WO2000024906A1

WO2000024906A1 - Sequence encoding cysdv coat protein

Info

Publication number: WO2000024906A1
Application number: PCT/GB1999/003469
Authority: WO
Inventors: Robert Henry Arnold Coutts; Ioannis Livieratos
Original assignee: Imperial College Of Science, Technology And Medicine
Priority date: 1998-10-28
Filing date: 1999-10-19
Publication date: 2000-05-04
Also published as: GB0107378D0; AU6223399A; GB2357508A

Abstract

This application relates to isolated Cucurbit Yellow Stunting Disorder Virus Coat Protein, or a fragment, homologue or derivative thereof.

Description

SEQUENCE ENCODING CYSDV COAT PROTEIN

Background of the Present Invention

The present invention relates to a sequence. In particular, the present invention relates to the cucurbit yellow stunting disorder virus (CYSDV) coat protein and nucleic acids encoding it. Moreover, the invention relates to the use of CYSDV coat protein and/or the CYSDV coat protein gene in detecting CYSDV infection in plants and in producing resistance to such infections.

Background Art

CYSDV is a serious pathogen of all cucurbits, including melon and cucumber, which has emerged relatively recently in cucurbit growing regions such as the Mediterranean regions of Europe. Like certain other closteroviruses which infect commercial crops, CYSDV is transmitted by whitefly. The vector for CYSDV is Bemisia tabaci, the tobacco whitefly. Recently, β. tabaci has become prevalent over Trialeurodes vaporariorum, the whitefly vector responsible for transmitting the beet pseudo-yellows virus (BPYV) since the early 1980s. This has lead to increase in the occurrence of CYSDV infection (see Ceiix et al., (1996) Phytopathology 86:1370- 1376). S. tabaci is also becoming more prevalent in the USA (Wisler ef al., (1998) Plant Disease 82:270-279), where the population is estimated to have multiplied 1, 600-fold between the mid-1970s and the mid-1990s.

BPYV and CYSDV present identical symptoms in cucurbit infection. In order to develop resistant plant strains and to monitor the spread of CYSDV infection, it is necessary to differentiate between the viruses.

One way in which various cucurbit viruses have been distinguished in the past has been by their mode of transmission. Different viral species are transmitted by different whitefly vectors. CYSDV is transmitted mostly by the sweet potato whitefly {Bemisia tabaci), and perhaps to a lesser extent by the silverleaf whitefly (β. argentifolii), whereas BPYV is thought to be transmitted by the greenhouse whitefly (7.

et al., (1996) Mol Plant Pathol. vol 86:1167-1172; Wisler et al., (1998) Plant Disease 82:270-280). Attempts to identify viruses using this knowledge relied on complicated and time-consuming backtransmission experiments with the whiteflies and their various host species (for example see Jorda- Gutierrez ef al., (1993) Plant Pathol. 42:722-727).

Another way in which these cucurbit viruses have been distinguished in the past is by using RNA studies. Viral RNA molecules can be extracted from plant tissue, and examined by electrophoresis and ethidium bromide staining. Some viral species' RNA genomes are monopartite, and others are bipartite. These differences may show a distinctive pattern which can be used to infer the prescence of a particular virus in the original sample. However, these rather subtle differences can be difficult to see, and some viral species may produce multiple dsRNAs within infected plants (Dodds et al., (1987) intervirology 27:177-188). The sensitivity of this RNA-based approach can be increased by combining it with hybridisation studies. Viral cDNA probes are hybridised to RNA extracts from plant tissue, and the prescence or absence of this viral sequence in the test sample is scored. However, there are problems in producing cDNAs from the dsRNA viral genome, and RNA extraction can be very inefficient, often requiring hundreds of grams of plant tissue (Coffin and Coutts (1992) Intervirology 33:197-203).

More recently, PCR-based methods for identifying these viruses have been developed (for example Coutts and Coffin (1996) Virus Genes 13:179-181). These rely on prior sequence knowledge of viral gene product(s). This knowledge allows PCR primers to be designed which are intended to specifically amplify viral ORF(s). Drawbacks associated with this approach include the generation of PCR artifacts from contaminating plant RNAs which can confound the investigation, and the relatively labour-intensive methods of purifying RNA from plant tissues.

