Methods and means for obtaining modified phenotypes

[0001] The invention relates to methods for reducing the phenotypic expression of a nucleic acid of interest in plant cells by providing aberrant RNA molecules, preferably unpolyadenylated RNA molecules comprising at least one target specific nucleotide sequence homologous to the nucleic acid of interest, preferably a sense strand, into the nucleus of plant cells. The target-specific unpolyadenylated RNA molecules may be provided by introduction of chimeric DNAs which when transcribed under control of conventional promoter and 3 end formation and polyadenylation regions yield RNA molecules wherein at least the polyadenylation signal may be removed by the autocatalytic activity of a self-splicing ribozyme comprised within the transcribed RNA molecules. Also provided are plant cells comprising such RNA molecules or chimeric DNA encoding such RNA molecules, as well as plants. Similar methods and means for reducing the phenotypic expression of a nucleic acid by co-suppression in eukaryotic cells are provided.

[0002] Post-transcriptional gene silencing (PTGS) or co-suppression, is a common phenomenon associated with transgenes in transgenic plants. PTGS results in sequence-specific removal of the silenced transgene RNA as well as homologous endogenous gene RNA or viral RNA. It is characterized by low steady-state mRNA levels with normal (usually high) rates of nuclear transcription of transgenes being maintained. There are a number of common features or characteristics for PTGS. PTGS is

[0003] sequence-specific;

[0004] systemically transmissible;

[0005] often associated with the presence of multiple copies of transgenes or with the use of strong promoters;

[0006] frequently correlated with the presence of repetitive DNA structures, including inverted repeat T-DNA insertion patterns;

[0007] often accompanied by de novo DNA methylation in the transcribed region, and

[0008] may be meiotcally reset.

[0009] A number of hypothetical models have been proposed to explain PTGS (see e.g. Wassenegger and Plissier, 1998). Typically, these models suggest the involvement of a host encoded enzyme (RNA-directed RNA polymerase (RdRP)) which is proposed to use aberrant RNA as templates to synthesize small copy RNA molecules (cRNA). These cRNAs would then hybridize with the target mRNA to form duplex structures, thereby rendering the mRNA susceptible to degradation by endoribonucleases. So far, there has been no direct evidence that RdRP is involved in PTGS in plants.

[0010] An important question arising from the existing models is what type of RNA is the aberrant RNA that would be used as a template by RdRP, and in which cellular compartment RdRP would function.

[0011] Several reports have described the accumulation of unproductive or unpolyadenylated transgene RNA in plants which are post-transcriptionally silenced (Lee et al. 1997; van Eldik et al. 1998; Covey et al., 1997; van Houdt et al., 1997; Metzlaff et al.; 1997).

[0012] The following documents relate to methods and means for regulating or inhibiting gene expression in a cell.

[0013] U.S. Pat. No 5,190,131 and EP 0 467 349 A1 describe methods and means to regulate or inhibit gene expression in a cell by incorporating into or associating with the genetic material of the cell a non-native nucleic acid sequence which is transcribed to produce an mRNA which is complementary to and capable of binding to the mRNA produced by the genetic material of that cell.

[0014] EP 0 223 399 A1 describes methods to effect useful somatic changes in plants by causing the transcription in the plant cells of negative RNA strands which are substantially complementary to a target RNA strand. The target RNA strand can be a mRNA transcript created in gene expression, a viral RNA, or other RNA present in the plant cells. The negative RNA strand is complementary to at least a portion of the target RNA strand to inhibit its activity in vivo.

[0015] EP 0 240 208 describes a method to regulate expression of genes encoded for in plant cell genomes, achieved by integration of a gene under the transcriptional control of a promoter which is functional in the host and in which the transcribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous gene(s) one wishes to regulate.

[0016] EP 0 647 715 A1 and U.S. Pat. Nos 5, 034,323, 5,231,020 and 5,283,184 describe methods and means for producing plants exhibiting desired phenotypic traits, by selecting transgenotes that comprise a DNA segment operably linked to a promoter, wherein transcription products of the segment are substantially homologous to corresponding transcripts of endogenous genes, particularly endogenous flavonoid biosynthetic pathway genes.

[0017] Waterhouse et al. 1998 describe that virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and anti-sense RNA. The sense and antisense RNA may be located in one transcript that has self-complementarity.

[0018] Hamilton et al. 1998 describes that a transgene with repeated DNA, i.e. inverted copies of its 5 untranslated region, causes high frequency, post-transcriptional suppression of ACC-oxidase expression in tomato.

[0019] WO 98/53083 describes constructs and methods for enhancing the inhibition of a target gene within an organism which involve inserting into the gene silencing vector an inverted repeat sequence of all or part of a polynucleotide region within the vector.

[0020] WO 95/34688 describes methods for cytoplasmic inhibition of gene expression and provides genetic constructs for the expression of inhibitory RNA in the cytoplasm of eukaryotic cells. The inhibitory RNA may be an anti-sense or a co-suppressor RNA. The genetic constructs are capable of replicating in the cytoplasm of a eukaryotic cell and comprise a promoter region, which may be a plant virus subgenomic promoter in functional combination with the RNA encoding region.

[0021] WO095115394 and U.S. Pat. No 5,908,779 describe a method and construct for regulating gene expression through inhibition by nuclear antisense RNA in (mouse) cells. The construct comprises a promoter, antisense sequences, and a cis-or trans-ribozyme which generates 3-ends independently of the polyadenylation machinery and thereby inhibits the transport of the RNA molecule to the cytoplasm.

[0022] The present invention provides a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, the method comprising the step of providing to the nucleus of that plant cell aberrant RNA comprising a target-specific nucleotide sequence, preferably unpolyadenylated RNA comprising a target specific nucleotide sequence, particularly by producing aberrant RNA such as unpolyadenylated RNA by transcription of a chimeric DNA comprised within the plant cell, the chimeric DNA comprising a plant-expressible promoter operably linked to a target specific DNA region encoding that RNA and optionally further comprising a DNA region involved in 3 end formation and polyadenylation, preceded by a self-splicing ribozyme encoding DNA region.

[0023] The invention also provides a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, the method comprising the step of introducing into the nuclear genome of the plant cell a chimeric DNA to generate a transgenic plant cell, the chimeric DNA comprising the following operably linked parts:

[0024] a plant-expressible promoter region, preferably a constitutive promoter or an inducible promoter or a tissue-specific promoter;

[0025] a target-specific DNA region encoding a target-specific nucleotide sequence, preferably a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to the nucleic acid of interest or comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to the complement of said nucleic acid of interest;

[0026] a DNA region encoding a self-splicing ribozyme, preferably a self-splicing ribozyme comprising a cDNA copy of a self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNA, Cherry small circular viroid-like RNA or hepatitis delta virus RNA, particularly a DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and

[0027] a) a DNA region involved in 3 end formation and polyadenylation;

[0028] wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed. Optionally, a transgenic plant may be regenerated from the transgenic plant cell. Preferably, the DNA region encoding a self-splicing ribozyme is located immediately upstream of the DNA region involved in 3 end formation and polyadenylation.

[0029] It is another objective of the invention to provide a chimeric DNA molecule for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, comprising

[0030] a plant-expressible promoter region, preferably a constitutive promoter or an inducible promoter or a tissue-specific promoter;

[0031] a target-specific DNA region encoding a target-specific nucleotide sequence, preferably a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to the nucleic acid of interest or comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to the complement of said nucleic acid of interest;

[0032] a DNA region encoding a self-splicing ribozyme, preferably a self-splicing ribozyme comprising a cDNA copy of a self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNA, Cherry small circular viroid-like RNA or hepatitis delta virus RNA, particularly a DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and

[0033] a DNA region involved in 3 end formation and polyadenylation;

[0034] wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed. Preferably, the DNA region encoding a self-splicing ribozyme is located immediately upstream of the DNA region involved in 3 end formation and polyadenylation.

