WO2002063039A2 - Gene silencing gene - Google Patents

Abstract

Provided are isolated nucleic acid molecules which comprise an sde3 nucleotide sequence which encodes an SDE3 polypeptide capable of mediating Post Transcriptional Gene Silencing (PTGS) of a transgene in a plant into which the nucleic acid is introduced. The SDE3 polypeptide is believed to encode an RNA helicase-like polypeptide. The invention has utility in both enhancing and suppressing the PTGS of transgenes in plants, and methods and materials are provided to achieve this.

Description

GENE SILENCING GENE

TECHNICAL FIELD

The present invention relates generally to methods and materials for use in modulating post transcriptional gene silencing.

BACKGROUND ART

Eukaryotic cells suppress foreign genetic elements, including transgenes, through a process that operates at the RNA level and is referred to here as posttranscriptional gene silencing (PTGS) (Kooter et al . , 1999; Plasterk and Ketting, 2000). In higher plants this system provides protection against viruses (Mourrain et al . , 2000; Ratcliff et al . , 1997; Ratcliff et al . , 1999) whereas in C. elegans (Ketting et al . , 1999), Drosophlla (Jensen et al . , 1999) and Chlamydomonas (Wu-Scharf et al . , 2000) the targeted elements are transposons. Expression of transgenes is suppressed by PTGS in higher plants (Kooter et al . , 1999), Chlamydomonas (Wu-Scharf et al . , 2000), Neurospora crassa (Cogoni et al . , 1996), C. elegans (Ketting and Plasterk, 2000) and Drosophila (PalBhadra et al . , 1997) . Presumably, in PTGS of transgenes, the foreign DNA or the corresponding RNA is perceived by the cell as if it were a virus or a transposon.

A remarkable feature of PTGS is its ability to provide nucleotide sequence-specific protection against many different types of foreign genetic element. In effect it is a type of immune system that operates at the nucleic acid level. However, unlike antibody- mediated immunity, the specificity of the system is not genetically programmed. Instead it seems that the foreign genetic element is the source of the specificity determinant in PTGS through a process that is likely to involve an RNA-dependant RNA polymerase (RdRP) homologue in higher plants (Dalmay et al . , 2000b; Mourrain et al . , 2000), N. crassa (Cogoni and Macino, 1999a) and C. elegans (Smardon et al . , 2000). Other proteins that are involved include an EIF2C homologue in higher plants (Fagard et al . , 2000), N. crassa (Catalanotto et al . , 2000) and C. elegans (Tabara et al . , 1999). Proteins with homology to RNAse D/ReqQ are involved in N. crassa (Cogoni and Macino, 1999b) and C. elegans (Ketting et al . , 1999)).

Double stranded (ds) RNA is also a likely component of the PTGS mechanism. It is a potent activator of PTGS in plants (Chuang and Meyerowitz, 2000; Smith et al . , 2000) and animals (Fire et al . , 1998; Wianny and Zernicka-Goetz, 2000) . In Drosophila dsRNA is processed into small 21-23nt RNAs (Zamore et al . , 2000) that are incorporated as guide RNAs into an RNase complex (Hammond et al . ,

2000) . These short RNAs, which are also found in plants exhibiting PTGS (Hamilton and Baulcombe, 1999), would anneal with complementary RNAs and thereby ensure that the RNase specifically targets the RNA species that are homologous to the original dsRNA.

PTGS in virus-infected plants is targeted against the viral genome and we this finding has been exploited with virus vectors carrying fragments of host genes as a means of inactivating host gene expression (Baulcombe, 1999; Burton et al . , 2000; Ruiz et al . , 1998) . The PTGS is targeted against the RNA of the host gene so that the symptoms in the infected plant reflect the function of the encoded protein. This approach complements genetic approaches to assigning gene function (Baulcombe, 1999) and may be used to investigate PTGS. For example, in virus-mediated PTGS of a GFP transgene, there is a virus-dependent initiation stage of PTGS. A later stage that accounts for maintenance of PTGS is transgene- rather than virus-dependent (Ruiz et al . , 1998) and is associated with methylation of the GFP transgene (Jones et al . , 1999). In contrast, the virus-mediated PTGS of endogenous genes does not exhibit the progression from initiation to maintenance (Jones et al . , 1999; Ruiz et al . , 1998), does not become virus-independent and is not associated with DNA methylation.

With PTGS mediated by viruses, viroids and inverted repeat transposons the dsRNA component of the PTGS process may be produced directly as a replication intermediate or by transcription. However, in other examples of PTGS, the foreign genetic element may produce single stranded RNA that the RdRP converts to a ds form. The precise mechanism whereby the single stranded RNA template of the RdRP is differentiated from native RNA species is not known.

Indeed, as is clear from the foregoing, it has not yet been established how, and in what order, the various components act in the PTGS mechanism. The characterisation and/or isolation of any of these various components could potentially be used to modulate PTGS, and would thus provide a contribution to the art.

DISCLOSURE OF THE INVENTION

The present inventors have used of virus-mediated gene silencing for genetic analysis of PTGS, focussing on SDE3 which is one of at least four Silencing DEfective ( SDE) loci in Arabidopsis encoding proteins required for PTGS (Dalmay et al . , 2000b). They have demonstrated that SDE3 is required for PTGS mediated by transgenes (in this case GFP) but not by a TRV vector construct and therefore that SDE3, like the previously described RNA polymerase encoded by SDE1 , acts at a stage in the mechanism that is circumvented when PTGS is mediated by TRV. Further results show that SDE3 represents one of a group of RNA helicase-like proteins in which the other members are encoded by gbll O in mouse and homologous genes in humans and Drosophila. These proteins are similar to, but clearly distinct from the SMG-2 (C. elegans) RNA helicase involved in nonsense mediated RNA degradation and PTGS in C. elegans (Domeier et al . , 2000) .

In the light of this finding, the inventors have hypothesized that the role of SDE3 is to assist in the production of a dsRNA activator of PTGS - a role which, in the context of virus-induced PTGS, is performed by virus-encoded protein. However, those skilled in the art will appreciate that the teaching herein can be used to modulate PTGS irrespective of the precise explanation for the underlying mechanisms involved.

Various aspects of the invention will now be discussed in more detail .

According to a first aspect of the present invention there is provided a nucleic acid obtainable from the SDE3 locus of a plant. Such a nucleic acid will generally encode a polypeptide which is capable of mediating PTGS of a transgene in a plant into which the nucleic acid is introduced.

The alteration in the PTGS may be assessed by comparison with a plant in which the nucleic acid, or an orthologue thereof, is not present. It may be preferable to use a sample of plants in each case. PTGS may be measured directly, or inferred from other factors. The change in PTGS may be associated with a qualitative change in the intensity of PTGS, or in the timescale over which it occurs (e.g. if and when the plant in question 'recovers'), or in the manner in which it can be initiated (e.g. in the absence of an initiator of viral origin) . Experiments in which PTGS is assessed are set out in the Examples hereinafter.

Apart from the change in PTGS it is preferred that other characteristics of the plant are substantially unchanged by the polypeptide, which is to say that the polypeptide acts specifically on a transgene-based PTGS response in the plant.

Preferably the polypeptide encoded by the nucleic acid is an RNA helicase-like protein, most preferably of the Upflp-like type (see discussion hereinafter), such as SDE3 of Arabidopsis .

Nucleic acid molecules according to the present invention may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of other nucleic acids of the species of origin. Where used herein, the term "isolated" encompasses all of these possibilities. The nucleic acid molecules may be wholly or partially synthetic. In particular they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively they may have been synthesised directly e.g. using an automated synthesiser.

Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and modified nucleic acids or nucleic acid analogs (e.g. peptide nucleic acid). Where a DNA sequence is specified, e.g. with reference to a figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed. Where genomic nucleic acid sequences of the invention are disclosed, nucleic acids comprising any one or more introns or exons from any of those sequences are also embraced. Where a nucleic acid of the invention is referred to herein, the complement of such nucleic acid will also be embraced by the invention.

Thus in one embodiment of this aspect of the invention, there is disclosed a nucleic acid encoding the SDE3 polypeptide of Annex I.

More preferably the nucleic acid includes the sequence shown as the SDE3 genomic sequence or SDE3 cDNA sequence in Annex II, or a sequence degeneratively equivalent to either of these. Where genomic nucleic acid sequences of the invention are disclosed, nucleic acids comprising any one or more introns or exons from any of those sequences are also embraced. Where a nucleic acid of the invention is referred to herein, the complement of such nucleic acid will also be embraced by the invention.

In a further aspect of the present invention there are disclosed nucleic acids which are variants of the sequences of the first aspect .

An SDE3 variant nucleic acid molecule shares homology with, or is identical to, all or part of the coding sequence discussed above. Generally, variants may encode, or be used to isolate or amplify nucleic acids which encode, polypeptides which modify transgene silencing in a plant into which they are introduced, and hence alter expression in that plant, and/or which will specifically bind to an antibody raised against the SDE3 polypeptide of Annex I. The gene silencing function may be assessed as set out in the Examples below.

Variants of the present invention can be artificial nucleic acids (i.e. containing sequences which have not originated naturally) which can be prepared by the skilled person in the light of the present disclosure. Alternatively they may be novel, naturally occurring, nucleic acids, which may be isolatable using the sequences of the present invention.

Thus a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequence provided. The fragments may encode particular functional parts of the polypeptide .

Equally the fragments may have utility in probing for, or amplifying, the sequence provided or closely related ones. Suitable lengths of fragment, and conditions, for such processes are discussed in more detail below.

Also included are nucleic acids which have been extended at the 3' or 5 ' terminus .

Sequence variants which occur naturally may include alleles or other homologues (which may include polymorphisms or mutations at one or more bases) .

Artificial variants (derivatives) may be prepared by those skilled in the art, for instance by site directed or random mutagenesis, or by direct synthesis. Preferably the variant nucleic acid is generated either directly or indirectly (e.g. via one or amplification or replication steps) from an original nucleic acid having all or part of the sequences of the first aspect. Preferably it encodes an SDE3 orthologue.