Recently it has been shown that it is possible to differentiate CYSDV and BPYV- infected material using an RT-PCR assay which is based on the sequence of the different viral HSP70 genes (Livieratos er al., (1998) Plant Pathology 47:362-369). Use of the CYSDV CP sequence as disclosed herein may significantly improve the efficacy of this technique. However, this assay is complex to perform, and must be conducted in a specialised laboratory. It cannot be conducted in the field. Moreover, since it is impossible to culture CYSDV, isolation of CYSDV polypeptides and antibody synthesis for use in an immunoassay has not been practicable.

Identification of viral infection in cucurbits is therefore presently a very labour intensive task, requiring nucleic acid extraction from relatively large quantities of plant material. Results obtained from such experiments can be difficult to interpret and prone to artifacts.

The present invention seeks to overcome these problems.

Summary of Aspects of the Present Invention

Aspects of the present invention are set out in the claims and are described below.

In brief, some of the aspects of the current invention relate to:

1 The nucleotide sequence of the CYSDV coat protein, and its use in detecting the prescence of CYSDV, and its use in the production of transgenic organisms, and its use in producing CYSDV coat protein, and fragments, homologues or derivatives thereof.

2 The amino acid sequence of the CYSDV coat protein.

3 Isolated CYSDV coat protein, and its use in producing antibodies with immunoreactivity towards CYSDV coat protein, or with immunoreactivity towards fragments, homologues or derivatives thereof, and its use in screening such antisera for such reactivity.

4 Antisera with immunoreactivity towards CYSDV coat protein, or with immunoreactivity towards a fragment, homologue or derivative thereof. 5 Transgenic organisms expressing CYSDV coat protein or a fragment, homologue or derivative thereof.

6 Methods for detecting the prescence of CYSDV by nucleic acid hybridisation, or by immunodetection with an antibody which has immunoreactivity towards CYSDV coat protein, or a fragment, homologue or derivative thereof.

The current invention is distinct from the prior art as it utilises serological tests which can identify certain plant virus infections quickly, reliably and from much smaller tissue samples than current nucleic acid-based techniques. The greater precision in the test results, and the saving of labour costs in conducting them, make this invention potentially very commercially useful.

There are a number of advantages associated with the current invention. For example, it is expected that the CYSDV Coat Protein (CYSDV CP) sequence disclosed in this application will result in marked improvement of the detection of CYSDV. Since this application discloses the sequence of CYSDV CP, and demonstrates methods of utilising this CYSDV CP sequence in PCR assays, identification of these viruses by such methods will be significantly improved.

In accordance with the present invention, we have cloned and characterised the CYSDV coat protein gene. By expression of the CYSDV coat protein gene purified CYSDV coat protein is obtained and can be used to raise antisera capable of specific detection of CYSDV in infected material. Moreover, transgenic plants expressing CYSDV coat protein can be obtained which are resistant to CYSDV infection.

Thus, according to a first aspect of the present invention, therefore, there is provided an isolated CYSDV coat protein, or a fragment or derivative thereof, which fragment or derivative retains at least an antigenic determinant of CYSDV coat protein. General Definitions

Some commonly used terms are defined here; other definitions may appear as required in the text below.

'Antigenic determinant' means that the derivative in question retains at least one antigenic function of CYSDV coat protein. Antigenic functions include possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured CYSDV coat protein polypeptide or fragments thereof. Thus CYSDV coat protein as provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants, glycosylation variants and other covalent derivatives of CYSDV coat protein which retain the physiological and/or physical properties of CYSDV coat protein. Exemplary derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Such a moiety may be a detectable moiety such as an enzyme or a radioisotope, or an adjuvant molecule capable of increasing the immune response to CYSDV in an organism.

Derivatives which retain common antigenic determinants can be fragments of CYSDV coat protein. Fragments of CYSDV coat protein comprise individual domains thereof, as well as smaller polypeptides derived from the domains. Preferably, smaller polypeptides derived from CYSDV coat protein according to the invention define a single epitope which is characteristic of CYSDV coat protein. Fragments may in theory be almost any size, as long as they retain one characteristic of CYSDV coat protein. Preferably, fragments may be between 5 and 150 amino acids in length. Longer fragments are regarded as truncations of the full-length CYSDV coat protein and generally encompassed by the term "CYSDV coat protein".