[0035] It is yet another objective of the invention to provide plant cells and plants comprising a nucleic acid of interest which is normally capable of being phenotypically expressed, further comprising a chimeric DNA, preferably stably-integrated into the nuclear genome, comprising

[0036] a plant-expressible promoter region, preferably a constitutive promoter or an inducible promoter or a tissue-specific promoter;

[0037] a target-specific DNA region encoding a target-specific nucleotide sequence, preferably a target-specific DNA region comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to the nucleic acid of interest or comprising a nucleotide sequence of at least 10 consecutive nucleotides having at least about 70% sequence identity to about 100% sequence identity to the complement of said nucleic acid of interest;

[0038] a DNA region encoding a self-splicing ribozyme, preferably a self-splicing ribozyme comprising a cDNA copy of a self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNA, Cherry small circular viroid-like RNA or hepatitis delta virus RNA, particularly a DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2 or a ribozyme-effective part thereof; and

[0039] a DNA region involved in 3 end formation and polyadenylation;

[0040] wherein said chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed.

[0041] The invention also provides a method for identifying a phenotype associated with the expression of a nucleic acid of interest in a plant cell, the method comprising:

[0042] 1) selecting within the nucleic acid of interest a target sequence of at least 10 consecutive nucleotides;

[0043] 2) introducing a chimeric DNA into the nucleus of a suitable plant host cell comprising the nucleic acid of interest, the chimeric DNA comprising the following operably linked DNA fragments:

[0044] a) a plant-expressible promoter region;

[0045] b) a target-specific DNA region comprising a nucleotide sequence of at least about 70% to about 100% sequence identity to said target sequence or to the complement of said target sequence; followed by

[0046] c) a DNA region encoding a self-splicing ribozyme located immediately upstream of

[0047] d) a DNA region involved in 3 end formation and polyadenylation;

[0048] 3) observing the phenotype by a suitable method.

[0049] Yet another objective of the invention is to provide a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, the method comprising the step of providing to the nucleus of said eukaryotic cell aberrant RNA, preferably unpolyadenylated RNA, comprising a target specific nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest, paritucularly by producing aberrant RNA such as unpolyadenylated RNA by transcription of a chimeric DNA comprised within the eukaryotic cell, the chimeric DNA comprising a plant-expressible promoter operably linked to a target specific DNA region encoding that RNA and optionally further comprising a DNA region involved in 3 end formation and polyadenylation, preceded by a self-splicing ribozyme encoding DNA region.

[0050] Still another objective of the invention is to provide a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, comprising the step of introducing into the nuclear genome of the eukaryotic cell a chimeric DNA to generate a transgenic plant cell, comprising the following operably linked parts:

[0051] a) a promoter region functional in the eukaryotic cell;

[0052] b) a target-specific DNA region comprising nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest;

[0053] c) a DNA region encoding a self-splicing ribozyme; and

[0054] d) a DNA region involved in 3 end formation and polyadenylation

[0055] wherein the chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed.

[0056] The invention also provides a eukaryotic cell comprising a nucleic acid of interest, normally capable of being phenotypically expressed, further comprising a chimeric DNA comprising the following operably linked parts:

[0057] a promoter region functional in the eukaryotic cell;

[0058] a target-specific DNA region comprising nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest;

[0059] a DNA region encoding a self-splicing ribozyme; and

[0060] a DNA region involved in 3 end formation and polyadenylation

[0061] wherein said chimeric DNA when transcribed in the eukaryotic cell produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed, as well as non-human eukaryotic organisms comprising or consisting essentially of such eukaryotic cells.

[0062] FIG. 1. Schematic representation of the ribozyme-containing GUS chimeric gene (pMBW267 and pMBW259) the control construct (pMBW 265) and the GUS chimeric gene used for supertransformation (pBPPGH). 35S-P: CaMV 35S promoter; GUS: region encoding -glucuronidase; SAT: cDNA copy of the satellite RNA of Barley Yellow Dwarf Virus (BYDV) in positive strand orientation () or in minus strand orientation (); Ocs-T: region from the octopine synthase gene from Agrobacterium involved in 3 end formation and polyadenylation; 3 Sat: cDNA copy of the 3 end of the satellite RNA of BYDV; 5 SAT: cDNA copy of the 5 end of the satellite RNA of BYDV; PP2-P: 1.3 kb promoter region of a gene encoding the cucurbit phloem protein PP2; Nos-T: region from the nopaline synthase gene from Agrobacterium involved in 3 end formation and polyadenylation; C: autocatalytic cleavage site in the RNA molecule transcribed from the chimeric gene.

[0063] Although gene-silencing, either by anti-sense RNA or through co-suppression using sense RNA, is a commonly observed phenomenon in transgenic-research, the intentional generation of gene-silenced transgenic eukaryotic cells and transgenic organisms, particularly plant cells and plants, still faces a number of problems. In particular the efficiency of gene-silencing is still amenable to improvement, both in number of transgenic lines exhibiting the phenomenon as well as in the level of reduction of transcription and ultimately the phenotypic expression of particular nucleic acid of interest in a particular transgenic line.

[0064] A number of improved methods for gene-silencing have already been described, e.g. the simultaneous use in one cell of anti-sense and sense RNA targeted to the nucleic acid of interest, preferably co-located on one transcript exhibiting self-complementarity. Novel methods for increasing the efficiency of gene-silencing, preferably gene-silencing through co-suppression in a eukaryotic cell or organism, preferably plant cell or plant, and means therefore, are described in the different embodiments provided by the specification and claims.

[0065] The current invention is based on the unexpected observation by the inventors, that the provision or the introduction of aberrant target-specific RNA, preferably unpolyadenylated target-specific RNA, particularly an aberrant target-specific RNA comprising a nucleotide sequence essentially identical to the nucleic acid of interest in sense orientation, into the nucleus of a cell of a eukaryotic organism, particularly a cell of plant, resulted in an efficient reduction of the expression of the nucleic acid of interest, both in the level of reduction as well as in the number of transgenic lines exhibiting gene-silencing. The understanding of hypothetical mechanisms through which gene-silencing, particularly PTGS, is supposed to proceed did not allow to predict that among all variables potentially involved in initiation and maintenance of gene-silencing, the selection of this one parameter-i.e. providing aberrant, preferably unpolyadenylated RNA would have been sufficient to significantly increase the efficiency of gene-silencing, particularly gene-silencing through co-suppression.

[0066] In one embodiment of the invention, a method is provided for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, comprising the step of providing aberrant RNA such as unpolyadenylated RNA which includes a target-specific nucleotide sequence to the nucleus of that plant cell. Conveniently, the aberrant RNA such as unpolyadenylated RNA including the target-specific nucleotide sequence may be produced by transcription of a chimeric DNA or chimeric gene comprised within the plant cell, preferably incorporated, particularly stably integrated into the nuclear genome of the plant cell. In a particularly preferred embodiment, the aberrant RNA is unpolyadenylated RNA which still exhibits other modifications characteristic of mRNA, such as, but not limited to, the presence of a cap-structure at the 5 end.

[0067] As used herein, the term expression of a gene refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly to a promoter region, 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 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 protein or polypeptide.

[0068] A nucleic acid of interest is capable of being expressed, when said nucleic acid, when introduced in a suitable host cell, particularly in a plant cell, can be transcribed (or replicated) to yield an RNA, and/or translated to yield a polypeptide or protein in that host cell.

[0069] The term gene means any DNA fragment comprising a DNA region (the transcribed DNA region) that is transcribed into a RNA molecule (e.g., a mRNA) in a cell operably linked to suitable regulatory regions, e.g., a plant-expressible promoter. A gene may thus comprise several operably linked DNA fragments such as a promoter, a 5 leader sequence, a coding region, and a 3 region comprising a polyadenylation site. A plant gene endogenous to a particular plant species (endogenous plant gene) is a gene which is naturally found in that plant species or which can be introduced in that plant species by conventional breeding. 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.

[0070] As used herein, phenotypic expression of a nucleic acid of interest refers to any quantitative trait associated with the molecular expression of a nucleic acid in a host cell and may thus include the quantity of RNA molecules transcribed or replicated, the quantity of post-transcriptionally modified RNA molecules, the quantity of translated peptides or proteins, the activity of such peptides or proteins.

