g., an m?RNA) in a cell, operably linked to suitable regulatory regions, e.g., a plant-expressible promoter. A gene may thus comprise several operably linked fragments such as a promoter, a 5' leader sequence, a coding region, and a 3' nontranslated sequence, comprising a polyadenylation site. A plant gene endogenous to a particular plant species or virus (endogenous plant or virus gene) is a gene which is naturally found in that plant species or virus, or which' can be introduced in that plant species by breeding techniques such as conventional breeding techniques. A chimeric gene is any gene which is not normally found in a plant species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.

The term "expression of a gene" refers to the process wherein a DNA or -RNA region which is operably linked to appropriate regulatory regions, particularly to a promoter, is transcribed into an ?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. In addition to the above defined elements, 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.

For the purpose of this invention the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a^! percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of two sequences is performed using the Needleman and Wunsch algorithm (1970) using a gap creation penalty = 50 (nucleotides) / 8 (amino acids), a gap extension penalty = 3 (nucleotides) / 2 (amino acids), and a scoring matrix "nwsgapdna" (nucleotides) or "Blosum62" (amino acids). Alternatively, 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.

Sequence alignments and scores for percent sequence identity between two sequences may be determined using computer programs, such as provided by the Wisconsin Package, Version 10.2, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin 53711, USA or Intelligenetics™ Suite (Intelligenetics Inc., CA). For example the progams GAP or BestFit may be used to align two sequences. Alternatively, percent similarity or identity scores are also obtained when searching against databases, such as nucleotide databases FASTA, TFASTA, TFASTX (using algorithms similar to BestFit), BLASTN, TBLASTN, TBLASTX or protein databases FASTA, FASTX, BLASTP, BLASTX.

BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local alignment algorithm of Smith and Waterman (1981).

GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992).

Sequences are indicated as "essentially similar" when they have a sequence identity of at least about 75%, particularly at least about 80%, more particularly at least 85%, quite particularly at least 90%, especially about 95%, more especially about 100%. It is clear that when -RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.

The following non-limiting Examples describe the construction of a chimeric gene comprising a viral RNA vector derived from a satellite viruses, and uses thereof. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989), in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson et al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

T?hroughout the description and Examples, reference is made to the following sequences:

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)

EXAMPLES

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:

- a CaMV 35S promoter fragment (SEQ ID No 3 from nucleotide 325 to nucleotide 1166)

- a cDNA copy of the satellite tobacco mosaic virus up to the Agel restriction site (SEQ ID No 3 from nucleotide 1172 to nucleotide 1504) - a fragment of the tomato phytoene desaturase gene (SEQ ID No 3 from nucleotide 1505 to nucleotide 1699)

- a cDNA copy of the satellite tobacco mosaic virus downstream of the Agel restriction site (SEQ ID No 3 from nucleotide 1700 to nucleotide 2431)

- a 3' nopaline synthase region (SEQ ID No 3 from nucleotide 1700 to nucleotide 2431).

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. REFERENCES

An et al. (1996) Plant Journal 10:107-121

Aoyama and Chua (1997) Plant Journal 11 :605-612

Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, Vol. l and 2, USA. Brown (1998) Molecular Biology LabFax, Volumes I and II, Second Edition, Academic Press (UK) Dhaese et al. 1983; The EMBO Journal 2 : 419-426 Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press Gielen et al. (1984) The EMBO Journal 3: 835-846 Gleba et al. (2004) Current Opinion in Plant Biology 7: 182-188 Gossele et al. (2002) Plant Journal 32: 859-866 Hapster et al., 1988 Mol. Gen. Genet. 212, 182-190 Hudspeth et al., 1989 Plant Mol Biol 12: 579-589 Keller et al., 1988 EMBO J. 7: 3625-3633 Keller et al., 1989 Genes Devel. 3: 1639-1646

McPherson et al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany

Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453 R.D.D. Croy (1993) Plant Molecular Biology Labfax jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY Severin and Schoeffl (1990) PMB 15: 825-833 Velten et al. 1985 Nucleic Acids Research 13: 6981-6998 Walkey (1985) Applied Virology, William Heinemann Ltd, London Waterhouse et al. (1998) Proc. Natl. Acad. Sci USA 95: 13959-13964 Wilbur and Lipmann (1983) Proc. Nat. Acad. Sci. USA 80: 726 Peleman et al., 1989 Gene 84: 359-369