Derivatives of CYSDV coat protein also comprise allelic variations or mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain at least one feature characteristic of CYSDV coat protein. Thus, conservative amino acid substitutions may be made substantially without altering the nature of CYSDV coat protein, as may truncations from the 5' or 3' ends. Deletions and substitutions may moreover be made to the fragments of CYSDV coat protein comprised by the invention. CYSDV coat protein mutants may be produced from a DNA encoding CYSDV coat protein which has been subjected to in vitro mutagenesis resulting e.g. in an addition, variation, modification, replacement, substitution, exchange and/or deletion of one or more amino acids. For example, substitutional, deletional or insertional variants of CYSDV coat protein can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of CYSDV coat protein.

Conserved substitutions may be made according to the following table which indicate some possible conservative substitutions, where amino acids on the same block in the second column and preferably in the same line in the third column may be substituted for each other. For some instances other conserved substitutions may be made.

The fragments, mutants and other derivative of CYSDV coat protein preferably retain substantial homology with CYSDV coat protein. As used herein, "homology" means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function. Preferably, homology is used to refer to sequence identity. Thus, the derivatives of CYSDV coat protein preferably retain substantial sequence identity with the sequence of SEQ ID No. 2.

"Substantial homology", where homology indicates sequence identity, means more than 75% sequence identity and most preferably a sequence identity of 90% or more. Amino acid sequence identity may be assessed by any suitable means, including the BLAST technique mentioned below.

CYSDV Coat Protein and Nucleotide Sequence Coding Therefor

According to one aspect of the present invention, there is provided CYSDV coat protein in isolated form.

"Isolated" means that the protein or derivative is free of one or more components of its natural environment. Isolated CYSDV coat protein therefore includes CYSDV coat protein in a recombinant cell culture. CYSDV coat protein present in an organism expressing a recombinant CYSDV coat protein gene, whether the CYSDV coat protein is "isolated" or otherwise, is also included within the scope of the present invention.

It will be understood that the CYSDV coat protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated.

A CYSDV coat protein of the present invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the present invention.

Polypeptides of the present invention may be modified for example by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote their secretion from a cell as discussed below.

According to a further aspect of the present invention, there is provided a nucleic acid encoding CYSDV coat protein. In addition to being useful for the production of recombinant CYSDV coat protein, these nucleic acids are also useful as probes, thus readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding CYSDV coat protein. The nucleic acid may be unlabelled or labelled with a detectable moiety. Furthermore, nucleic acid according to the invention is useful e.g. in a method determining the presence of CYSDV coat protein-specific nucleic acid, said method comprising hybridising the DNA (or RNA) encoding (or complementary to) CYSDV coat protein to test sample nucleic acid, and determining the presence of nucleotide sequence coding for CYSDV coat protein.

The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).

In another aspect, the invention provides nucleic acid sequence that is complementary to, or hybridises under stringent conditions to, a nucleic acid sequence encoding CYSDV coat protein.

The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein).

The invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) CYSDV coat protein.

In still another aspect of the invention, there is provided a replicable vector comprising the nucleic acid encoding CYSDV coat protein operably linked to control sequences recognised by a host transformed by the vector. Furthermore the invention provides host cells transformed with such a vector and a method of using a nucleic acid encoding CYSDV coat protein to effect the production of CYSDV coat protein, comprising expressing CYSDV coat protein nucleic acid in a culture of the transformed host cells and, if desired, recovering CYSDV coat protein from the host cell culture.

Thus, the present invention relates to isolated CYSDV coat protein and derivatives thereof encoded by the above-described nucleic acids. Preferably, the isolated CYSDV coat protein nucleic acid is in isolated form. This includes nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of CYSDV coat protein nucleic acid or in crude nucleic acid preparations, such as preparations of CYSDV from infected tissue. The isolated nucleic acid of the present invention may therefore be present in other than in the form or setting in which it is found in nature.

Thus, in accordance with the present invention, there are provided isolated nucleic acids, e.g. DNAs or RNAs, encoding CYSDV coat protein, or fragments thereof. In particular, the invention provides a DNA molecule encoding CYSDV coat protein, or a fragment thereof. By definition, such a DNA comprises a coding single stranded DNA, a double stranded DNA of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself. An exemplary nucleic acid encoding CYSDV coat protein is represented in SEQ ID No. 1. It will be recognised by those skilled in the art that the nucleic acids of the invention include fragments, mutants or derivatives of the nucleic acid sequence encoding the CYSDV coat protein, and also include nucleic acid sequences which are complementary to at least a part of the nucleic acid sequence encoding the CYSDV coat protein, or a fragment, mutant or derivative thereof.