[0071] A phenotypic trait associated with the phenotypic expression of a nucleic acid of interest refers to any quantitative or qualitative trait, including the trait mentioned, as well as the direct or indirect effect mediated upon the cell, or the organism containing that cell, by the presence of the RNA molecules, peptide or protein, or posttranslationally modified peptide or protein. The mere presence of a nucleic acid in a host cell, is not considered a phenotypic expression or a phenotypic trait of that nucleic acid, even though it can be quantitatively or qualitatively traced. Examples of direct or indirect effects mediated on cells or organisms are, e.g., agronomically or industrial useful traits, such as resistance to a pest or disease; higher or modified oil content etc.

[0072] As used herein, reduction of phenotypic expression refers to the comparison of the phenotypic expression of the nucleic acid of interest to the eukaryotic cell in the presence of the RNA or chimeric genes of the invention, to the phenotypic expression of the nucleic acid of interest in the absence of the RNA or chimeric genes of the invention. The phenotypic expression in the presence of the chimeric RNA of the invention should thus be lower than the phenotypic expression in absence thereof, preferably be only about 25%, particularly only about 10%, more particularly only about 5% of the phenotypic expression in absence of the chimeric RNA, especially the phenotypic expression should be completely inhibited for all practical purposes by the presence of the chimeric RNA or the chimeric gene encoding such an RNA.

[0073] A reduction of phenotypic expression of a nucleic acid where the phenotype is a qualitative trait means that in the presence of the chimeric RNA or gene of the invention, the phenotypic trait switches to a different discrete state when compared to a situation in which such RNA or gene is absent. A reduction of phenotypic expression of a nucleic acid may thus, a.o., be measured as a reduction in transcription of (part of) that nucleic acid, a reduction in translation of (part of) that nucleic acid or a reduction in the effect the presence of the transcribed RNA(s) or translated polypeptide(s) have on the eukaryotic cell or the organism, and will ultimately lead to altered phenotypic traits. It is clear that the reduction in phenotypic expression of a nucleic acid of interest, may be accompanied by or correlated to an increase in a phenotypic trait.

[0074] As used herein a nucleic acid of interest or a target nucleic acid refers to any particular RNA molecule or DNA sequence which may be present in a eukaryotic cell, particularly a plant cell.

[0075] As used herein aberrant RNA refers to polyribonucleotide molecules which have characteristic differing from mRNA molecules normally found in that cell. The different characteristics include but are not limited to the absence or removal of a 5 cap structure, presence of persistent introns e.g. introns which have been modified in their splice sites so as to prevent splicing, or the absence of the polyA tail normally found associated with the mRNA (unpolyadenylated RNA).

[0076] The term target-specific nucleotide sequence as used herein, refers to a nucleotide sequence (either DNA or RNA nucleotide sequence depending on the context) which can reduce the expression of the target nucleic acid of interest by gene-silencing. Preferably, only the expression of the target nucleic acid or gene, or nucleic acids or genes comprising essentially similar nucleotide sequence is reduced.

[0077] Preferably the target-specific nucleotide sequence comprises a nucleotide sequence corresponding to the sense nucleotide sequence of the nucleic acid or gene of interest. In other words, a target-specific sense nucleotide sequence may be essentially similar to part of an RNA molecule transcribed or produced from the nucleic acid or gene of interest or to parts of the nucleic acid or gene of interest controlling the production of that transcribed or produced RNA molecule, when read in the same 5 to 3 direction as the transcribed or produced RNA molecule.

[0078] Preferably, the target specific nucleotide sequence corresponds to part of a nucleic acid region from which RNA is produced, particularly a region which is transcribed and translated. It is particularly preferred that the target sequence corresponds to one or more consecutive exons, more particularly is located within a single exon of a coding region. However, the target specific nucleotide sequence may also be corresponding to untranslated regions of the RNA molecule produced from the nucleic acid or gene of interest. Moreover, in the light of a recent publication by Mette et al. (1999), it is expected that the target specific nucleotide sequence may also correspond to the regions controlling the production or transcription of RNA from the nucleotide or gene of interest, such as the promoter region.

[0079] The length of the sense target-specific nucleotide sequence may vary from about 10 nucleotides (nt) up to a length equaling the length (in nucleotides) of the target nucleic acid. Preferably the total length of the sense nucleotide sequence is at least 10 nt, preferably 15 nt, particularly at least about 50 nt, more particularly at least about 100 nt, especially at least about 150 nt, more especially at least about 200 nt, quite especially at least about 550 nt. It is expected that there is no upper limit to the total length of the sense nucleotide sequence, other than the total length of the target nucleic acid. However for practical reason (such as e.g. stability of the chimeric genes) it is expected that the length of the sense nucleotide sequence should not exceed 5000 nt, particularly should not exceed 2500 nt and could be limited to about 1000 nt.

[0080] It will be appreciated that the longer the total length of the sense nucleotide sequence is, the less stringent the requirements for sequence identity between the total sense nucleotide sequence and the corresponding sequence in the target nucleic acid or gene become. Preferably, the total sense nucleotide sequence should have a sequence identity of at least about 75% with the corresponding target sequence, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially be identical to the corresponding part of the target nucleic acid. However, it is preferred that the sense nucleotide sequence always includes a sequence of about 10 consecutive nucleotides, particularly about 20 nt, more particularly about 50 nt, especially about 100 nt, quite especially about 150 nt with 100% sequence identity to the corresponding part of the target nucleic acid. Preferably, for calculating the sequence identity and designing the corresponding sense sequence, the number of gaps should be minimized, particularly for the shorter sense sequences.

[0081] As used herein, sequence identity with regard to nucleotide sequences (DNA or RNA), refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences. The alignment of the two nucleotide sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window-size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can, e.g., be conveniently performed using the programs of the Intelligenetics Suite (Intelligenetics Inc., CA). Sequences are indicated as essentially similar when such sequence have a sequence identity of at least about 75%, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical. It is clear than 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.

[0082] It is expected however, that the target-specific nucleotide sequence may also comprise a nucleotide sequence corresponding to the antisense nucleotide sequence of the nucleic acid or gene of interest. In other words, a target-specific antisense nucleotide sequence may be essentially similar to the complement of part of an RNA molecule transcribed or produced from the nucleic acid or gene of interest or to the complement of parts of the nucleic acid or gene of interest controlling the production of that transcribed or produced RNA molecule, when read in the same 5 to 3 direction as the transcribed or produced RNA molecule.

[0083] The requirements for antisense target-specific nucleotide sequences with regard to length, similarity etc. are expected to be essentially similar as for sense target-specific nucleotide sequences as specified herein.

[0084] It will be clear to the person skilled in the art that the unpolyadenylated RNA molecule may comprise more than one target-specific nucleotide sequence and particularly that the unpolyadenylated RNA molecule may comprise sense and antisense target-specific nucleotide sequences wherein the sense and antisense nucleotide sequences are essentially complementary to each other and capable of forming an artificial hairpin structure as described in Waterhouse et al., 1998 or in PCT-application PCT/IB99/00606 (incorporated by reference).

[0085] As indicated above, introduction of target-specific unpolyadenylated RNA into the nucleus of a plant cell can conveniently be achieved by transcription of a chimeric DNA encoding RNA introduced into the nucleus, preferably stably integrated into the nuclear genome of a plant cell.

[0086] In a preferred embodiment of the invention, the target-specific unpolyadenylated RNA may be produced in the nucleus of a plant cell by transcription of a chimeric DNA encoding a first target-specific RNA, which may be further processed by the action of a ribozyme also present, and preferably also encoded by a chimeric gene, in the plant cell to yield a second unpolyadenylated target-specific RNA. It will be clear for the person skilled in the art that the RNA processing need not be subsequently but can occur simultaneously. In a particularly preferred embodiment the ribozyme is a self-splicing ribozyme which is comprised within the generated target specific RNA transcript.

[0087] Thus, in a particularly preferred embodiment of the invention, a method is 10 provided for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, the method comprising the step of introducing into the nuclear genome of the plant cell a chimeric DNA to generate a transgenic plant cell, the chimeric DNA comprising the following operably linked parts:

[0088] (a) a plant-expressible promoter region;

[0089] (b) a target-specific DNA region;

[0090] (c) a DNA region encoding a self-splicing ribozyme; and

[0091] (d) a DNA region involved in 3 end formation and polyadenylation wherein the chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed.