The term "variant" nucleic acid as used herein encompasses all of these possibilities. When used in the context of polypeptides or proteins it indicates the encoded expression product of the variant nucleic acid.

Some of the aspects of the present invention relating to variants will now be discussed in more detail.

Homology (i.e. similarity or identity) may be as defined using sequence comparisons are made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows:

Gapopen (penalty for the first residue in a gap) : -12 for proteins / -16 for DNA Gapext (penalty for additional residues in a gap) : -2 for proteins / -4 for DNA

KTUP word length: 2 for proteins / 6 for DNA.

Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares at least about 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology with the SDE3 sequence.

Thus a variant polypeptide in accordance with the present invention may include within the SDE3 sequence shown in Annex I, a single amino acid or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80, 90, 100, 200, 300, or 400 changes. In addition to one or more changes within the amino acid sequence shown, a variant polypeptide may include additional amino acids at the C-terminus and/or N-terminus.

Naturally, regarding nucleic acid variants, changes to the nucleic acid which make no difference to the encoded polypeptide (i.e. "degeneratively equivalent") are included within the scope of the present invention.

Thus in a further aspect of the invention there is disclosed a method of producing a derivative nucleic acid comprising the step of modifying the coding sequence of an SDE3 nucleic acid.

Changes to a sequence, to produce a derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.

Changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; codon usage; other sites which are required for post translation modification; cleavage sites in the encoded polypeptide; motifs in the encoded polypeptide (e.g. binding sites). Leader or other targeting sequences (e.g. hydrophobic anchoring regions) may be added or removed from the expressed protein to determine its location following expression. All of these may assist in efficiently cloning and expressing an active polypeptide in recombinant form (as described below) .

Other desirable mutation may be random or site directed mutagenesis in order to alter the activity (e.g. specificity) or stability of the encoded polypeptide.

Changes may be by way of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.

Also included are variants having non-conservative substitutions. As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide 's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.

In a further aspect of the present invention there is provided a method of identifying and/or cloning an SDE3 nucleic acid variant (e.g. orthologue) from a plant which method employs a probe or primer of the present invention. Target plants include (but are not limited to) crop plants such as rice, maize, wheat, barley, alfalfa, chickpea, bean and pea.

An oligonucleotide for use in probing or amplification reactions comprise or consist of about 48, 36 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16-30 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 100 's or even 2000 or more nucleotides in length.

A primer of the present invention is distinctive to SDE3 in the sense that it is based on any region present in the SDE3 sequence of Annex I, but not in any region of sequences of the prior art shown in Figure 6. 'Based on' in this sense can mean that the primer encodes an SDE3 region, or is the complement of a primer which does. Such primers will have utility not only in manipulating the Annex I SDE3 sequence, but also in isolating those sequences which are expected to be more closely related to it.

In one embodiment, nucleotide sequence information provided herein may be used in a data-base (e.g. of expressed sequence tags, or sequence tagged sites) search to find homologous sequences, such as those which may become available in due course, and expression products of which can be tested for activity as described below.

In a further embodiment, a variant in accordance with the present invention is also obtainable by means of a method which includes:

(a) providing a preparation of nucleic acid, e.g. from plant cells,

(b) providing a nucleic acid molecule which is a probe as described above,

(c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue (e.g. orthologue, parologue) if present by its hybridisation with said nucleic acid molecule.

Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.

Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector. If genomic DNA is used the probe may be used to identify untranscribed regions of the gene (e.g. promoters etc.), such as is described hereinafter. Probing may optionally be done by means of so-called "nucleic acid chips" (see Marshall & Hodgson (1998) Nature Biotechnology 16: 27-31, for a review) .

Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.

For instance, screening may initially be carried out under conditions, which comprise a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (e.g. Standard Saline Citrate ("SSC") = 0.15 M sodium chloride; 0.15 M sodium citrate; pH 7) concentration.

Alternatively, a temperature of about 50°C or less and a high salt (e.g. "SSPE" = 0.180 mM sodium chloride; 9 mM disodium hydrogen phosphate; 9 mM sodium dihydrogen phosphate; 1 mM sodium EDTA; pH 7.4). Preferably the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50°C and a salt concentration of about 2 X SSPE. These conditions will allow the identification of sequences which have a substantial degree of homology (similarity, identity) with the probe sequence, without requiring the perfect homology for the identification of a stable hybrid.

Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na₂HP0₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na₂HP0₄, pH 7.2,

6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.

It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched. Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989): T_m = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex

As an illustration of the above formula, using [Na+] = [0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the T_ra is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention. Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR (see below) or RN'ase cleavage. The identification of successful hybridisation is followed by isolation of the nucleic acid which has hybridised, which may involve one or more steps of PCR or amplification of a vector in a suitable host.

Thus one embodiment of this aspect of the present invention is nucleic acid including or consisting essentially of a sequence of nucleotides complementary to a nucleotide sequence hybridisable with any encoding sequence provided herein. Another way of looking at this would be for nucleic acid according to this aspect to be hybridisable with a nucleotide sequence complementary to any encoding sequence provided herein. Of course, DNA is generally double-stranded and blotting techniques such as Southern hybridisation are often performed following separation of the strands without a distinction being drawn between which of the strands is hybridising. Preferably the hybridisable nucleic acid or its complement encode a product able to influence a PTGS characteristic of a plant, particularly transgene initiated PTGS.

In a further embodiment, hybridisation of nucleic acid molecule to a variant may be determined or identified indirectly, e.g. using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) . PCR requires the use of two primers to amplify target nucleic acid, so preferably two primers as described above are employed. Using RACE PCR, one 'random' primer may be used (see "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990) ) .

Thus a method involving use of PCR in obtaining nucleic acid according to the present invention may be carried out as described above, but using a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR, at least one of which is a primer of the present invention as described above. In each case above, if need be, clones or fragments identified in the search can be extended. For instance if it is suspected that they are incomplete, the original DNA source (e.g. a clone library, mRNA preparation etc.) can be revisited to isolate missing portions e.g. using sequences, probes or primers based on that portion which has already been obtained to identify other clones containing overlapping sequence.

The methods described above may also be used to determine the presence of one of the nucleotide sequences of the present invention within the genetic context of an individual plant, optionally a transgenic plant, which may be produced as described in more detail below. This may be useful in plant breeding programmes e.g. to directly select plants containing alleles which are responsible for desirable traits in that plant species, either in parent plants or in progeny (e.g. hybrids, Fl, F2 etc.). Thus use of markers which can be designed by those skilled in the art on the basis the sequence information disclosed herein form one part of the present invention.

As used hereinafter, unless the context demands otherwise, the term ^ΛSDE3 nucleic acid' is intended to cover any of the nucleic acids of the invention described above, including functional variants.

In one aspect of the present invention, the SDE3 nucleic acid described above is in the form of a recombinant and preferably replicable vector.

^λVector' is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells).

A vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

The vector may further include SDEl-derived sequence (see Dalmay et al, 2000b) . SDE1 is believed to encode an RNA-dependant RNA polymerase (RdRP) homologue. SDE3 may work in conjunction with SDE1 in the formation of a double stranded RNA mediator of PTGS. Thus in this and other aspects of the present invention, the SDE3 is preferably used in conjunction with SDEl (particularly heterologous SDEl) .

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell

By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

Thus this aspect of the invention provides a gene construct, preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as SDE3 or a variant thereof, optionally in addition to an SDEl or variant sequence.

Generally speaking, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular

Cloning : a Labora tory Manual : 2nd edition, Sambrook et al , 1989, Cold Spring Harbor Laboratory Press (or later editions of this work) .

Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis (see above discussion in respect of variants) , sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John

Wiley & Sons, 1992. The disclosures of Sambrook et al . and Ausubel et al. are incorporated herein by reference.

In one embodiment of this aspect of the present invention, there is provided a gene construct, preferably a replicable vector, comprising an inducible promoter operatively linked to a nucleotide sequence provided by the present invention.

The term "inducible" as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. As described in the Examples below, one preferred embodiment of the invention uses an 'amplicon' as described in WO98/36083. Such an amplicon may comprise a promoter sequence operably linked to DNA for transcription in a plant cell of an RNA molecule that includes plant virus sequences that confer on the RNA molecule the ability to replicate in the cytoplasm of a plant cell following transcription. The transcripts replicate as if they are viral RNAs, and comprise a targeting sequence corresponding to the gene of interest ('the target gene'). In the present invention the target gene may be based on an SDE3 sequence (to silence any endogenous copies related thereto) . Alternatively or additionally, amplicons may contain a 'target gene' which it is desired to express at high levels, provided that these are expressed in a context where SDE activity (and hence the PTGS of the target gene) has been suppressed.

Also of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148) .

Suitable promoters which operate in plants include the Cauliflower Mosaic Virus 35S (CaMV 35S) . Other examples are disclosed at pg 120 of Lindsey & Jones (1989) 'Plant Biotechnology in Agriculture' Pub. OU Press, Milton Keynes, UK. The promoter may be selected to include one or more sequence motifs or elements conferring developmental and/or tissue-specific regulatory control of expression. Inducible plant promoters include the ethanol induced promoter of Caddick et al (1998) Nature Biotechnology 16: 177-180. It may be desirable to use a strong constitutive promoter such as the ubiquitin promoter, particularly in monocots.

If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to antibiotics or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate) .

The present invention also provides methods comprising introduction of such a construct into a host cell, particularly a plant cell.

In a further aspect of the invention, there is disclosed a host cell containing a heterologous construct according to the present invention, especially a plant or a microbial cell.

The term "heterologous" is used broadly in this aspect to indicate that the gene/sequence of nucleotides in question (an SDE gene) have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention. A heterologous gene may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the

« inserted sequence may be additional to the endogenous gene or other sequence .

Nucleic acid heterologous to a plant cell may be non-naturally occurring in cells of that type, variety or species. Thus the heterologous nucleic acid may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant. A further possibility is for a nucleic acid sequence to be placed within a cell in which it or a homolog is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.