The preferred sequence encoding CYSDV coat protein is that having substantially the same nucleotide sequence as the coding sequences in SEQ ID No. 1 , with the nucleic acid having the same sequence as the coding sequence in SEQ ID No. 1 being most preferred. As used herein, nucleotide sequences which are substantially the same share at least about 75% identity, more preferably at least about 90% identity.

The term "homologue" with respect to the nucleotide sequence of the present invention and the amino acid sequence of the present invention may be synonymous with allelic variations of the sequences. In particular, the term "homology" as used herein may be equated with the term "identity". Here, sequence homology with respect to the nucleotide sequence of the present invention and the amino acid sequence of the present invention can be determined by a simple "eyeball" comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has at least 75% identity to the sequence(s). Relative sequence homology (i.e. sequence identity) can also be determined by commercially available computer programs that can calculate % homology between two or more sequences. A typical example of such a computer program is CLUSTAL.

% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).

Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.

However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package (see below) the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux er a/., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for soem applications it is preferred to use the GCG Bestfit program.

Although the final % homology can be measured in terms of identity, in some cases, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. As indicated, for some applications, sequence homology (or identity) may be determined using any suitable homology algorithm, using for example default parameters.

By way of example, BLAST (Basic Local Alignment Search Tool) is an heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements. The BLAST programs were tailored for sequence similarity searching, for example to identify homologues to a query sequence. The programs are not generally useful for motif-style searching. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.

Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html.

Advantageously, "substantial homology" when assessed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more. The default threshold for EXPECT in BLAST searching is usually 10.

Should Gap Penalties be used when determining sequence identity, then preferably the following parameters are used:

Other computer program methods to determine identify and similarity between the two sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387 and FASTA (Atschul et al 1990 J Molec Biol 403-410).

The nucleic acids of the invention, whether used as probes or otherwise, are preferably substantially homologous to the nucleic acid sequence encoding CYSDV coat protein as shown in SEQ ID No. 1. The terms "substantially" and "homologous" are used as hereinbefore defined with reference to the CYSDV coat protein polypeptide.

Preferably, nucleic acids according to the invention are fragments of the CYSDV coat protein-encoding sequence, or derivatives thereof as hereinbefore defined in relation to polypeptides. Fragments of the nucleic acid sequence of a few nucleotides in length, preferably 5 to 150 nucleotides in length, are especially useful as probes.

Exemplary nucleic acids can alternatively be characterised as those nucleotide sequences which encode a CYSDV coat protein and hybridise to the DNA sequences set forth SEQ ID No. 1 , or a selected fragment of said DNA sequence. Preferred are such sequences encoding CYSDV coat protein which hybridise under high-stringency conditions to the sequence of SEQ ID No. 1.

Derivatives of said nucleotide sequences also encompass sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.

Preferably, the term "derivative" also encompasses sequences that are complementary to sequences that are capable of hydridising under stringent conditions (e.g. 65°C and OJxSSC {1xSSC = 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0}) to the nucleotide sequences presented herein. Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68°C. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0J Na+ pyrophosphate and 0J mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - OJx SSC, 0.1 % SDS.

Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridisation temperature in 1x SSC, 0J % SDS.

Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0J % SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skill in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the probe also play a role.

Advantageously, the invention moreover provides nucleic acid sequence which are capable of hybridising, under stringent conditions, to a fragment of the sequence of SEQ ID No. 1. Preferably, the fragment is between 15 and 50 bases in length. Advantageously, it is about 25 bases in length.

Given the guidance provided herein, the nucleic acids of the invention are obtainable according to methods well known in the art. For example, a DNA of the invention is obtainable by chemical synthesis. Particularly preferred is the use of RACE PCR (Coffin and Coutts (1992) Intervirology 33:197-203).

Chemical methods for synthesis of a nucleic acid of interest are known in the art and include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports. These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.

As used herein, a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases set forth in SEQ ID No. 1. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised. The nucleotide sequences are usually based on conserved or highly homologous nucleotide sequences or regions of CYSDV coat protein. The nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known. Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The preferred method of labelling a DNA fragment is by incorporating a³²P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually end-labelled with g³²P-labelled ATP and polynucleotide kinase. However, other methods (e.g. non- radioactive) may also be used to label the fragment or oligonucleotide, including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation.