[0092] The method may optionally further comprise the step of regenerating a the transgenic plant cell into a transgenic plant.

[0093] As used herein, a ribozyme is a catalytic RNA molecule that has the intrinsic ability to break and form covalent bonds in ribonucleic acids at specific sites in the absence of a cofactor other than a divalent cation.

[0094] As used herein a self-splicing ribozyme or self-cleaving ribozyme is a ribozyme capable of autocatalysis at a specific site within that ribozyme. Preferred self-splicing ribozymes are self-splicing ribozymes with a so-called hammerhead structure. However, it is expected that self-cleaving ribozymes with another conformation such as the hairpin self-cleaving structures encountered in the minus strand of replication intermediates of e.g. the nepoviruses can also be used to the same effect.

[0095] Particularly preferred self-splicing ribozymes are those involved in the replication of small circular plant pathogenic RNAs, such as but not limited to the self-splicing ribozyme from avocado sunblotch viroid, peach latent mosaic viroid, Chrysanthemum chlorotic mottle viroid, carnation stunt associated viroid, Newt satellite 2 transcript, Neurospora VS RNA, barley yellow dwarf virus satellite RNA,arabis mosaic virus satellite RNA, chicory yellow mottle virus satellite RNA S1, lucerne transient streak virus satellite RNA, tobacco ringspot virus satellite RNA, subterranean clover mottle virus satellite RNA, solanum nodiflorum mottle virus satellite RNA, velvet tobacco mottle virus satellite RNAvSCMoV or Cherry small circular viroid-like RNAcscRNA1. Table 1 lists different variant ribozymes suitable for the invention, as well as a reference to their nucleotide sequence.

[0096] The DNA regions encoding self-splicing ribozymes may be cDNA copies of part of the mentioned plant pathogenic RNAs comprising the ribozyme, or may be synthetic DNA. Also comprised are variants such as mutants including substitutions, deletions or insertions of nucleotides within the ribozyme nucleotide sequence in such a way that the autocatalytic capacity of the ribozymes is not substantially altered.

[0097] Preferably, the DNA region encoding the self-splicing ribozyme is located immediately upstream of the DNA region encoding the 3 end formation and polyadenylation signal. However, having read the specification, the person skilled in the art will immediately realize that the DNA region encoding the self-splicing ribozyme may be comprised within the chimeric gene encoding the unpolyadenylated RNA at other locations, provided that a sufficiently large second RNA comprising a target-specific nucleotide wherein the polyadenylation site is removed may be generated.

TABLE 1
Different self-cleaving ribozymes
				Accession
RNA species	Reference			Nr	() strand	() strand
Avocado sunblotch viroid	Symons	1981	Nucleic Acids	JO2020	hammerhead	hammerhead
			Res. 9 6527-6537
Avocado sunblotch viroid variant C-10	Rakowski and Symons	1989	Virology 173 352-356	M31100	hammerhead	hammerhead
Avocado sunblotch viroid variant B-1	Rakowski and Symons	1989	Virology 173 352-356	M31086	hammerhead	hammerhead
Avocado sunblotch viroid variant A-2	Rakowski and Symons	1989	Virology 173 352-356	M31085	hammerhead	hammerhead
Avocado sunblotch viroid variant B-2	Rakowski and Symons	1989	Virology 173 352-356	M31087	hammerhead	hammerhead
Avocado sunblotch viroid variant C-2	Rakowski and Symons	1989	Virology 173 352-356	M31092	hammerhead	hammerhead
Avocado sunblotch viroid variant B-3	Rakowski and Symons	1989	Virology 173 352-356	M31088	hammerhead	hammerhead
Avocado sunblotch viroid variant C-3	Rakowski and Symons	1989	Virology 173 352-356	M31093	hammerhead	hammerhead
Avocado sunblotch viroid variant B-4	Rakowski and Symons	1989	Virology 173 352-356	M31089	hammerhead	hammerhead
Avocado sunblotch viroid variant C-4	Rakowski and Symons	1989	Virology 173 352-356	M31094	hammerhead	hammerhead
Avocado sunblotch viroid variant B-5	Rakowski and Symons	1989	Virology 173 352-356	M31090	hammerhead	hammerhead
Avocado sunblotch viroid variant C-5	Rakowski and Symons	1989	Virology 173 352-356	M31095	hammerhead	hammerhead
Avocado sunblotch viroid variant B-6	Rakowski and Symons	1989	Virology 173 352-356	M31091	hammerhead	hammerhead
Avocado sunblotch viroid variant C-6	Rakowski and Symons	1989	Virology 173 352-356	M31096	hammerhead	hammerhead
Avocado sunblotch viroid variant C-7	Rakowski and Symons	1989	Virology 173 352-356	M31097	hammerhead	hammerhead
Avocado sunbiotch viroid variant C-8	Rakowski and Symons	1989	Virology 173 352-356	M31098	hammerhead	hammerhead
Avocado sunbiotch viroid variant C-9	Rakowski and Symons	1989	Virology 173 352-356	M31099	hammerhead	hammerhead
Avocado sunblotch viroid ASBVd-B	Semancik and Szychowski	1994	J. Gen Virol.	S74687	hammerhead	hammerhead
			75 1543-1549
Avocado sunblotch viroid ASBVd-V	Semancik and Szychowski	1994	J. Gen Virol.	S73861	hammerhead	hammerhead
			75 1543-1549
Peach latent mosaic viroid PLMVd.1	Hernandez and Flores	1992	Proc. Natl. Acad. Sci.	M83545	hammerhead	hammerhead
			89 3711-3715
Peach latent mosaic viroid PLMVd.2	Hernandez and Flores	1992	Proc. Natl. Acad. Sci.		hammerhead	hammerhead
			89 3711-3715
Peach latent mosaic viroid Peach-Italy	Schamloul et al.	1995	Acta Hortic.		hammerhead	hammerhead
			386 522-530
Peach latent mosaic viroid Cherry-Canada	Hadini et al.	1997	Plant Dis. 81, 154-158		hammerhead	hammerhead
Peach latent mosaic viroid variant gds2	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005294	hammerhead	hammerhead
Peach latent mosaic viroid variant gds21	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005295	hammerhead	hammerhead
Peach latent mosaic viroid variant gds15	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005296	hammerhead	hammerhead
Peach latent mosaic viroid variant gds23	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005297	hammerhead	hammerhead
Peach latent mosaic viroid variant gds18	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005298	hammerhead	hammerhead
Peach latent mosaic viroid variant gds1	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005299	hammerhead	hammerhead
Peach latent mosaic viroid variant gds3	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005300	hammerhead	hammerhead
Peach latent mosaic viroid variant gds19	Ambros et al.	1998	J. Virol. 72 7397-7406	A.J005301	hammerhead	hammerhead
Peach latent mosaic viroid variant gds13	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005302	hammerhead	hammerhead
Peach latent mosaic viroid variant gds6	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005303	hammerhead	hammerhead
Peach latent mosaic viroid variant gds16	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005304	hammerhead	hammerhead
Peach latent mosaic viroid variant esc8	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005305	hammerhead	hammerhead
Peach latent mosaic viroid variant esc16	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005306	hammerhead	hammerhead
Peach latent mosaic viroid variant esc5	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005307	hammerhead	hammerhead
Peach latent mosaic viroid variant esc12	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005308	hammerhead	hammerhead
Peach latent mosaic viroid variant eec10	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005309	hammerhead	hammerhead
Peach latent mosaic viroid variant esc14	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005310	hammerhead	hammerhead
Peach latent mosaic viroid variant ls4b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005311	hammerhead	hammerhead
Peach latent mosaic viroid variant ls16b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005312	hammerhead	hammerhead
Peach latent mosaic viroid variant ls17b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005313	hammerhead	hammerhead
Peach latent mosaic viroid variant ls1	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005314	hammerhead	hammerhead
Peach latent mosaic viroid variant ls18b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005315	hammerhead	hammerhead
Peach latent mosaic viroid variant ls11	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005316	hammerhead	hammerhead
Peach latent mosaic viroid variant ls8	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005317	hammerhead	hammerhead
Peach latent mosaic viroid variant ls19b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005318	hammerhead	hammerhead
Peach latent mosaic viroid variant ls5b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005319	hammerhead	hammerhead
Peach latent mosaic viroid variant ls11b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005320	hammerhead	hammerhead
Peach latent mosaic viroid variant ls6b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005321	hammerhead	hammerhead
Peach latent mosaic viroid variant ls14b	Ambros et al.	1998	J. Virol. 72 7397-7406	AJ005322	hammerhead	hammerhead
Crysanthemum chlorotic mottle viroid	Navarro and Flores	1997	Proc. Natl. Acad. Sci.	Y14700	hammerhead	hammerhead
			94 11262-11267
Barley yellow dwarf virus satellite RNA	Miller et al.	1991	Virology 183 711-720	M63666	hammerhead	hammerhead
Arabis mosaic virus satellite RNA	Kaper et al.	1988	Biochem. Biophys.	M21212	hammerhead	hairpin
			Res. Com.
			154 318-325
Chicory yellow mottle virus satellite RNA S1	Rubino et al.	1990	J. Gen Virol.	D00721	hammerhead	hairpin
			71 1897-1903
Lucerne translent streak virus satellite	Keese et al.	1983	FEBS Lett.	X01985	hammerhead	hammerhead
RNA LTSV-N			159 185-190
Lucerne translent streak virus satellite	Keese et al.	1983	FEBS Lett.	X01984	hammerhead	hammerhead
RNA LTSV-A			159 185-190
Lucerne translent streak virus satellite	Abouhaidar and Paliwal	1988	J. Gen. Virology	D00341	hammerhead	hammerhead
RNA LTSV-C			69 2369-2373
Tobacco ringspot virus satellite RNA.1	Buzayan et al.	1986	Virology 151,	M14879	hammerhead	hairpin
			186-199
Tobacco ringspot virus satellite RNA.2	Buzayan et al.	1987	Virology 160, 95-99	M17439	hammerhead	hairpin
Subterraneanclover mottle virus satellite RNA.1	Davies et al.	1990	Virology 177,	M33001	hammerhead
			216-224
Subterraneanclover mottle virus satellite RNA.2	Davies et al.	1990	Virology 177,	M33000	hammerhead
			216-224
Solanum nodiflorum mottle virus RNA	Haseloff and Symons	1982	Nucleic Acids	J02386	hammerhead
			Res. 10 3681-3691
Velvet tobacco mottle virus	Haseloff and Symons	1982	Nucleic Acids		hammerhead
circular viroid-like RNA-1			Res. 10 3681-3691
Velvet tobacco mottle virus	Haseloff and Symons	1982	Nucleic Acids	J02439	hammerhead
circular viroid-like RNA-1			Res. 10 3681-3691
Cherry small circular viroid-like RNA	Di Serio et al.	1997	J. Virol.	Y12833	mod.	mod.
			71 6603-6610		hammerhead
Carnation small viroid-like RNA-1	Hernandez et al.	1992	Nucleic Acids	X68034	hammerhead	hammerhead
			Res. 20 6323-6329
Carnation small viroid-like RNA-2	Hernandez et al.	1992	Nucleic Acids		hammerhead	hammerhead
			Res. 20 6323-6329
Notophtalmus viridescens (Newt)	Epstein et al.	1986	J. Cell. Biol.	X04478	hammerhead
satellite 2 transcript			103 1137-1144
Neurospora VS RNA	Saville and Collins	1990	Cell 61 685-696	M32974	VS RNA
					selfcleavage
Schistosome satellite DNA	Ferbeyre et al.	1998	Mol. Cell. Biol.	AF036739
			18 3880-3888