The host cell (e.g. plant cell) is preferably transformed by the construct, which is to say that the construct becomes established within the cell, altering one or more of the cell's characteristics and hence phenotype e.g. with PTGS of a target transgene.

Nucleic acid can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A- 270355, EP-A-0116718, NAR 12(22) 8711 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Cul ture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Physiol . 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U. S . A . 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech . Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has also been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (see e.g. Hiei et al . (1994) The Plant Journal 6, 271-282)). Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP- A-486233) .

It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Thus a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a construct as described above into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce a nucleic acid according to the present invention into the genome .

The invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention (e.g. comprising an SDE sequence) especially a plant or a microbial cell. In the transgenic plant cell (i.e. transgenic for the nucleic acid in question) the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome. There may be more than one heterologous nucleotide sequence per haploid genome.

Generally speaking, following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Cul ture and Soma tic Cell Genetics of Plants , Vol I, II and III, Laboratory Procedures and Their Applica tions, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al . (1992) Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Na ture Biotechnology 14 page 702).

Plants which include a plant cell according to the invention are also provided.

In addition to the regenerated plant, the present invention embraces all of the following: a clone of such a plant, selfed or hybrid progeny and descendants (e.g. FI and F2 descendants) and any part of any of these. The invention also provides parts of such plants e.g. any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on, or which may be a commodity per se e.g. grain.

The present invention also encompasses the expression product of any of SDE nucleic acid sequences disclosed above, plus also methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells. A preferred polypeptide includes the SDE3 amino acid sequence shown in Annex I. However a polypeptide according to the present invention may be an variant (allele, fragment, derivative, mutant or homologue etc.).

Use of such a polypeptide as an RNA-helicase is also embraced by the invention.

Also encompassed by the present invention are polypeptides which although clearly related to a functional SDE polypeptide (e.g. they are immunologically cross reactive with SDE3 polypeptide) no longer have SDE3 function.

Following expression, the recombinant product may, if required, be isolated from the expression system. Generally however the polypeptides of the present invention will be used in vivo (in particular in planta ) .

Purified SDE3 or variant protein, produced recombinantly by expression from encoding nucleic acid therefor, may be used to raise antibodies employing techniques which are standard in the art.

Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal. As an alternative or supplement to immunising a mammal, antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes. Thus, the present invention provides a method of identifying or isolating an SDE polypeptide, including screening candidate peptides or polypeptides with a polypeptide including the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind an SDE3 peptide, polypeptide or fragment, variant or variant thereof or preferably has binding specificity for such a peptide or polypeptide, such as having an amino acid sequence identified herein. Specific binding members such as antibodies and polypeptides including antigen binding domains of antibodies that bind and are preferably specific for an SDE3 peptide or polypeptide or mutant, variant or derivative thereof represent further aspects of the present invention, as do their use and methods which employ them.

Candidate peptides or polypeptides for screening may for instance be the products of an expression library created using nucleic acid derived from an plant of interest, or may be the product of a purification process from a natural source.

As set out in the Examples hereinafter, SDE3-based materials may be used to either enhance or suppress PTGS of transgenes, with a corresponding affect on their expression. For instance, materials (e.g. constructs such as those discussed above) which have the effect of increasing SDE activity in a plant (e.g. expressing it in cells in which it was not previously present, or in which it was present at a lower level) can be used to inhibit transgene expression in that plant.

Thus the invention further provides a method of influencing or affecting the nature or degree of PTGS of a transgene in a plant, the method including the step of causing or allowing expression of a heterologous SDE3 nucleic acid sequence as discussed above within the cells of the plant. Preferably the influence is exerted selectively i.e. such that virus-induced PTGS is not affected.

The step may be preceded by the earlier step of introduction of the nucleic acid into a cell of the plant or an ancestor thereof.

Likewise, materials which have the effect of reducing SDE activity in a plant (e.g. silencing it, or impairing transcription, or competitively or non-competitively inhibiting its activity) can be used to enhance transgene expression in that plant.

The sequence information disclosed herein may be used for the down- regulation of SDE3 (to enhance expression of transgenes) e.g. using anti-sense technology (see e.g. Bourque, (1995), Plant Science 105, 125-149); sense regulation [co-suppression] (see e.g. Zhang et al . , (1992) The Plant Cell 4, 1575-1588) . Further options for down regulation of gene expression include the use of ribozymes, e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g. Jaeger (1997) ^λThe new world of ribozymes' Curr Opin Struct Biol 7:324-335.

In using anti-sense genes or partial gene sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al, 1987; Smith et al, (1988) Na ture 334, 724- 726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al . , (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Flavell, (1994) PNAS USA 91, 3490-3496.

A preferred alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by either Viral Induced Gene Silencing, or using Amplicons (Angell & Baulcombe (1997) The EMBO Journal 16,12:3675- 3684) or other viral-based methods (W095/34668; Biosource) .

Thus the invention further provides a method of suppressing the nature or degree of PTGS of a transgene in a plant, the method including the step of causing or allowing expression of an SDE3 heterologous nucleic acid sequence as discussed above within the cells of the plant. This aspect preferably involves use of a sequence encoding a disabled SDE3 polypeptide, or a sequence which inhibits the activity of SDE3 in the plant. The term 'suppressing' in this context does not imply a requirement that PTGS be totally negated. The suppression may be partial, for instance in terms of localisation (e.g. restricted to an area in which the SDE construct is infiltrated) temporally (e.g. the plant recovers from the SDE- blocking effect) or intensity (i.e. PTGS continues at a reduced level) . The term is used herein where convenient because those skilled in the art well understand this.

Thus the present invention provides methods of or for suppressing PTGS, and enhancing expression of transgene nucleic acids (to produce heterologous polypeptides) , in cells, which employ the SDE3-based materials disclosed herein, optionally in conjunction with SDEl-based materials. Likewise use of SDE3-based materials disclosed herein to enhance the activity of SDEl-based materials is also embraced.

Target transgenes for enhanced expression may encode, inter alia , genes of bacterial, fungal, plant or animal origin. The polypeptides may be utilised in planta (to modify the characteristics of the plant e.g. with respect to pest susceptibility, vigour, tissue differentiation, fertility, nutritional value etc.) or the plant may be an intermediate for producing the polypeptides which can be purified therefrom for use elsewhere. Such proteins include, but are not limited to retinoblastoma protein, p53, angiostatin, and leptin. Likewise, the methods of the invention can be used to produce mammalian regulatory proteins. Other sequences of interest include proteins, hormones, growth factors, cytokines, serum albumin, haemoglobin, collagen, etc.

The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

Figure Legends Figure 1. SDE3 affects susceptibility to CMV but not to TRV

RNA was isolated from leaves of TRV (A and B) or CMV (C and D) infected wild type (wt[GxA]), sdel[GxA] and sde3[GxA] plants. Northern blot analysis was carried out using ³²P-labeled probes corresponding to TRV (A), to CMV (C) and to rRNA (B and D) . The RNA species detected were TRV RNAl and 2 (TRVl and TRV2), CMV RNAl, 2, 3 and 4 (CMV1, CMV2, CMV3 and CMV4) and rRNA.

Figure 2. CMV encodes for a stronger suppressor than TRV

(A) RNA was isolated from leaves of non-inoculated [GxA] (1) or [G] (2) and TRV-or CMV- inoculated [GxA] (3 and 4, respectively) plants. Northern blot analysis was carried out using ³²P-labeled probes corresponding to the full-length GFP RNA sequence (top) or to rRNA (bottom) . The RNA species detected were the genomic (G) or subgenomic (sgl and sg2) RNA of PVX, GFP mRNA (GFP) and rRNA. (B)The images of [GxA] plants were produced under UV light in a dissecting microscope, two weeks after inoculation with water

(mock), TRV or CMV. The red fluorescence is due to chlorophyll.

Figure 3. SDE3 is not required for TRV-induced gene silencing

(A) Photographs were taken of TRV: PDS-infected wt[G] and sde3[GxA] plants, three weeks after inoculation (1 and 4) . Panels 2 and 5 show the same plant as 1 and 4, respectively, focusing on the newly emerging leaves. The white areas are a result of photobleaching due to the PTGS of the PDS gene. Panels 3 and 6 show non-infected wt[G] and sde3[GxA].

(B) Plant were infected with TRV:GFP and images were produced under UV light in a dissecting microscope and the red fluorescence is due to chlorophyll. The photographs were taken of TRV: GFP infected wt[G], sdel[GxA] and sde3[GxA] at 10 (panels 1,4 and 7) or 20 (panels 2,5 and 8) days after inoculation. Panels 3,6 and 9 show non-infected wt[G], sdel[GxA] and sde3[GxA].

Figure 4. SDE3 is required for persistent silencing of GFP RNA was isolated from the emerging leaves of mock inoculated (lanes 1,2 and 3) or TRV: GFP inoculated wt[G], sdel [GxA] and sde3 [GxA] plants. Samples were taken from the TRV: GFP inoculated plants at 10 (lanes 4,5 and 6) or 20 (lanes 7,8 and 9) days after inoculation. Northern blot analysis was carried out using ³²P-labeled probes corresponding to the full-length GFP RNA sequence (top) or rRNA (bottom) .

Figure 5. Mapping of SDE3

The sde3 phenotype cosegregated with PUV4 and CAT3a markers on chromosome 1. Seven markers were generated throughout this region and the mapping population was increased to 1200 plants. There were seven recombination events between M103 and SDE3 and seven between SDE3 and M121, indicating that SDE3 is located between these two markers. Another nine markers were generated to make a high resolution map around SDE3. There was one recombination event between SDE3 and both M148 and M154, locating SDE3 in a 100 kb region . This sequence is represented in BAC clones T25N20 and F3F20. The bottom part shows the organization of SDE3. The arrow indicates that SDE3 is in a complementary orientation on T25N20 in the database. The numbers indicating the translation start point, the start codon, the exon-intron boundaries, the stop codon, the translation stop point and the positions of the mutation in different mutant alleles , are according to the numbering of T25N20.