It is envisaged that the nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such mutants can be used e.g. to produce a CYSDV coat protein mutant that has an amino acid sequence differing from the CYSDV coat protein sequences as found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridise to produce secondary mRNA structure such as loops or hairpins.

Manipulation and Expression of CYSDV Coat Protein Sequences

The nucleic acid encoding native or mutant CYSDV coat protein can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

The term "construct" - which is synonymous with terms such as "transgene", "conjugate", "cassette" and "hybrid" - includes the nucleotide sequence according to the present invention directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the SM-intron or the ADH intron, which may interconnect the promoter sequences and the nucleotide sequences of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In each case, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.

The construct may contain or express a marker which allows for the selection of the genetic construct in, for example, a bacterium, preferably of the genus Bacillus, such as Bacillus subtilis, or plants, such as melon, cucumber etc., into which it has been transferred. Various markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance - e.g. resistance to G418, hygromycin, bleomycin, kanamycin, gentamycin or any other suitable marker mentioned below or known to those skilled in the art.

Preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.

Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.

Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another class of organisms for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome. However, the recovery of genomic DNA encoding CYSDV coat protein is more complex than that of exogenousiy replicated vector because restriction enzyme digestion is required to excise CYSDV coat protein DNA. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.

Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1 , or HIS3 gene.

Since the replication of vectors is conveniently done in E. coli, an £ coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from £ coli plasmids, such as pBR322, Bluescript© vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both £. coli replication origin and £ coli genetic marker conferring resistance to antibiotics, such as ampicillin.

Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to CYSDV coat protein nucleic acid. Such a promoter may be inducible or constitutive. The promoters are operably linked to DNA encoding CYSDV coat protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native CYSDV coat protein promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of CYSDV coat protein DNA. The term "operably linked" refers to the components in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is iigated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

Promoters suitable for use with prokaryotic hosts include, for example, the b- lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding CYSDV coat protein, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding CYSDV coat protein.

Preferred expression vectors are bacterial expression vectors which comprise a promoter of a bacteriophage such as phagex or T7 which is capable of functioning in the bacteria. In one of the most widely used expression systems, the nucleic acid encoding the fusion protein may be transcribed from the vector by T7 RNA polymerase (Studier et al, Methods in Enzymol. 185; 60-89, 1990). In the £ coli BL21 (DE3) host strain, used in conjunction with pET vectors, the T7 RNA polymerase is produced from the l-lysogen DE3 in the host bacterium, and its expression is under the control of the IPTG inducible lac UV5 promoter. This system has been employed successfully for over-production of many proteins. Alternatively the polymerase gene may be introduced on a lambda phage by infection with an int- phage such as the CE6 phage which is commercially available (Novagen, Madison, USA), other vectors include vectors containing the lambda PL promoter such as PLEX (Invitrogen, NL), vectors containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE) , or vectors containing the tac promoter such as pKK223-3 (Pharmacia Biotech) or PMAL (new England Biolabs, MA, USA).

Moreover, the CYSDV coat protein gene according to the invention preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body. The peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.

Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or a-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, those phosphate isomerase, phosphoglucose isomerase or glucokinase genes, the S. cerevisiae GAL 4 gene, the S. pombe nmt 1 gene or a promoter from the TATA binding protein (TBP) gene can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05-GAP hybrid promoter). A suitable constitutive PHO5 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide - 173 and ending at nucleotide -9 of the PH05 gene.

An expression vector includes any vector capable of expressing CYSDV coat protein nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing CYSDV coat protein expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe which may be based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.

It will be appreciated that, in addition to the naturally-occurring CYSDV coat protein gene, the present invention extends to any nucleic acid sequence which encodes the CYSDV coat protein. Every such sequence, represented in the IUPAC format, is set forth in SEQ ID No. 3.

Antisera Directed Against CYSDV Coat Protein Sequences

According to a further aspect of the invention, there is provided an antibody which binds specifically to CYSDV coat protein, such as in a preparation of CYSDV infected material.