[0098] The use of ribozymes in transgenic organisms to generate RNA molecules with 5 and or 3 termini of interest has been documented in the art. Rubio et aL. 1999, describe broad-spectrum protection against Tombusviruses elicited by defective interfering (DI) RNAs in transgenic plants. To produce RNAs with authentic 5 and 3 termini identical to those of native DI RNA, the DI RNA sequence transcribed from a DNA cassette was flanked by ribozymes. Transgenic Nicotiana benthamiana plants were better protected than non-Transgenic transgenic plants against infection by tomato bushy stunt virus and related tombusviruses. DI RNAs interfere drastically with virus accumulation through effective competition with the parental virus for transacting factors required for replication. Egli and Braus, 1994 describe uncoupling of mRNA 3 cleavage and polyadenylation by expression of a hammerhead ribozyme in yeast. Eckner et al. 1991 described that test gene transcripts which could obtain a mature histone 3 end by the RNA cleaving activity of a cis-acting ribozyme, thus circumventing the cellular 3 end processing machinery were found to be transport deficient and accumulated in the nuclear compartment. However, these documents in the art are not related to methods for inhibiting phenotypic expression by homology dependent gene-silencing, particularly by PTGS.

[0099] A particularly preferred self-splicing ribozyme is the ribozyme comprised with the Barley yellow dwarf virus (BYDV) satellite RNA, quite particularly the satellite RNA found in BYDV isolates of the RPV serotype.

[0100] It has been found that reduction of the phenotypic expression of the nucleic acid of interest using a chimeric gene according to the invention was most efficient using a cDNA copy of the ribozyme comprised within the minus strand of BYDV satellite RNA. Therefore, ribozymes which show an autocatalytic activity similar to the autocatalytic activity of the ribozyme comprised within the minus strand of BYDV satellite RNA are especially suited for the methods of the invention. Autocatalytic activity of ribozymes can be compared with the autocatalytic activity of the () strand of BYDV satellite RNA as described by Miller et al. 1991.

[0101] The ribozyme motif within the () strand of BYDV satellite RNA has been identified as the nucleotide sequence of SEQ ID No 1 from the nucleotide at position 194 to the nucleotide at position 236. The ribozyme motif within the () strand of BYDV satellite RNA has been identified as the nucleotide sequence of SEQ ID No 2 from the nucleotide at position 310 to the nucleotide at position 322 followed by the nucleotide sequence of SEQ ID No. 2 from the nucleotide at position 1 to the nucleotide at position 89.

[0102] It goes without saying that more than one DNA region encoding a ribozyme may be comprised within the chimeric gene. These ribozymes may be clustered, e.g. they may all be located the region immediately proceeding DNA region encoding the '3 end formation and polyadenylation signal.

[0103] However, it is expected that more than one DNA region encoding a ribozyme may be comprised within the chimeric gene in such a way that upon self-cleavage more than one unpolyadenylated RNA molecules each comprising a target-specific nucleotide sequence is generated. Such a chimeric DNA could thus comprise:

[0104] a plant expressible promoter

[0105] a first target-specific DNA region

[0106] a DNA region encoding a first self-splicing ribozyme

[0107] a second target-specific DNA region

[0108] a DNA region encoding a second self-splicing ribozyme

[0109] a DNA region encoding a 3 end formation and polyadenylation signal.

[0110] The first and second self-splicing ribozyme may be identical, essentially similar or different. Likewise, the first and second target-specific DNA region encoding the RNA with a target-specific nucleotide sequence may be identical, essentially similar or different.

[0111] For practical reasons, it is thought that the number of DNA regions encoding a ribozyme within a single chimeric gene should not exceed five.

[0112] In a preferred embodiment, the nucleic acid of interest, whose phenotypic expression is targeted to be reduced, is a gene incorporated in the genome of a eukaryotic cell, particularly a plant cell. It will be appreciated that the means and methods of the invention can be used for the reduction of phenotypic expression of a gene which belongs to the genome of the cell as naturally occurring, (an endogenous gene), as well as for the reduction of phenotypic expression of a gene which does not belong to the genome of the cell as naturally occurring, but has been introduced in that cell (a transgene). The transgene can be introduced stably or transiently, and can be integrated into the nuclear genome of the cell, or be present on a replicating vector, such as a viral vector.