Figure 6. Sequence alignments of SDE3 and other RNA helicase-like proteins

The sequence of SDE3 was aligned with other RNA helicase-like proteins Upflp from yeast (accession number SWALL: P30771) , AtUpflp from Arabidopsis (accession number SWALL :BAB10240) SMG-2 from C. elegans (accession number SWALL:076512 ) and homologues of a protein in mouse (mmgbllO ; accession number SWALL: P23249) and Drosophila (dmgbllO ; accession number SWALL: Q9VZP4) using CLUSTALW. These proteins represent the best matches to SDE3 in a database search using BLASTP. The output of CLUSTALW was shaded with GeneDoc, the shading shows residues which are similar in SDE3, mmgbllO and dmgbllO; and also residues which are similar in Upflp, AtUpflp and SMG-2. Residues in white on black are similar in all six sequences. Motifs that have previously been used to define the Upflp group of RNA helicases (Linder and Daugeron, 2000) are shown beneath the aligned sequences. Residues which are the repeated SQ motifs in the carboxy terminal domain are also shaded. It is likely that the predicted AtUpflp and dmgbllO sequences do not account for introns that were not recognised in the annotation of the genome sequence.

Sequence Annexes

Annex I shows the SDE3 polypeptide.

Annex II shows the Genomic DNA sequence of SDE3 from Arabidopsis C24.

General Experimental Procedures

Transgenic plants and mutagenesis

These were as described in Dalmay et al . , 2000a. Briefly, the green fluorescent ( GFP) insert in pPVX204 (Baulcombe et al . , 1995) was replaced with the mGFP5 insert from pBin-35S-mGFP5 (Haseloff et al., 1997). This new potato virus X (PVX)-GFP5 sequence was inserted between the cauliflower mosaic virus 35S promoter and the transcriptional terminator of the nopaline synthase gene and transferred into the binary vector pSLJ755/5. The PVX rep: GFP construct was obtained by deleting a Bglll fragment (positions 965- 2694) from the full-length PVX:GFP. pBin-35S-mGFP5 (Haseloff, et al., 1997) was used without any change. Transformation of

Arabidopsis thaliana ecotype C24 with Agrobacterium tumefaciens GV3101 carrying 35S-PVX-GFP or 35SGFP constructs was performed as described by (Bechtold, et al., 1993). The transformants were selected in vitro on medium supplemented with 10 mg/L L- Phosphinotricin (Melford) or 50 mg/L kanamycin, respectively.

The wt [A], wt [G]and wt [GxA] Arabidopsis (C24 ecotype) were described (Dalmay et al . , 2000a) as Amp243, GFP142 and GFP142xAmp243, respectively. The wt [A] contains a 35S-PVX:GFP and wt [G] carries a 35S-GFP transgene. Both lines are homozygous and have a single copy of the respective transgene. The wt [GxA] is the progeny of a cross between the two lines above and it is homozygous for both of the transgenes. The mutagenesis of wt [GxA] and screen for loss of PTGS plants were described (Dalmay et al . , 2000b).

GFP imaging

GFP expression was monitored using a MZ12 dissecting microscope (Leica, Heidelberg, Germany) coupled to an epifluorescence module. Photograph were taken using Kodak Ektachrome Panther (400 ASA) film.

RNA analysis

RNA gel blot analysis was performed as described previously (Mueller et al . , 1995). DNA fragments were labeled by random priming incorporation of ³²P-dCTP (Amersham) . After hybridisation, the signal present in the membranes was analysed and quantified using the Fujix Bio-Imaging Analyzer Bas 1000 (Fuji Photo Film Co., Ltd., Fuji, Japan) equipment. Polymerase chain reaction amplified full-length GFP DNA was used for the GFP specific probe. TRV RNA was detected with a probe made of a BstEII and Smal fragment (5345- 6792) of a TRVl clone (pTR7116) (Hamilton and Baulcombe, 1989) and pCa7 (Ratcliff et al . , 2000) which contains the full sequence of RNA2. The CMV probe was pKl, pK2 and pK3 each of them contain the full sequence of RNA 1,2 and 3, respectively (Boccard and Baulcombe, 1992)

Genetic mapping and DNA sequence analysis

A set of CAPS markers described by (Konieczny and Ausubel, 1993) was used to detect polymorphism between the Columbia and Landsberg ecotypes and to map sde loci to the 10 chromosome arms. However, several of these markers did not show polymorphism between C24 and Landsberg ecotypes and in some instances it was necessary to generate alternative markers. The sde3 mutation was mapped into a 2 Mb region between PUV4 (l.lcM) and CAT3a (27.8 cM) . There were 13 (PUV4) and 24 (CAT3a) recombinants in 140 plants. Seven markers were generated throughout this region and the mapping population was increased to 1200 plants. SDE3 was localized into a 550 kb region between markers M103 and M121 (7 recombinants with both markers) . Using another 9 markers SDE3 was finally mapped between M148 and M154, with 1 recombination between the gene and both markers. This region is about 100 kb and represented in BAC clones T25N20 and F3F20. These BACs are sequenced and annotated by the Arabidopsis Sequencing

Project (www.arabidopsis.org). Primers were designed to amplify all 23 predicted ORFs within the genetically defined region. DNA from wild type and three different sde3 mutant plants was used to amplify these ORFs. The PCR products were digested with eight restriction enzymes (ffpall, Rsal , Alul , Ha el l l , Hinfl , Hind i , Ddel and Sau3AI) and run in 1.5% agarose gels. Only the PCR products amplifying T25N20.11 showed polymorphism between wt and mutants, after digestion with Ddel ( sde3-l ) and Ddel or Haelll ( sde3-2) .

Eight overlapping DNA fragments were generated by PCR from the wt, sde3-l , sde3-2, and sde3-3 plants at the position in BAC T25N20 positions 33000-37500 kb and sequenced directly using the Big Dye Terminator Mix (PE Applied Biosystem) . The sequencing reactions were resolved on an ABI377 automated sequencer (Applied Biosystem, La Jolla, CA, USA) . The regions where mutation was found were sequenced on both strands from three independent PCR reactions from both the wt and the mutant plants .

The 5' and 3' ends of the SDE3 cDNA were determined by rapid amplification of cDNA ends (RACE) by using the Marathon cDNA amplification kit (Clontech) . RACE products were cloned into pGEM-T plasmid (Promega) and sequences of 10 independent 3' and 5' end clones were determined. The cDNA library, obtained by the Marathon cDNA amplification kit (Clontech) , was used as a template to PCR amplify eight overlapping fragments which were directly sequenced. The number and location of introns was determined by comparing the sequence data obtained from the genomic DNA with the data obtained from the cDNA. Other protein sequences homologous to SDE3 were identified using the BLASTP program (Altschul et al . , 1990). Protein sequences were obtained and collated using the Wisconsin package (Wisconsin Package Version 10.0, Genetics Computer Group (GCG) , Madison, Wisconsin) . Sequence alignments were produced using CLUSTALW

(Thompson et al . , 1994) and were displayed using Genedoc (Nicholas and Nicholas, 1997) .

Wild-type and recombinant viruses The TRV vector carrying the phytoene desaturase fragment is similar to TRV-.GFP described previously (Ratcliff et al . , 1999) This vector is described in more detail elsewhere (Ratcliff et al . , 2000). The insert in RNA2 of the TRV: phytoene desaturase vector is 1770 nucleotides of the arabidopsis phytoene desaturase sequence.

Example 1 - attenuation of PTGS in plant lines carrying sde3

The sde3 locus was identified through its loss of PTGS phenotype in Arabidopsis plants carrying a PVX: GFP amplicon [A] and a 35S:GFP reporter gene [G] (Dalmay et al . , 2000b). We refer to plants carrying both loci in the homozygous condition as [GxA] . In wild type [GxA] plants the [A] locus mediated PTGS of the [G] locus whereas, in plants carrying sde mutations, the PTGS was attenuated or absent. In plants carrying the sdel mutation there was complete loss of PTGS whereas, in sde3 plants, the PTGS was reduced in the true leaves and flowers but was as strong as in wild type plants in the hypocotyl and cotyledons (Dalmay et al . , 2000b).

Example 2 - SDE3 effects on the susceptibility to different viruses

We have shown previously that virus-induced PTGS is a manifestation of an antiviral defense in plants (Ratcliff et al . , 1997; Ratcliff et al . , 1999). If the sde3 mutation results in loss of this defense mechanism, the plants should exhibit hyper-susceptibility to virus infection and viral RNAs should accumulate at a higher level in mutant than in wild type (wt) plants. To test this possibility we infected plants with tobacco rattle virus (TRV) and cucumber mosaic virus (CMV) . Viral RNA accumulation was monitored by northern analysis. The results in Figure 1 reveal that, with sde3, the mutations had no effect on susceptibility to TRV; the level of viral RNA was the same in sde3 (panel A, tracks 4-6) and wt plants (panel A, tracks 7-9) . Figure 1 also confirms our previous finding that sdel had no effect on TRV accumulation (panel A, tracks 1-3) . TRV symptoms were also unaffected by sde3. In contrast, with CMV the virus accumulation was five times higher in the sdel and sde3 plants than in wild type (Figure 1 panel C) and symptoms were more severe in the flowering stem.

It has been suggested previously that the effect of sde and similar mutations on virus accumulation would be masked if the virus encodes a suppressor of PTGS (Mourrain et al . , 2000). To investigate this possibility we inoculated TRV and CMV to [GxA] plants. Virus-encoded suppressors of PTGS would cause an increase in the levels of the PVX: GFP RNA from locus [A] and GFP RNA from locus [G] (Brigneti et al . , 1998; Voinnet et al . , 1999). The increase in these RNAs would lead to enhanced GFP fluorescence in the presence of viral suppressors of PTGS.