The term "antibody", as used herein with reference to the present invention, refers to a complete antibody or an antibody fragment or an antibody component, as well as any combination thereof, capable of binding to the selected target - namely the CYSDV coat protein identified herein above, or a fragment, homologue or derivative thereof.

Antibody fragments and components include Fv, ScFv, dsFv, Fab, F(ab), Fab', F(ab)2, F(ab')2, Facb, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such Fv and ScFv, possess advantageous properties for analytical applications.

Preferably the antibody is linked to a detectable moiety.

Any suitable detectable moiety can used. The moiety can be directly detectable - such as a radiolabelled moiety, a moiety comprising a dye that is capable of producing a visually detectable signal (which need not necessarily be detectable by means of the naked eye) or a luminescent moiety. The moiety can be indirectly detectable - such as an enzyme moiety that is capable of acting on a substrate that is itself capable of generating a detectable signal or a moiety that is itself recognised by a labelled antibody.

The term "linked" includes direct attachment - such as through a direct bond, e.g. an ionic bond or a covalent bond. In addition, the term "antibody" refers to both conventionally produced antisera and monoclonal and engineered antibody molecules.

Polyclonal antibodies (antisera) may be prepared by conventional means which comprise inoculating a host animal, for example a mouse, rat or a rabbit, with a polypeptide of the invention or peptide fragment thereof and recovering immune serum.

In order that such antibodies may be made, the invention also provides polypeptides of the invention or fragments thereof haptenised to another polypeptide for use as immunogens in suitable animals.

Monoclonal and engineered antibodies include complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, Fab' and F(ab')2, monoclonal antibodies, engineered antibodies including chimeric, CDR- grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.

The antibodies according to the invention are especially indicated for diagnostic and therapeutic applications. Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in vivo. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient. Moreover, the may be fluorescent labels or other labels which are visualisable on tissue samples removed from patients.

Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0239400 (Winter)] and, optionally, framework modification [see international patent application WO 90/07861 (Protein Design Labs)].

Antibodies according to the invention may be produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

Therefore, the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. £ coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. fetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanoi, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells which are bacterial cells or yeast ceils is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges. Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody- producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma ceils with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.

The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256:495-497; US 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are incorporated herein by reference.

The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of plant cells infected with CYSDV by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion- exchange chromatography, chromatography over DEAE-celluiose and/or (immuno) affinity chromatography, e.g. affinity chromatography with a CYSDV coat protein molecule or with Protein-A. The invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention. The preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.

The invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to a CYSDV coat protein molecule, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with a purified CYSDV coat protein molecule, an antigenic carrier containing a purified CYSDV coat protein molecule or with cells bearing CYSDV coat protein, antibody- producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example spleen cells of Balb/c mice immunised with cells bearing CYSDV coat protein or the protein itself are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.

Preferred is a process for the preparation of a hybridoma cell line, characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally with a preparation containing CYSDV coat protein and a suitable adjuvant several times, e.g. four to six times, over several months, e.g. between two and four months, and spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000. After the fusion the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The invention also concerns recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to a CYSDV coat protein molecule as described hereinbefore. By definition such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to a CYSDV coat protein molecule can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification (s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody. Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence is also a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.

The invention therefore also concerns recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed to CYSDV coat protein fused to a human constant domain g, for example g1 , g2, g3 or g4, preferably g1 or g4. Likewise the invention concerns recombinant DNAs comprising an CYSDV coat protein fused to a human constant domain k or I, preferably k.

In another embodiment the invention pertains to recombinant DNAs coding for a recombinant polypeptide wherein the heavy chain variable domain and the light chain variable domain are linked by way of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a cleavage site and/or a peptide spacer and/or an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications. Thus, effector molecules which are toxins or enzymes, especially enzymes capable of catalysing the activation of prodrugs, are particularly indicated. The DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.