[0113] In another preferred -embodiment, the nucleic acid of interest, whose phenotypic expression is targeted to be reduced is a viral nucleic acid, particularly a viral RNA molecule, capable of infecting a eukaryotic cell, particularly a plant cell. In this case, the phenotype to be reduced is the replication of the virus, and ultimately, the disease symptoms caused by the infecting virus.

[0114] For the purpose of the invention, the term plant-expressible promoter means a promoter which is capable of driving 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. A whole range of plant expressible promoters, is available to direct the transcription of the chimeric genes of the invention. These include, but are not limited to strong promoters such as CaMV35S promoters (e.g., Harpster et al., 1988). In the light of the existence of variant forms of the CaMV35S promoter, as known by the skilled artisan, the object of the invention can equally be achieved by employing these alternative CaMV35S promoters and variants. It is also clear that other plant-expressible promoters, particularly constitutive promoters, such as the opine synthase promoters of the Agrobacterum Ti- or Ri-plasmids, particularly a nopaline synthase promoter, or subterranean clover virus promoters can be used to obtain similar effects. Also contemplated by the invention are chimeric genes to reduce the phenotypic expression of a nucleic acid in a cell, which are under the control of single subunit bacteriophage RNA polymerase specific promoters, such as a T7 or a T3 specific promoter, provided that the host cells also comprise the corresponding RNA polymerase in an active form.

[0115] It is a further object of the invention, to provide methods for reducing the phenotypic expression of a nucleic acid in specific cells, particularly specific plant cells by placing the chimeric genes of the invention under control of tissue-specific or organ-specific promoters. Such tissue-specific or organ-specific promoters are well known in the art and include but are not limited to seed-specific promoters (e.g., WO89/03887), organ-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.

[0116] In another embodiment of the invention, the expression of a chimeric gene to reduce the phenotypic expression of a target nucleic acid can be controlled at will by the application of an appropriate chemical inducer, by operably linking the transcribed DNA region of the chimeric genes of the invention to a promoter whose expression is induced by a chemical compound, such as the promoter of the gene disclosed in European Patent publication (EP) 0332104, or the promoter of the gene disclosed in WO 90/08826.

[0117] It will be clear to the person skilled in the art that the same effect in reducing the phenotypic expression of a nucleic acid in a plant cell may be achieved using a trans-splicing ribozyme to remove at least the polyadenylation site from the RNA transcript of a chimeric gene comprising a plant expressible promoter, a target-specific DNA region and a DNA region encoding a 3 end termination and polyadenylation signal to generate unpolyadenylated RNA comprising a target-specific nucleotide sequence.

[0118] As used herein a trans-splicing ribozyme is an RNA molecule capable of catalyzing the breakage or formation of a covalent bond within another RNA molecule at a specific site.

[0119] The trans-splicing ribozyme should be chosen or designed in such a way that it recognizes a specific site preceding, preferably immediately preceding the polyadenylation signal of the RNA transcript comprising a target-specific nucleotide sequence. Methods to design such trans-splicing ribozyme with endoribonuclease activity are known in the art (see e.g. Haselhoff and Gerlach, 1988, WO 89/05852)

[0120] The DNA region encoding a trans-splicing ribozyme may be comprised within the chimeric gene encoding the target-specific RNA. Upon transcription of the chimeric gene an RNA molecule comprising the trans-splicing ribozyme and the target-specific nucleotide sequence may then generated, wherein the trans-splicing ribozyme is capable of cleaving a specific site preceding the polyadenylation site of another similar RNA molecule , to generate unpolyadenylated target-specific RNA molecules.

[0121] The trans-splicing ribozyme may also be provided by expression of another chimeric gene encoding an RNA molecule comprising the trans-splicing ribozyme in the same plant cell, according to methods and means available in the art (see e.g. Vaish et al. 1998; Bramlage et al. 1998).

[0122] Alternative methods may exist to provide unpolyadenylated target-specific RNA to the nucleus of a plant cell. Such methods include e.g. transcription of a chimeric gene, integrated in the nuclear genome of a plant cell comprising a target-specific DNA region, by an DNA-dependent RNA polymerase different from RNA polymerase II, such that RNA transcripts are generated independent from the normal processing mRNA machinery (including intron-splicing, capping and polyadenylation). This can be achieved e.g. by operably linking the target-specific DNA region to a promoter region, recognized by a single subunit RNA polymerase from a bacteriophage, such as but not limited to the T7 polymerase, and a DNA region comprising a terminator for such a polymerase. In this case, the plant cell needs to be provided with a chimeric gene encoding the corresponding RNA polymerase. Providing unpolyadenylated target-specific RNA to the nucleus of a plant cell can also be achieved e.g. by operably linking the target-specific DNA region to a promoter region, recognized by a eukaryotic RNA polymerase I or III, and a DNA region comprising a terminator for such a polymerase. The means and methods for constructing such chimeric genes and plant cells are described in detail in WO 97/49814 (incorporated by reference). Another alternative to provide unpolyadenylated target-specific RNA to the nucleus of a plant cell may include transcription of a chimeric gene comprising a target-specific DNA region operably linked to a plant-expressible promoter and linked to a DNA region comprising a 3 end formation signal but not a polyadenylation signal.

[0123] Although not intending to limit the invention to a specific mode of action, it is expected that the trigger of the homology-dependent gene-silencing mechanisms of the cell, particularly the co-suppression mechanism, is the accumulation of target-specific RNA into the nucleus of that cell. Providing unpolyadenylated RNA to the nucleus of the cell may be one mechanism of causing accumulation of target-specific RNA in a nucleus of a cell, but other aberrations such as the absence of a cap-structure or the presence of persistent introns etc. may constitute alternative ways to cause the accumulation of target-specific RNA in the nucleus of a cell.

[0124] Moreover, it is expected that other aberrations in the target-specific RNA molecules in addition to the absence of the polyA tail, including the absence of a cap-structure, or the presence of persistent introns or the presence of abnormal secondary structures, particularly the presence of giant hairpin structures, may have a cumulative effect on the inhibition of the normal transit of the RNA from the nucleus to the cytoplasm and hence have a cumulative or synergystic effect on the reduction of the phenotypic expression of a nucleic acid of interest.

[0125] The recombinant DNA comprising the chimeric gene to reduce the phenotypic expression of a nucleic acid of interest in a host cell, may be accompanied by a chimeric marker gene, particularly when the stable integration of the transgene in the genome of the host cell is envisioned. The chimeric marker gene can comprise a marker DNA that is operably linked at its 5 end to a promoter, functioning in the host cell of interest, particularly a plant-expressible promoter, preferably a constitutive promoter, such as the CaMV 35S promoter, or a light inducible promoter such as the promoter of the gene encoding the small subunit of Rubisco; and operably linked at its 3 end to suitable plant transcription 3 end formation and polyadenylation signals. It is expected that the choice of the marker DNA is not critical, and any suitable marker DNA can be used. For example, a marker DNA can encode a protein that provides a distinguishable colour to the transformed plant cell, such as the A1 gene (Meyer et al., 1987), can provide herbicide resistance to the transformed plant cell, such as the bar gene, encoding resistance to phosphinothricin (EP 0,242,246), or can provide antibiotic resistance to the transformed cells, such as the aac(69 gene, encoding resistance to gentamycin (WO94/01560).

[0126] A recombinant DNA comprising a chimeric gene to reduce the phenotypic expression of a gene of interest, can be stably incorporated in the nuclear genome of a cell of a plant. Gene transfer can be carried out with a vector that is a disarmed Ti-plasmid, comprising a chimeric gene of the invention, and carried by Agrobacterium. This transformation can be carried out using the procedures described, for example, in EP 0 116 718.

[0127] Alternatively, any type of vector can be used to transform the plant cell, applying methods such as direct gene transfer (as described, for example, in EP 0 233 247), pollen-mediated transformation (as described, for example, in EP 0 270 356, WO85/01856 and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example, in EP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example, in U.S. Pat. No. 4,536,475), and the like.

[0128] Other methods, such as microprojectile bombardment as described for corn by Fromm et al. (1990) and Gordon-Kamm et al. (1990), are suitable as well.