These predicted effects of suppressors of PTGS were evident in CMV- infected plants (Figure 2A tracks 1 and 4 and Figure 2B) . Presumably the CMV-encoded 2b protein, which is a suppressor of PTGS (Brigneti et al . , 1998), was responsible for these effects. In contrast, the TRV infected plants failed to exhibit GFP fluorescence (Figure 2B) and the levels of PVX: GFP and GFP RNA were as low in TRV-infected [GxA] as in non infected plants (Figure 2A, tracks 1 and 3) . From these results we can rule out that TRV encodes a strong suppressor of PTGS in Arabidopsis that would mask the effect of mutations on virus-mediated PTGS. Therefore our finding that sde3 has no effect on TRV accumulation indicates that the encoded protein, like SDEl (Dalmay et al . , 2000b), is not required for TRV infection.

Example 3 - role of SDE3 in TRV-mediated gene silencing

To further investigate the role of SDE3 we infected plants with a TRV vector carrying an insert from the endogenous phytoene desaturase (PDS) gene. In wt plants this virus causes PTGS to be targeted against the endogenous PDS RNA resulting in low levels of photoprotective carotenoids and photobleaching in the leaves. These symptoms appeared at approximately 10 dpi and persist for 5-7 days (Figure 3A) . At later times the PTGS phenotype was lost and the newly emerging leaves were fully green. This transient PTGS most likely reflects the kinetics of TRV accumulation in infected plants. Initially, in the phase when there is abundant virus accumulation the PTGS is strong. In the later stages of the infection process the virus levels are low (Ratcliff et al . , 1999).

We predicted that, if SDE3 is required for virus-mediated PTGS, the photobleaching symptoms of PTGS would be delayed or more transient than on wt plants. However, in 3 independent experiments with 10 infected plants, the photobleaching on sde3 plants developed at the same rate and was as persistent as on wt plants (Figure 3A) . These results therefore provide confirmation of the susceptibility data (Figure 2) indicating that SDE3 is not required for TRV-mediated PTGS.

Example 4 - role of SDE3 in transgene silencing

In a second assay for TRV-mediated PTGS we inoculated plants with TRV: GFP. At 10 dpi on wt[G] plants this virus induced PTGS of the GFP transgene in the regions around the veins (Figure 3B 1) . By 20 dpi the silencing had spread throughout the plant leaves (Figure 3B 2) and, unlike the PDS silencing, this effect persisted for the life of the plant. In both the early and late stages of this process the absence GFP fluorescence was associated with a reduced steady state level of GFP RNA (Figure 4 lanes 1, 4 and 7) .

When TRV:GFP was inoculated to the sdel and sde3 mutants of [GxA], the initial stages of PTGS were exactly the same as on the wt[G] lines - GFP expression was lost from the regions around the veins (Figure 3B 4 and 7) and GFP RNA levels were ten fold lower in the infected leaves (Figure 4 lanes 5-6) than in equivalent tissue of the mock inoculated plants (Figure 4 lanes 2-3) . However the PTGS of GFP in the sdel and sde3 plants did not persist at later times. By 20 dpi the GFP expression in the newly emerging leaves of the TRV: GFP-infected plants (Figure 3B 5 and 8) was as extensive as on the non inoculated plants (Figure 3B 3, 6, and 9) and the GFP RNA was as abundant as in mock inoculated plants (figure 4 lanes 2, 3, 8 and 9) . Thus, on wt plants, there was a different pattern of TRV- induced PTGS of GFP and PDS. However on the sdel and sde3 plants the PTGS of PDS and GFP followed similar kinetics.

The difference between TRV-mediated PTGS of GFP and PDS is likely to be due to the ability of the GFP transgene to participate in PTGS as well as provide an RNA target (Jones et al . , 1999; Ruiz et al . , 1998). In contrast, PDS only provides a target. Initially, with both GFP and PDS, the TRV-mediated PTGS is virus-dependent and unaffected by sdel or sde3. However in the PTGS of GFP there is a later stage when PTGS becomes dependent on the GFP transgene rather than the virus and when the SDEl and SDE3 loci are required. This later stage would not be evident with PDS because there is no stage when PTGS of this gene is independent of TRV: PDS. Therefore, these data with TRV: GFP confirm that SDE3, like SDEl , is not required for TRV-dependent PTGS (Dalmay et al . , 2000b). These data also confirm that SDE3 is required for transgene-dependent PTGS.

Example 5 - cloning and analysis of SDE3

To clarify the role of SDE3 in the PTGS mechanism we used genetic markers to identify the DNA coding sequence at the SDE3 locus. First we assigned SDE3 to chromosome 1 between PUV4 (1.1 cM) and CAT3a (27.8 cM) . We then used a series of markers in the interval of approximately 2 Mbp and a mapping population of 1200 plants from a cross of SDE3 [Gx.A] x wt Landsberg ecotype to assign SDE3 to the interval M148-M154. Within this interval there are 23 predicted open reading frames. We were not able to generate a more precise map position of the SDE3 locus due to the low frequency of recombination in this interval. However, using primers designed to PCR amplify each of the open reading frames from wt and three mutant plants, we identified only one gene with polymorphisms compared to the wt plant.

Sequence analysis of cDNA generated by 5' and 3' RACE indicates that this candidate gene has three introns. This gene has a 14 bp deletion in sde3-l and a 20 bp deletion in sde3-2 that is replaced by a 38 bp insertion. The third allele { sde3-3) had a point mutation which introduced an early stop codon (Figure 5) . The occurrence of three independent mutations in the same gene is confirmation that this gene is the SDE3 locus.

BLASTP database searches revealed that the 113363Da SDE3 has motifs that are conserved in RNA helicase-like proteins (Koonin, 1992). In SDE3 these motifs are typical of a class of RNA helicases that also includes the yeast protein Upflp (Linder and Daugeron, 2000) . However a mouse protein encoded by gbll O (Movl O) (Hamann et al . , 1993; Mooslehner et al . , 1991) rather than Upflp was the closest homologue of SDE3 as identified by BLASTP. All of these SDE3 homologues have RNA helicase motifs that are quite distinct from those of the DEAD, DEAH and Ski2p types of RNA helicase (Linder and Daugeron, 2000) .

Several features (Figure 6) indicate that there are two groups of SDE3-like helicase. For example, in the amino terminal region of Upflp and its homologue in C. elegans (SMG-2), there is a conserved cysteine-rich region. In addition, near the carboxy terminal of these proteins, there are multiple SQ doublets (Leeds et al . , 1992; Page et al . , 1999). The presence of these domains defines one of the two groups that includes a predicted Arabidopsis protein of unknown function (accession number SWALL:BAB10240 ) but not SDE3. We refer to the SWALL :BAB10240 protein as AtUpflp because it is the likely functional homologue of Upflp/SMG-2 in Arabidopsis .

The second group of Upflp-like helicases includes SDE3 and proteins from mouse, humans and Drosophila (Figure 6, and data not shown) . This group is defined by the absence of the cysteine-rich and SQ domains. In addition there are conserved motifs, between 620 and 740 of the aligned sequences (Figure 6) , in which the SDE3 group is distinct from Upflp and its close homologues. Motif searches failed to identify a function of these conserved motifs.

A role for SDE3 in synthesis of double stranded RNA?

As described above, the SDE3 locus encodes an RNA helicase-like protein (Figure 6) that is required for PTGS of GFP in the true leaves of [GxA] plants. In addition, in plants infected with TRV GFP, this gene was required for persistent PTGS of a GFP transgene (Figure 3B) . Both of these are examples of PTGS in which the transgene is an active participant. In contrast SDE3 was not required for PTGS of an endogenous PDS gene in TRV: PDS infected plants (Figure 3A) . Similarly the initial stages of the TRV:GFP- induced PTGS of GFP were indistinguishable in wt and sde3 plants (Figure 3B) . From these findings we conclude that SDE3 facilitates PTGS of transgenes and that it is not essential for TRV-mediated PTGS.

We proposed previously that the role of the SDEl RdRP was to convert aberrant single stranded RNA of a transgene into a double stranded form (Dalmay et al . , 2000b). The double stranded RNA would be processed into short 21-23nt RNAs (Hamilton and Baulcombe, 1999; Zamore et al . , 2000) that guide RNase to the targets of PTGS (Hammond et al . , 2000). The proposed role of the RdRP in production of dsRNA was based in part on the finding that SDEl is not required for TRV:PDS-mediated PTGS of PDS (Dalmay et al . , 2000b). It seemed likely that the TRV-encoded RdRP could synthesize a double stranded replication intermediate and thereby compensate in PTGS for the absence of SDEl.

In the light of the present application, it can be seen that SDE3 is also not required for TRV: PDS-mediated PTGS of PDS (Figure 3A) and therefore it is likely that this protein, like SDEl, is involved in production of dsRNA. If that is the case, an SDE1/SDE3 complex would be similar to replicases of RNA viruses that have RdRP and helicase components and synthesize double stranded RNA (Matthews, 1991) . In some instances the RdRP and helicase domains of the replicase are part of the same virus-encoded protein. However, in other examples, as would be the case in an SDE1/SDE3 complex, these two domains are on separate proteins. It is possible that the role of the helicase in both types of RdRP complex is to allow access of the polymerase molecule to its template by unfolding structured regions of the template RNA.

A role for SDEl and SDE3 in antiviral defense ? As PTGS represents a type of antiviral defense it might be expected that the sde plants would be hyper-susceptible to virus-infection. However, in most examples tested, that is not the case; the sdel and sde3 plants were as susceptible to tobacco mosaic virus, TRV and turnip crinkle virus as were the wt plants (Figure 1 and unpublished data) . Of the tested viruses only CMV is affected by sde3 or sdel/sgs2 (Figure 1) (Mourrain et al . , 2000). As discussed below, viruses may show different responses to sdel and sde3 because they have evolved different strategies for overcoming disease resistance mechanisms.

TMV and TCV may represent one of these evolutionary strategies. These viruses may have lost the ability to produce the aberrant single stranded RNA that we have proposed is required for SDEl/SDE3-dependent PTGS. Alternatively, or additionally, these viruses may encode suppressors of PTGS (Voinnet et al . , 1999). The action of these proteins would suppress PTGS in both the wild type and mutant plants so that there would be no discernable effect of the mutations .