Claims

1. A method for introducing inhibitory RNA into cells of a plant comprising the steps of: - providing a transgenic plant comprising stably integrated into the genome of the cells of said plant a chimeric gene, said chimeric gene comprising the following operably linked fragments: i) a plant expressible promoter; ii) a DNA region which when transcribed yields a viral ?RNA vector, said viral ?RNA vector being derived from a satellite RNA virus and comprising a) the N-terminal amino acids from the coat protein of said satellite virus; b) 10 contiguous nucleotides of nucleotide 1365 to 1394 of SEQ ID No l; c) a nucleotide sequence capable of reducing the expression of a gene of interest in cells of said plants; iii) a transcription termination signal; and - infecting said transgenic plants With a corresponding helper virus.

2. The method according to claim 1, wherein said nucleotide sequence capable of reducing the expression of said gene of interest comprises a nucleotide sequence of at least 27 nucleotides in length, said 27 nucleotides having at least 75% sequence identity to a nucleotide sequence within the target gene of said plant cell, or the complement thereof.

3. The method according to claim 2, wherein said gene of interest is an endogenous gene or a transgene.

4. A method according to any of the preceding claims, wherein the nucleotide sequence encoding the N-terminal amino acids of said coat protein comprises nucleotides 162 to 328 of SEQ ID No 2.

5. A method according to any of the preceding claims, wherein the ten contiguous nucleotides are the nucleotides from position 1365 to position 1374 of SEQ ID No 1.

6. A method according to any of the preceding claims, wherein the RNA vector comprises nucleotides 1365 to 1385 of SEQ ID No 1.

7. A method according to any of the preceding claims, wherein the RNA vector comprises nucleotides 1365 to 1386 of SEQ ID No 1.

8. A method according to any of the preceding claims, wherein the RNA vector comprises nucleotides 1365 to 1391 of SEQ ID No 1.

9. A method according to any of the- preceding claims, wherein the ?RNA vector i ■ " comprises nucleotides 1365 to 1394 of SEQ ID No 1.

10. A method according to any of the preceding claims, wherein the viral RNA vector is derived from satellite tobacco mosaic virus (STMV).

11. A method according to any of the preceding claims, wherein said plant expressible promoter is a constitutive promoter.

12. A method according to any of the preceding claims, wherein said plant expressible promoter is a tissue-specific promoter.

13. Use of a kit for introducing inhibitory RNA into a plant cell, said kit comprising a cDNA copy of a viral vector hs^:. described in any of claims 1 to 12, operably linked to a plant expressible prombter and a .transcription termination signal, and a corresponding helper virus, characterized in that said cDNA copy of said viral RNA vector is stably integrated into the genome of said plant cells.

14. A plant cell comprising stably integrated into its genome a cDNA copy of a viral vector as described in any of claims 1 to 12 operably linked to a plant expressible promoter and a transcription termination signal.

15. The plant cell according to claim 14, further comprising a correponding helper virus.

16. A plant consisting essentially of plant cells according to claim 14.

17. A method for inducible initiation of gene silencing in a plant, comprising the steps of

- providing a transgenic plant containing a chimeric gene as described in any one of claims 1 to 12; and

- infecting said plant with a corresponding helper virus.

18. A method for imaging movement of a virus or a satellite virus in a plant comprising the step of a.) providing a transgenic plant comprising a first chimeric gene encoding a scorable marker, preferably a β-glucuronidase or a green fluorescent protein, operably linked to expression signals, in such a way that said chimeric gene is expressed at least when said virus is present and further comprising a second chimeric gene as described in any one of claims 1 to 12, wherein said second chimeric gene is characterized in that said a nucleotide sequence capable of reducing the expression of a gene of interest is a nucleotide sequence capable of reducing the expression of said scorable marker; b.) infecting said plant with a corresponding helper virus; and c.) scoring the movement of the virus by imaging the reduction in expression of said scorable marker.