[0129] Cells of monocotyledonous plants, such as the major cereals, can also be transformed using wounded and/or enzyme-degraded compact embryogenic tissue capable of forming compact embryogenic callus, or wounded and/or degraded immature embryos as described in WO92/09696. The resulting transformed plant cell can then be used to regenerate a transformed plant in a conventional manner.

[0130] The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric gene for reduction of the phenotypic expression of a nucleic acid of interest of the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert.

[0131] The means and methods of the invention can also be used for the reduction of gene expression by co-suppression in eukaryotic cells and organisms.

[0132] In one embodiment the invention provides a method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, comprising the step of providing unpolyadenylated RNA comprising a target specific sense nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity to about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest, to the nucleus of the eukaryotic cell.

[0133] In another embodiment, a method is provided for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, comprising the step of introducing into the nuclear genome of the eukaryotic cell a chimeric DNA to generate a transgenic plant cell, DNA comprising the following operably linked parts:

[0134] (e) a promoter region functional in the eukaryotic cell;

[0135] (f) a target-specific DNA region comprising nucleotide sequence of at least 10 consecutive nucleotides with at least about 70% sequence identity about 100% sequence identity to the nucleotide sequence of the nucleic acid of interest;

[0136] (g) a DNA region encoding a self-splicing ribozyme; and

[0137] (h) a DNA region involved in 3 end formation and polyadenylation

[0138] wherein the chimeric DNA when transcribed produces a first RNA molecule comprising a target specific nucleotide sequence and a self-splicing ribozyme, which when cleaved by autocatalysis produces a second RNA molecule comprising a target specific nucleotide sequence wherein the 3 end of the first RNA molecule comprising the polyadenylation site has been removed.

[0139] Different preferred embodiments and definitions described in connection with the reduction of gene expression by homology dependent gene silencing in plant cells and plants also apply mutatis mutandis to the means and methods described for reduction of gene expression by co-suppression in eukaryotic cells and organisms. As used herein eukaryotic cells comprise plant cells, animal cells and human cells and cells from yeasts and fungi as well as cultures of such cells.

[0140] It is a further object of the invention to provide eukaryotic cells, preferably plant cells and organisms (preferably plants) comprising the chimeric genes for the reduction of the phenotypic expression of a target nucleic acid as described in the invention.

[0141] The methods and means of the invention can thus be used to reduce phenotypic expression of a nucleic acid in a eukaryotic cell or organism, particularly a plant cell or plant, for obtaining shatter resistance (WO 97/13865), for obtaining modified flower colour patterns (EP 522 880, U.S. Pat. No. 5,231,020), for obtaining nematode resistant plants (WO 92/21757, WO 93/10251, WO 94/17194), for delaying fruit ripening (WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO 93/07275, WO 92/04456, U.S. Pat. No. 5,545,366), for obtaining male sterility (WO 94/29465, WO89/10396, WO 92/18625), for reducing the presence of unwanted (secondary) metabolites in organisms, such as glucosinolates (WO97/16559) or chlorophyll content (EP 779 364) in plants , for modifying the profile of metabolites synthesized in a eukaryotic cell or organisms by metabolic engineering e.g. by reducing the expression of particular genes involved in carbohydrate metabolism (WO 92/11375, WO 92/11376, U.S. Pat. No. 5,365,016, WO 95/07355) or lipid biosynthesis (WO 94/18337, U.S. Pat. No. 5,530,192), for delaying senescence (WO 95/07993), for altering lignification in plants (WO 93/05159, WO 93/05160), for altering the fibre quality in cotton (U.S. Pat. No. 5,597,718), for increasing bruising resistance in potatoes by reducing polyphenoloxidase (WO 94/03607), etc.

[0142] The methods of the invention will lead to better results and/or higher efficiencies when compared to the methods using conventional sense or antisense nucleotide sequences and it is believed that other sequence-specific mechanisms regulating the phenotypic expression of target nucleic acids might be involved and/or triggered by the presence of the double-stranded RNA molecules described in this specification.

[0143] A particular application for reduction of the phenotypic expression of a transgene in a plant cell, inter alia, by antisense or sense methods, has been described for the restoration of male fertility, the latter being obtained by introduction of a transgene comprising a male sterility DNA (WO 94/09143, WO 91/02069). The nucleic acid of interest is specifically the male sterility DNA. Again, the processes and products described in this invention can be applied to these methods in order to arrive at a more efficient restoration of male fertility.

[0144] It will be appreciated that the methods and means described in the specification can also be applied in High Throughput Screening (HTS) methods, for the identification or confirmation of phenotypes associated with the expression of a nucleic acid sequence with hitherto unidentified function in a eukaryotic cell, particularly in a plant cell.

[0145] Such a method comprises the steps of:

[0146] 1. selecting a target sequence within the nucleic acid sequence of interest with unidentified or non-confirmed function/phenotype when expressed. Preferably, if the nucleic acid has putative open reading frames, the target sequence should comprise at least part of one of these open reading frames. The length of the target nucleotide sequence may vary from about 10 nucleotides up to a length equalling the length (in nucleotides) of the nucleic acid of interest with unidentified function.

[0147] 2. Introducing a chimeric DNA into the nucleus of a suitable host cell, comprising the nucleic acid of interest, wherein the chimeric DNA comprises a promoter region suitable for expression in the host cell, a DNA region encoding the target-specific nucleotide sequence, and a DNA region encoding a self-splicing ribozyme located immediately upstream of a DNA region involved in 3 end formation and polyadenylation.

[0148] 3. observing the phenotype by a suitable method. Depending on the phenotype expected, it may be sufficient to observe or measure the phenotype in a single cell, but it may also be required to culture the cells to obtain an (organized) multicellular level, or even to regenerate a transgenic organism, particularly a transgenic plant.

[0149] It is also clear that the methods and means of the invention are suited for the reduction of the phenotypic expression of a nucleic acid in all plant cells of all plants, whether they are monocotyledonous or dicotyledonous plants, particularly crop plants such as but not limited to corn, rice, wheat, barley, sugarcane, cotton, oilseed rape, soybean, vegetables (including chicory, brassica vegetables, lettuce, tomato), tobacco, potato, sugarbeet but also plants used in horticulture, floriculture or forestry. The means and methods of the invention will be particularly suited for plants which have complex genomes, such as polyploid plants.

[0150] It is expected that the chimeric RNA molecules produced by transcription of the chimeric genes described herein, can spread systemically throughout a plant, and thus it is possible to reduce the phenotypic expression of a nucleic acid in cells of a non-transgenic scion of a plant grafted onto a transgenic stock comprising the chimeric genes of the invention (or vice versa) a method which may be important in horticulture, viticulture or in fruit production.

[0151] The following non-limiting Examples describe the construction of chimeric genes for the reduction of the phenotypic expression of a nucleic acid of interest in a eukaryotic cell and the use of such genes. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994 ) Current Protocols in Molecular Biology, Current Protocols, USA. 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.

[0152] Throughout the description and Examples, reference is made to the following sequences:

[0153] SEQ ID No 1: cDNA copy of the () strand of BYDV RPV satellite RNA

[0154] SEQ ID No 2: cDNA copy of the () strand of BYDV RPV satellite RNA

[0155] SEQ ID No 3: oligonucleotide for PCR amplification (SatPR1)

[0156] SEQ ID No 4: oligonucleotide for PCR amplification (SatPR2)

[0157] SEQ ID No 5: oligonucleotide for PCR amplification (SatPR3)

[0158] SEQ ID No 6: oligonucleotide for PCR amplification (SatPR4)

[0159] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should not be construed, however, as limiting the broad scope of the invention.

[0160] 1: Experimental Procedures

[0161] 1.1 Chimeric DNA Constructs

[0162] Ribozyme-Containing GUS Gene Constructs and a Control Construct

[0163] The ribozyme sequences used are the plus strand or negative strand self-cleavage sequences of the satellite RNA of the barley yellow dwarf virus (BYDV) RPV serotype, which was isolated in CSIRO Plant Industry (SEQ ID 1 and 2; Miller et al., 1991).