The counter-defense strategy of TRV does not involve a strong suppressor of PTGS (Figure 2) (Voinnet et al . , 1999) and it is possible that this virus does not produce the putative aberrant RNAs required for SDE1/SDE3- dependent PTGS. An alternative possibility is that these RNAs are produced but that they are somehow hidden in the cell so that they do not participate in the ■SDEl/SDEJ-dependent branch of PTGS.

A third counterdefense strategy, exhibited by CMV, is different from that of the other viruses tested, as illustrated by the effect of sde3 or sgs∑f sdel on accumulation of this virus (Figure 1) . Thus, CMV is affected by the SDE1/SDE3- mechanism despite its ability to produce the 2b protein suppressor of PTGS (Brigneti et al . , 1998). Clearly this virus does produce the RNA species that are required for the SDE1/SDE3- mechanism. Perhaps the suppression of PTGS by the 2b protein is enough to allow accumulation and spread of CMV in the infected plant but not so complete that the sdel / sde3 phenotype is masked. By extrapolation of these ideas, it might be predicted that viruses other than CMV will also be affected by the sdel/sde3 mutations. They would be viruses that have the potential to activate the SDEl/ SDE3 mechanism through the production of aberrant RNA. In addition these viruses should have only limited ability to evade or suppress PTGS in Arabidopsis . It seems most likely that these attributes will often be associated with viruses that are not naturally adapted to infect this plant. If that is the case PTGS may be significant as a component of a general defense system, referred to as non host resistance, that accounts for the maxim that ^Λmost plants are resistant against most viruses' .

RNA helicases in PTGS

SDE3 is the third RNA helicase-like protein to be implicated in

PTGS. The others are SMG-2 from C. elegans (Domeier et al . , 2000), a member of the Upflp group of RNA helicases (Linder and Daugeron, 2000) and MUT6 in Chlamydomonas that encodes a DEAH helicase (Wu- Scharf et al . , 2000). SDE3 is more closely related to Upflp than MUT6 based on the sequence of RNA helicase motifs (Figure 6) . However it lacks a cysteine-rich region in the amino terminal region of Upflp and homologues, including SMG-2 (Page et al . , 1999) . This domain facilitates the nonsense mediated mRNA decay function of Upflp by interacting with a second protein, Nmd2p (He et al . , 1997) . The repeated SQ motifs in the carboxy terminal region of SMG-2 and Upflp are also absent from SDE3 (Figure 6) . It has been proposed that these motifs are the substrate of phophatidylinositol 3-kinase related kinase (Page et al . , 1999).

The predicted Arabidopsis protein AtUpflp has the cysteine rich region, the Upflp helicase motifs and the carboxy terminal SQ repeats (Figure 6) indicating that it, rather than SDE3, is the likely functional equivalent of Upflp.

Thus it is possible that SDE3 represents a subgroup of the Upflp- like RNA helicases in which the cysteine rich region and the carboxy terminal SQ repeats are absent. The SDE3 subgroup is also characterised by motifs on the amino terminal side of the RNA helicase motifs (Figure 6) . Other members of this group are the GB110 protein of mouse and homologues in humans and Drosophila . Presumably the absence of the cysteine rich region indicates that this subgroup of proteins does not interact with homologues of Nmd2p and, therefore, that they are available to interact with other as yet unidentified proteins.

It is notable that there are no RNA helicases in the SDE3 subgroup encoded in the genome of C. elegans, although this, organism is competent in PTGS. One explanation for this discrepancy is that SDE3-like proteins are regulators rather than essential cofactors of PTGS and are not used in C. elegans . Consistent with that idea we have reported that sde3 plants exhibit only partial loss of PTGS. PTGS of GFP in the cotyledons is as complete as in the wt plants and, in the true leaves, the PTGS defect is less pronounced than in sdel and sde2 plants. A second explanation is based on the idea, as discussed above, that SDE3 mediates conversion of single stranded RNA into a ds form. If that is the case, an SDE3 homologue would not be required in C. elegans because PTGS is normally induced by direct introduction of dsRNA (Fire et al . , 1998).

It is not known to what extent PTGS in mammals is used as defense against foreign genetic elements. Mouse embryos are competent in PTGS induced by dsRNA (Wianny and Zernicka-Goetz, 2000) but there is currently only limited information about other cell types or the possibility that PTGS can be induced by genetic elements that do not produce ds RNA (Bahramian and Zarbl, 1999) . The presence of SDE3 in mammals suggests that PTGS can operate in mammals as in plants. The possibility remains that SDE3 has multiple roles in the way that SMG-2 in C. elegans is involved in PTGS and nonsense mediated mRNA decay.

Example 6 - isolation of SDE homologues from other species

This may be most readily achieved using PCR primers derived from the Arabidopsis gene sequence shown above. It may be preferred to use areas of the polypeptide which are distinctive to SDE3 (not well conserved with other proteins) in order to generate degenerate primers . In particular it may desirable to identify the SDE 3 orthologue in crop plants. PCR primers based on the arabidopsis sequence can be used to amplify the orthologous DNA from the crop plant and identity of the amplified sequence as the orthologue can be confirmed by comparison with the arabidopsis homologues of SDE3.

Example 7 - assessing gene silencing potentiation by SDE3

Overexpression of SDE3, or homologues thereof, may be used to enhance to gene silencing of a selected target gene. The following assays may be used to assess the effectiveness of SDE3, or a homologue thereof, in a given system (optionally in conjunction with heterologous SDEl) . In this and the following Examples the methods are exemplified using SDE3 and a particular transgene (GFP) in particular lines. However those skilled in the art will be able to readily modify the methods, in the light of this disclosure, to suit a system of choice.

In one embodiment, SDE3 is transferred into the 35S promoter expression cassette of an Agrobacterium binary plasmid (pBinl9) , taking care to ensure that none of the transcribed sequences are similar to the transcribed sequences of the target gene (which may, for test purposes, be a 35S.GFP transgene in N. benthamiana line 16c) . An agrobacterium liquid culture containing the binary plasmid is infiltrated into young leaves of the test plant (line 16c) to determine whether transient expression of SDE3 potentiates gene silencing of the target gene. Normally there is no silencing of the GFP transgene in this line unless the overexpressed gene has sequence similarity to the GFP transgene and is in a pBinl9-based plasmid.

In an alternative assay, the SDE3 is overexpressed from pBinl9 in conjunction with GFP that is being overexpressed from a pSLJ binary plasmid.

In a third variation of these experiments SDE3 is overexpressed from pBin 19 together with GFP that is also being overexpressed from a pBinl9 binary plasmid. Enhance gene silencing from SDE3 would be manifested in that the initiation and spread of PTGS should be quicker than in plants infiltrated with the pBinl9 GFP plasmid alone.

If preferred, an SDE3 homologue from N. benthamiana may be used.

Example 8 - assessing effect of SDE3 on transgene expression

In the light of the Examples above it appears that SDE3 may be required in certain instances of transgene silencing. Thus use of plants with a mutation in sde3 (i.e sde3) , or in which SDE3 is down-regulated, may permit these lines to express transgenes at a higher level than the equivalent wild type plants.

Plants carrying an sde3 mutation may be transformed with a transgene (e.g. GUS) construct and the expression levels assessed in many independent lines (preferably up to 50) . If the transgene expression is more consistent and higher in the sde3 mutants than in equivalent wild type plants it will indicate that an sde3 background is appropriate for transgene expression.

In principle an SDE3-based approach to improved expression may have advantages over other silencing-suppressing systems based involving virus encoded suppressors of gene silencing (see e.g. Anandalakshsmi et al, 1998; Brigneti et al, 1998; Kasschau and Carrington, 1998; Voinnet et al, 1999; GB application 9927609.9)

These systems, as a side-effect, may be hyper-susceptible to virus infection. As shown above, sde3 mutant plants are not necessarily more susceptible to virus infection than wild type plants.

Example 9 - use of sde3 mutations for high level transgene expression in crop plants

Sde3 homologues, mutations and transgenes identified in accordance with the Examples above may be used to generate crop plants in which expression of the SDE3 orthologue is blocked or eliminated by mutation.

Having identified the SDEl orthologue, primers are generated to different parts of the gene. Additionally, randomly mutagenised lines of the crop plant may be produced by fast neutron mutagenesis. Fast neutrons cause deletion mutations that vary from one to several hundreds of base pairs. The Ml families would be screened using the SDE 3 (orthologue) primers to identify plants carrying a mutation in the SDE 3 (orthologue) . The selfed progeny of these mutant plants would be screened for the homozygous mutation in SDE3 (orthologue) using the PCR primers described above. These homozygous plants would then be tested to confirm their ability to support high level transgene expression using a strategy that is parallel to the strategy used in example 8.

Example 10 - use of virus induced silencing of SDE3 for consistent high level transgene expression in crop plants

SDE3 does not affect virus induced gene silencing but it does affect transgene induced gene silencing. Thus lines may be generated in which SDE3 expression is blocked by virus induced gene silencing or amplicons. These lines will then be suitable for consistent high level transgene expression in crop plants.

The effect of blocked expression may be assayed as follows, as exemplified with Nicotiana benthamiana . The SDE 3 homologue in N. benthamiana is identified as described in Example 6 and a fragment of this gene (approximately 500 nucleotides) is introduced into a PVX vector. These constructs are inoculated into N. benthamiana line 16c in which silencing of the GFP transgene has already been induced. Virus-induced silencing of the SDE3 (orthologue) inhibits maintenance of the gene silencing and the plants revert to high level expression of GFP.

In a further embodiment, silencing may be achieved using amplicons. A PVX amplicon is generated carrying the SDE3 (orthologue) . This construct is transformed into the corresponding crop plant, and the transgenic plants tested for the ability to support consistent high level transgene expression as described above.

Example 11 - amplicon super plus for extremely high level transgene expression. Amplicon gene silencing of transgenes is a combined effect resulting from the presence of replicating virus RNA and a transgene (Dalmay et al, 2000a) . In the absence of a functional SDE3 (orthologue) in a plant, there would be no gene silencing in the amplicon lines and the expression of any genes in the amplicon should be at an extremely high level.

'Amplicons', as described in WO98/36083, comprise a promoter operably linked to a viral replicase, or a promoter sequence operably linked to DNA for transcription in a plant cell of an RNA molecule that includes plant virus sequences that confer on the RNA molecule the ability to replicate in the cytoplasm of a plant cell following transcription. The transcripts replicate as if they are viral RNAs, and comprise a targeting sequence corresponding to the gene of interest ('the target gene').

In the present context, the target sequence may be a gene which it is desired to express at a high level. This can be exemplified using a PVX amplicon in which a GFP reporter gene is inserted adjacent to and on the 5' side of a fragment of an appropriate

SDE3 (orthologue) . The construct would be able to mediate silencing of SDE3 (orthologue) but it would not initiate transgene silencing targeted against itself. Expression of the GFP from this contruct (PVX:GFP[SDE3 (orthologue) ] would be as high as in lines expressing PVX: GFP amplicon in the background of Hcpro or other viral-based suppressors of gene silencing discussed above. HcPro causes suppression of a host defense mechanism by the Hc-protease encoded in the potyviral genome ( Pruss, G., Ge, X., Shi, X. M., Carrington, J. C. & Vance, V. B. (1997) Plant Cell 9, 859-868. 10) .

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Polypeptide sequence of SDE3 from Arabidopsis C24.

MSVSGYKSDDEYSVIADKGEIGFIDYQNDGSSGCYNPFDEGPVWSVPFPFKKEKPQSVTVGETSFD SFTVKNTMDEPVDLWTKIYASNPEDSFTLSILKPPSKDSDLKERQCFYETFTLEDRMLEPGDTLTIW VSCKPKDIGLHTTWTVDWGSDRVERWFLLAEDKISSSLTSNRPYSRSRRAPKKDFAVDDYVKGSR PSKWERSFRNRLPLYEIPKEIREMIENKEFPDDLNEGLTARNYANYYKTLLIMEELQLEEDMRAYD MENVSMKRRGIYLSLEVPGLAERRPSLVHGDFIFVRHAYDDGTDHAYQGFVHRVEADEVHLKFASEF HQRHTAGSVYNVRFTYNRINTRRLYQAVDAAEMLDPNFLFPSLHSGKRMIKTKPFVPISPALNAEQI CSIEMVLGCKGAPPYVIHGPPGTGKTMTLVEAIVQLYTTQRNARVLVCAPSNSAADHILEKLLCLEG VRIKDNEIFRLNAATRSYEEIKPEIIRFCFFDELIFKCPPLKALTRYKLWSTYMSASLLNAEGVKR GHFTHILLDEAGQASEPENMIAVSNLCLTETVWLAGDPRQLGPVIYSRDAESLGLGKSYLERLFEC DYYCEGDENYVTKLVKNYRCHPEILDLPSKLFYDGELVASKEETDSVLASLNFLPNKEFPMVFYGIQ GCDEREGNNPSWFNRIEISKVIETIKRLTANDCVQEEDIGVITPYRQQVMKIKEVLDRLDMTEVKVG SVEQFQGQEKQVIIISTVRSTIKHNEFDRAYCLGFLSNPRRFNVAITRAISLLVIIGNPHIICKDMN WNKLLWRCVDNNAYQGCGLPEQEEFVEEPFKQEGSSNGPQYPPEAEWNNSGELNNGGANENGEWSDG WNNNGGTKEKNEWSDGWNSNGGGTKKKDEWSDGWDNNGGTNGINQEGSSNAPQDPQEAEWNDSGEVK NGGTKEKDVRSDGWNNNGGKNEKEECCDGWKDGGSGEEIKNGGKFETRGDFVGKEEDEWSDGWK

Annex II

Genomic DNA sequence of SDE3 from Arabidopsis C24. Transcription starts at 240 Transcription terminates at 4048 Translation starts at 579 Translation stops at 3880 1^st intron: 1362-1449 2^nd intron: 1615-1729 3^rd intron: 3185-3274

1 tcttatttat ttgattaaat atgcatctgt ccaattataa ttattacggc tccaaaggtc 61 aattcttaga attaccgttg taattaacaa cgattgggct taacatatac ctcttaggcc 121 cataacatat acaaaaccca aaaggcccaa atgtttgatg cacgtgcttt acttgagcga

181 attaattccc aatttcagca attttaattt ttctacgact cttcagtctt cactcactct

241 tttcatgttt cttctccttt gaagcctgcc tgcgttagtc tggcttcatt gcttctccat

301 ttcttggtgt gatcgaatca aagagtgtaa cccattttgc tactgattca gtacgtatga

361 tcaattctct caatttcagt taatctcatg ctcaatttcg ttttctgtgt tagggttttg 421 ggtttttgtt atgctctgag tctagttcac gctactcgaa ttccaataca atcctcttag

481 cgtcatgttg tgatcattac attgtggttc aaaatgtgta atttgtttat aatcgatatg

541 tgttgtgttt atgttacaga ttgtcatcat catcagcaat gagtgtgagt ggatacaaat

601 cggatgacga atattctgta atcgcagaca agggagagat tggatttata gactatcaga

661 atgatggatc ttctggttgc tacaatccat ttgatgaagg tccagttgtt gtttctgttc 721 ctttcccatt taagaaagag aagcctcaat ctgtaactgt tggagagact tcttttgatt

781 ccttcactgt caagaacact atggatgagc ctgttgatct ctggactaag atttacgcat

841 ctaaccctga ggattctttt actctctcga tattgaaacc gccttcgaaa gattcagact

901 tgaaagagag acagtgtttt tatgaaactt ttactctgga agataggatg cttgagcctg

961 gtgacacttt gaccatttgg gtgtcttgta agcctaagga cattggttta cataccactg

1021 ttgttactgt tgactgggga agtgataggg ttgagcgggt tgttttcctt ttggctgagg

1081 ataagatttc gagttctctg acttctaaca ggccttactc tagaagtaga agggctccga 1141 agaaagattt tgctgtggat gattatgtga agggatcacg cccttcaaag gtggttgaac

1201 gtagttttag aaataggctt cctctgtatg aaattccaaa ggagattaga gagatgatcg

1261 agaataagga attccctgat gatttgaacg aaggtcttac agcaaggaac tatgctaact

1321 actataaaac tttactgatt atggaagaat tacaactcga ggtatcctca ttgtttcaga

1381 tttttgttaa taactcaata aacctcttgt agcatgaatc atgtttcttg taattttttg 1441 gttgtttagg aagacatgcg agcttatgat atggagaatg tttcaatgaa gagaaggggt

1501 atctatttgt ccctcgaagt tcctggcctt gctgagagaa gaccttccct tgtccatgga

1561 gacttcatct ttgttagaca tgcatatgat gatggaactg accacgctta tcaggttttg

1621 ttctcgcttt actttctcat actttcagat cactttctgc ttcacaactg caaatatgtt

1681 taatcattta tcttttagag gatacaaaat tgaaatcaat ttctcacagg gctttgtaca 1741 ccgcgtagag gcggatgaag tacatttgaa gtttgcctct gagttccacc aacgccacac

1801 agctgggagt gtctataatg tgaggttcac atacaatcga atcaacacta gaaggttgta

1861 tcaggctgtt gatgcggcag aaatgttgga tccaaacttc cttttcccat ccttgcactc

1921 tgggaaaagg atgataaaga caaaaccctt tgtgcccatt tcaccagctc ttaatgcaga

1981 gcagatttgt tccattgaaa tggttcttgg ctgcaaagga gcaccaccat atgtaatcca

2041 tggacctcct ggtactggga aaaccatgac attggtggag gctatagttc aactttatac

2101 aacacagaga aatgctcggg ttcttgtctg tgctccctcc aatagtgcag ctgatcacat

2161 tttagagaaa ctcctttgtt tagagggagt tcgcatcaag gacaatgaga ttttcaggct

2221 gaatgcagct actcgttcat atgaagaaat taagcctgag attatccgct tttgcttctt 2281 tgatgagtta attttcaagt gcccccctct caaagctttg actcgttaca aactcgttgt

2341 gtcaacatat atgagtgcat cgcttcttaa cgcggaaggt gtaaagcgtg gtcacttcac

2401 tcatattctc ttagatgagg ctggtcaagc ttcagagcct gaaaacatga ttgctgtatc

2461 aaatctctgc ttgacagaaa ctgtggttgt gcttgctgga gatccaaggc aattgggtcc

2521 ggttatatac tcaagagatg ctgagtcact cggcttgggg aagtcatact tggaaagatt 2581 gtttgagtgt gattattact gcgaagggga tgagaattat gtaacaaagc tagtcaagaa

2641 ctacagatgc cacccagaga ttcttgatct cccgtcaaag ctgttctatg atggagaact

2701 ggtagcttcc aaagaagaga cagactctgt tcttgcctcg ttaaacttcc ttccaaacaa

2761 agaatttccg atggttttct atgggatcca aggatgcgat gagagggaag gtaacaatcc

2821 gtcctggttc aaccgtatag agatcagcaa agttatagag acaatcaaga gattaacagc 2881 gaatgactgt gtgcaagaag aggatatcgg agttataact ccttacaggc agcaagtaat

2941 gaagatcaag gaggttcttg acaggttaga tatgactgaa gtcaaagttg gtagtgtaga

3001 gcaattccaa ggtcaagaga aacaggtcat catcatttca actgtaagat ctactatcaa

3061 acacaacgag ttcgatagag cctattgtct gggcttccta agcaatccaa ggaggtttaa

3121 tgtcgccata acccgtgcaa tctctctgct agtgatcatc ggaaacccac atatcatatg

3181 caaggtacca cttacttagt tgttctggac aaagacataa tgcgttgaca tcattcccaa

3241 tctctcaact aataaaacgt attatatgaa acaggacatg aactggaaca agcttttgtg

3301 gcgatgtgtg gataacaatg cttaccaagg atgcggttta ccagagcagg aggagtttgt

3361 ggaggaacca ttcaagcaag aaggaagcag caatgggcct caataccctc cagaagctga 3421 gtggaacaac agtggagagc tcaacaatgg tggtgcgaat gagaatggtg aatggtctga

3481 tgggtggaac aacaatggcg gcacaaagga aaaaaatgaa tggtctgatg gatggaacag

3541 caatggtggt gggacaaaga agaaagatga atggtctgat gggtgggaca acaatggcgg

3601 cacaaacggg atcaatcaag aaggaagcag caacgctccc caagaccctc aagaagctga

3661 gtggaacgat agtggcgagg tcaagaatgg tggcacaaag gagaaagatg tcaggtctga 3721 tgggtggaac aacaatggcg gaaaaaatga aaaagaagaa tgttgtgatg gttggaaaga

3781 cggcggcagt ggtgaagaga tcaagaatgg tggtaagttt gaaaccagag gtgattttgt

3841 aggcaaggaa gaagatgagt ggtctgatgg gtggaagtaa attaaaaaaa aaagtagagt

3901 aataagggtt aattgtaggt ggatggacaa aaggctattt tggatttgtt catattttct

3961 ttctatctct caacctttgg tttttgcaga gtctcgtttt taaatgcatg atttggtgtg 4021 tataaaatta aggaaactgt tttattaata gggctatcaa

Claims

1 An isolated nucleic acid molecule which comprises an sde3 nucleotide sequence encoding an SDE3 polypeptide which is capable of mediating PTGS of a transgene in a plant into which the nucleic acid is introduced.