[0164] The two ribozyme-containing GUS constructs (pMBW259 and pMBW267) and one control GUS construct (pMBW265) are schematically drawn in FIG. 1. pMBW259 contains two plus strand cleavage sites, while pMBW267 contains the negative strand cleavage site.

[0165] To make these constructs, a -glucuronidase (GUS) gene sequence was modified to contain a Ncol site around the translational start ATG and cloned into pART7 (Gleave, 1992) at the XhoI/EcoRI sites, forming pMBW258. The full-length BYDV-RPV satellite sequence was amplified by PCR using primers SatPR1 (SEQ ID No. 3) and SatPR4 (SEQ ID No. 6), digested with BamHI and cloned into pMBW258 at the BamHI site, and the resulting 35S-GUS-Sat-ocs cassette was excised and cloned into pART27 (Gleave, 1992), forming pMBW265. The same full-length satellite sequence was inserted into the BamHI site of pMBW258 but in the antisense orientation, and the resulting 35S-GUS-asSat-ocs was cloned into pART27 to give rise to pMBW267.

[0166] To make pMBW259, the 3 and 5 halts of the satellite RNA sequences were amplified by PCR using primer pairs SatPR3 (SEQ ID No. 5) and SatPR4 (SEQ ID No. 6), and using SatPR1 (SEQ ID No. 3), and SatPR2 (SEQ ID No 4), respectively. Fusion of the full-length sequence with the 3 half and the 5 half sequences were made through ligation between the EcoRV and Hpal ends of the three PCR fragments. This fusion mimics the natural multimeric forms of the satellite RNA, and therefore maintains the plus strand cleavae property of the native forms. The fusion sequence was cloned into pGEM-3Z (Promega) at the SacI/PstI sites, excised with HindIII/EcoRI, blunted, and inserted into pART7 at the SmaI site, into which the GUS sequence described above was then cloned at the XhoI/EcoRI sites. The resulting 3SS-GUS-Sat-ocs was inserted into pART27 at the NotI site, forming pMBW259.

[0167] The Super-Transforming GUS Construct

[0168] The BamHI fragment was excised from pIG121 Hm (Hiei et al., 1994) and cloned into pART7. The GUS-nos sequence was then excised by Acci, blunted, and inserted into pBluescript at the HincII site. The 1.3 kb promoter region of a cucurbit phloem protein PP2 gene was excised with NotI/HindIII from a lambda clone CPPI.3 and cloned into the above Bluescript plasmid. The resulting PP2-GUS-nos was excised with NotI/KpnI and inserted into pWBVec2 (Wang et al., 1998), giving rise to pBPPGH (FIG. 1).

[0169] 1.2 Tobacco Transformation

[0170] Nicotiana tobaccum cv. W38 was transformed and regenerated into whole plants essentially as described by Ellis et al. 1987. For constructs pMBW259, pMBW265 and pMBW267, 50 mg/L kanamycin was included in the media for selection of transformed tissue. For construct pBPPGH, 25 mg/L hygromycin B was used.

[0171] 1.3 GUS Assay

[0172] GUS gene expression was assayed histochemically or fluorometrically according to Jefferson et al. 1987.

[0173] GUS Expression in Transgenic Tobacco Transformed with a Single Type of the GUS Constructs

[0174] Transgenic plants containing pMBW259 and pMBW267 showed very low levels of GUS expression, as judged by lack of, or faint blue, GUS staining. Plants transformed with pMBW265 showed more GUS expression than with pMBW259 and pMBW267, but the level was much lower than plants transformed with pBPPGH. The best pMBW265 lines expressed 13.3% of the GUS activity by an average pBPPGH line.

[0175] GUS Expression in Super-Transformed Lines Containing pBPPGH and One of the Three Other Constructs of Example 1.

[0176] In order to promote silencing of a normal GUS gene by the presence of the ribozyme sequence near the 3 end of the GUS gene transcript, plants containing pMBW259, pMBW265 or pMBW267 and pBPPGH were constructed by re-transformation. Histochemical GUS assays of the super-transformants showed that the pMBW267 background gave substantially higher proportions of transformants than the pMBW259 or the pMBW265 background that showed low levels of GUS expression as indicated by the lack of strong and uniform blue staining. Super-transformants containing pBPPGH and pMBW265 showed the best GUS expression.

[0177] Table 2 shows the result of fluorometric GUS (MUG) assay of the super-transformats. The lines (E and F) containing pBPPGH and pMBW267 showed uniformly low GUS expression compared with the other lines. The best GUS expression came from the C lines which contain pBPPGH and pMBW265.

[0178] Among the three constructs tested, pMBW265 does not contain the full-length functional ribozyme sequences of the BYDV satellite RNA in a continuous stretch, and is therefore expected to produce mainly poly(A) RNA. pMBW259 contains two copies of the plus strand ribozyme sequence, and should give rise to RNA that have poly(A) tails removed by ribozyme cleavage. pMBW267 contain the negative strand ribozyme. The negative strand ribozyme was previously shown to be much (at least 10-fold) more efficient than the plus strand ribozyme (Miller et al., 1991), and therefore it is expected that pMBW267 produces poly(A) RNA more efficiently. Our experiment showed that the super-transformed lines having the pMBW267 background expressed uniformly low levels of GUS activity in comparison with the lines having the pMBW259 or the pMBW265 background. The highest GUS expressing lines were from the pMBW265 background, which does not produce polyA RNA.

TABLE 2
MUG assay of super-transformed tobacco lines*.
	Super-
	transformed	MUG
	lines	Readings
	A1	10.1
	A2	15.8
	A3	30.6
	A4	47.3
	A5	0.29
	A6	10.3
	A7	5.8
	A8	13.15
	A9	7.34
	A10	9.76
	A11	17.74
	A12	34.8
	A13	4.33
	A14	3.41
	A15	11.2
	A16	2.04
	A17	13.29
	A18	14.6
	A19	0.14
	A20	17.2
	A21	9.22
	A22	17.3
	B1	9.57
	B2	44.7
	B3	17.7
	B4	1.25
	B5	13.5
	B6	11.4
	B7	6.28
	B8	24.8
	B9	16.3
	B10	9.72
	B11	3.71
	B12	0.08
	B13	20.6
	B14	11.9
	B15	3.11
	B16	8.25
	B17	4.12
	B18	6.04
	C1	8.84
	C2	16.9
	C3	17.9
	C4	22.8
	C5	11.7
	C6	14.5
	C7	44.0
	C8	19.0
	C9	29.8
	C10	32.1
	C11	37.1
	C12	2.51
	C13	14.5
	C14	25.8
	C15	7.20
	C16	30.2
	C17	9.70
	C18	13.4
	C19	19.3
	C20	17.0
	D1	6.01
	D2	12.9
	D3	0.19
	D4	7.88
	D5	1.24
	D6	0.44
	D7	14.1
	D8	0.91
	D9	5.49
	D10	1.30
	D11	15.1
	D12	6.63
	D13	12.2
	D14	15.8
	D15	1.32
	D16	2.29
	D17	3.59
	D18	22.1
	D19	13.0
	D20	4.37
	E1	4.32
	E2	3.15
	E3	3.56
	E4	3.31
	E5	3.68
	E6	5.02
	E7	2.63
	E8	10.27
	E9	10.81
	E10	13.1
	E11	5.10
	E12	2.86
	E13	4.00
	E14	16.8
	E15	4.02
	E16	1.29
	E17	1.78
	E18	3.57
	E19	0.43
	E20	11.8
	F1	5.73
	F2	5.10
	F3	4.16
	F4	4.69
	F5	0
	F6	1.93
	F7	3.21
	F8	2.77
	F9	1.86
	F10	3.27
	F11	2.85
	F12	3.25
	F13	2.17
	F14	2.84
	F15	3.11
	F16	2.06
	F17	2.90
	F18	3.75
	F19	4.16
	F20	2.49
	*A and B, from super-transformation of two independent pMBW259 lines with pBPPGH; C and D, from super-transformation of two independent pMBW265 lines with pBPPGH; E and F, from super-transformation of two independent pMBW267 lines with pBPPGH.

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