2 A nucleic acid as claimed in claim 1 wherein the sde3 nucleotide sequence encodes an RNA helicase-like polypeptide.

3 A nucleic acid as claimed in claim 1 or claim 2 wherein the sde3 nucleotide sequence is derived from the SDE3 locus of Arabidopsis .

4 An isolated nucleic acid molecule as claimed in any one of the preceding claims which comprises an sde3 nucleotide sequence which:

(i) encodes the SDE3 polypeptide shown in Annex I, or (ii) encodes a variant SDE3 polypeptide which is a homologous variant of the SDE3 polypeptide shown in Annex I and which shares at least about 70% identity therewith,

5 A nucleic acid as claimed in claim 4 wherein the sde3 nucleotide sequence is the Arabidopsis sde3 genomic sequence or sde3 cDNA portion thereof shown by the exons of Annex II, or a sequence degeneratively equivalent to either of these.

6 A nucleic acid as claimed in claim 4 wherein the sde3 nucleotide sequence encodes a variant SDE3 polypeptide which is capable of mediating PTGS of a transgene in a plant into which the nucleic acid is introduced and/or which specifically binds to an antibody raised against the SDE3 polypeptide of Annex I.

7 A nucleic acid as claimed in claim 6 wherein the sde3 nucleotide sequence encodes a variant SDE3 polypeptide which is a derivative of the SDE3 polypeptide shown in Annex I by way of addition, insertion, deletion or substitution of one or more amino acids . 8 A nucleic acid as claimed in claim 6 wherein the sde nucleotide sequence consists of an allelic or other homologous or orthologous variant of the sde3 nucleotide sequence of claim 5.

9 An isolated nucleic acid which comprises a nucleotide sequence which is the complement of the sde3 nucleotide sequence of any one of the preceding claims.

10 An isolated nucleic acid for use as a probe or primer, said nucleic acid having a distinctive sde3 nucleotide sequence of at least about 18-24 nucleotides in length, which sequence is present in Annex II, or a sequence which is degeneratively equivalent thereto, or the complement of either but in any case does not encode a portion of a non-SDE3 sequence in Figure 6.

11 A process for producing a nucleic acid as claimed in claim 7 comprising the step of modifying a nucleic acid as claimed in claim 6.

12 A method for identifying or cloning a nucleic acid as claimed in claim 8, which method employs a nucleic acid as claimed in claim 10.

13 A method as claimed in claim 12, which method comprises the steps of:

(a) providing a preparation of nucleic acid from a plant cell;

(b) providing a nucleic acid molecule which is a nucleic acid probe as claimed in claim 10, (c) contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation, and, (d) identifying nucleic acid in said preparation which hybridises with said nucleic acid molecule probe.

14 A method as claimed in claim 12, which method comprises the steps of:

(a) providing a preparation of nucleic acid from a plant cell;

(b) providing a pair of nucleic acid molecule primers suitable for PCR, at least one of said primers being a primer of claim 10 ,

(c) contacting nucleic acid in said preparation with said primers under conditions for performance of PCR,

(d) performing PCR and determining the presence or absence of an amplified PCR product.

15 A method as claimed in claim 13 or claim 14 wherein the plant cell is selected from: rice, maize, wheat, barley, alfalfa, chickpea, bean and pea.

16 A recombinant SDE3 expression vector which comprises the nucleic acid of any one of claims 1 to 8 operably linked to a promoter for transcription in a host cell, wherein the promoter is optionally an inducible promoter.

17 A vector as claimed in claim 16 which further comprises a nucleotide sequence encoding an RNA-dependant RNA polymerase .

18 A vector as claimed in claim 17 wherein the RNA-dependant RNA polymerase is SDEl.

19 A vector as claimed in any one of claims 16 to 18 which is a plant vector.

20 A recombinant amplicon vector for use in enhancing the expression of a transgene in a plant into which said amplicon vector is introduced, which vector comprises (i) a plant active promoter operably linked to (ii) DNA for transcription in a plant cell of an RNA molecule that includes plant virus sequences that confer on the RNA molecule the ability to replicate in the cytoplasm of a plant cell in said plant following transcription, which viral RNA comprises an sde3 nucleotide sequence of any one of claims 1 to 9 or a part thereof, whereby replication of said RNA molecule suppresses the SDE3 activity in said plant thereby suppressing PTGS of the transgene.

21 A vector as claimed in claim 20 wherein the sde3 nucleotide sequence in the viral RNA comprises an approximately 100, 200, 300, 400, or 500 nucleotide fragment of the sde3 sequence of claim 5. 22 A vector as claimed in claim 20 or claim 21 further comprising the transgene the expression of which it is desired to enhance .

23 A vector as claimed in claim 22 wherein the transgene is present in the vector adjacent to and on the 5' side of the sde3 nucleotide sequence.

24 A method which comprises the step of introducing the vector of any one of claims 16 to 23 into a host cell, and optionally causing or allowing recombination between the vector and the host cell genome such as to transform the host cell.

25 A host cell containing or transformed with a heterologous vector of any one of claims 16 to 23.

26 A method for producing a transgenic plant, which method comprises the steps of: (a) performing a method as claimed in claim 24 wherein the host cell is a plant cell, (b) regenerating a plant from the transformed plant cell.

27 A transgenic plant which is obtainable by the method of claim 26, or which is a clone, or selfed or hybrid progeny or other descendant of said transgenic plant, which in each case includes a heterologous nucleic acid of any one of claims 1 to 8 or an amplicon vector of claim 20 to 23.

28 A part of propagule from a plant as claimed in claim 18 or claim 19, which in either case includes a heterologous nucleic acid of any one of claims 1 to 8 or an amplicon vector of claim 20 to 23.

29 An isolated polypeptide which is encoded by the SDE3 nucleotide sequence of any one of claims 1 to 8.

30 A polypeptide as claimed in claim 29 which is the SDE3 polypeptide shown in Annex I . 31 A method of making the polypeptide of claim 29 or claim 30, which method comprises the step of causing or allowing expression from a nucleic acid of any one of claims 1 to 8 in a suitable host cell.

32 Use the polypeptide of claim 29 or claim 30 as an RNA- helicase .

33 A polypeptide which comprises the antigen-binding site of an antibody having specific binding affinity for the polypeptide of claim 30.

34 A method for enhancing PTGS of a transgene in a plant by increasing SDE activity in the plant, the method including the step of causing or allowing expression of a heterologous sde3 nucleic acid of any one of claims 1 to 8 within the cells of the plant following an earlier step of introducing the sde3 nucleic acid into a cell of the plant or an ancestor thereof.

35 A method as claimed in claim 34 wherein the sde3 nucleic acid is expressed in conjunction with a nucleotide sequence encoding an RNA-dependant RNA polymerase.

36 A method as claimed in claim 35 wherein the RNA-dependant RNA polymerase is SDEl.

37 A method for suppressing PTGS of a transgene in a plant hence enhancing expression thereof by suppressing SDE activity in the plant, the method including the step of causing or allowing transcription of a heterologous nucleic acid comprising an sde3 nucleotide sequence of any one of claims 1 to 9 or a part thereof within the cells of the plant following an earlier step of introducing the nucleic acid into a cell of the plant or an ancestor thereof, whereby transcription of the heterologous nucleic acid suppresses the SDE3 activity in said plant thereby suppressing PTGS of the transgene. 38 A method as claimed in claim 37 which method comprises any of the following steps of:

(i) causing or allowing transcription in the plant from a nucleic acid comprising an sde3 nucleotide sequence as claimed in claim 9, or a part thereof, in the plant such as to reduce SDE3 activity by an antisense mechanism;

(ii) causing or allowing transcription in the plant from a nucleic acid comprising a part of the sde3 nucleotide sequence described in any one of claims 1 to 8 such as to reduce SDE3 activity by co- suppression;

(iii) introducing into the plant a nucleic acid encoding a ribozyme specific for an sde3 nucleotide sequence described in any one of claims 1 to 8, (iv) causing or allowing replication in the plant of a nucleic acid viral vector comprising an sde3 nucleotide sequence described in any one of claims 1 to 9, or a part thereof, such as to reduce SDE3 activity by virus induced gene silencing, (v) causing or allowing replication of a recombinant amplicon vector comprising an sde3 nucleotide sequence described in any one of claims 1 to 9 or a part thereof, such as to reduce SDE3 activity by amplicon induced silencing.

39 A method as claimed in claim 38 wherein an approximately 100, 200, 300, 400, or 500 nucleotide fragment of the sde3 nucleotide sequence is used.

40 A method as claimed in claim 38 or claim 39 wherein the recombinant amplicon vector is the vector of any one of claims 20 to 23.

41 A method as claimed in any one of claims 37 to 40 further comprising the step of purifying the polypeptide encoded by the transgene from the plant.