Antibodies according to the invention may be used in the preparation of diagnostic kits suitable for detection of CYSDV in infected tissue. The immunoassay employed in the kit may be any conventional form of immunoassay and advantageously identifies the presence of CYSDV colorimetrically, such that it may be used by inexperienced operators in the field. Samples to be Tested

The sample from the plant to be tested may comprise one or more tissue(s) for example; leaf, root, stem, tuber, fruit, seed, bud, floral tissue or reproductive tissues such as stigma or stamen, or any other part of the plant. More preferably, tissue may be taken from areas known or suspected to harbour high titres of virus for example; leaf vein, phloem, pith, xylem or other conductive tissue. Alternatively, cells may be prepared from any part of the plant and used either as a suspension, or immobilized on a suitable substrate. It would be expected that any material would be prepared in such a way as to retain any viral particles which may be present. Alternatively, polypeptides may be extracted from the plant material, and the resulting extract may assayed for the prescence of viral coat protein using the reagent(s) provided herein by Western blotting, slot blotting, dot blotting, immunoprecipitation, ELISA testing, or any other method known to those skilled in the art. Methods of obtaining and preparing such samples for use in the method of the invention are known to those skilled in the art or will be apparent from the present disclosure.

Transformed Hosts Transgenic Organisms Expressing CYSDV Coat Protein

The term "host cell" - in relation to the present invention includes any cell that could comprise a nucleotide sequence coding for a polypeptide according to the present invention and/or products obtained therefrom, wherein a promoter can allow expression of a nucleotide sequence according to the present invention when present in the host cell.

Thus, a further embodiment of the present invention provides host cells transformed or transfected with a polynucleotide of the present invention. Preferably said polynucleotide is carried in a vector for the replication and expression of said polynucleotides. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast, plant or any other suitable cells. In a further aspect of the invention, a nucleic acid encoding CYSDV coat protein may be used to generate transgenic plants which express CYSDV coat protein and thereby gain resistance to CYSDV infection. This technique is fully described in Namba et al., (1991) Gene 107:181-188 and Ling et al., (1991 ) Bio Technology 9:752, incorporated herein by reference.

In another embodiment, the invention comprises a method for producing transgenic plants which are in some degree resistant to infection by CYSDV. This method comprises: a) constructing a suitable transgene which incorporates at least part of the CYSDV coat protein, as claimed herein, in a form capable of being expressed within at least a subset of the target plant cells b) introducing this transgene into target plant cells by way of DNA transformation c) regenerating plantiets from the pool of transformed and non-transformed plant cells, and d) selecting from the regenerated plantiets those which sucessfully express the CYSDV transgene

This method is explained more fully below.

Transgene construction: manipulating DNA in vitro using PCR, restriction enzymes, ligases and other DNA modifying enzymes, and verifying these changes by restriction mapping, hybridisation and DNA sequencing etc. are techniques known to those skilled in the art. These topics are dealt with in texts such as (Stryer, L (1991) 'Bioche istr , Fourth edn. W.H. Freeman & Co.; Watson, (1987) 'Molecular Biology of the Gene', Fourth edn. Benjamin/Cummings Publishing Co. Inc.) and comprehensive sets of laboratory protocols are available elsewhere (e.g. (Sambrook et al., (1989) 'Molecular Cloning: a Laboratory Manua?, second edn. Cold Spring Harbor Laboratory Press); incorporated herein by reference).

Transgenes suitable for promoting the expression of CYSDV coat protein in plant cells in the manner of the invention may contain several DNA elements. These elements may include, but are not limited to, promoters, enhancer sequences, a 5' untranslated leader, coding sequence, intron(s), a 3' untranslated region (3'-UTR), polyadenylation signal(s) and transcriptional termination sequences. Promoter sequences may be from any suitable eukaryotic source, but most preferably are derived from a plant virus for example the 35S promoter of Cauliflower Mosaic Virus (CMV). It will be recognised by those skilled in the art that tissue-specific, developmentally regulated or chemically inducible promoters could also be employed, for example the tetracycline-responsive element. Increased translational efficiency may be imparted to the transgene by choosing an appropriate 5' untranslated leader sequence. This 5' element will have optimal sequence around the initiating methionine, similar to the mammalian Kozak sequence, and preferably is derived from the tobacco mosaic virus, or from the alfalfa mosaic virus. One or more introns may be incorporated into the DNA construct, thereby enhancing expression of the transgene. Preferably, the first intron of the maize alcohol dehydrogenase I {Adhl) gene may be used. In order to optimise the stability of the mRNA transcribed from the transgene, any 'instability motifs' known to those skilled in the art, such as the 'ATTTA instability motif, may be removed from the 3'-UTR. The coding sequence of the transgene may be optimised for expression in the host plant by reference to codon usage tables. For a given amino acid, there may be several different codons which each specify the same amino acid. However, the different codons do not occur equally, some are 'preferred' over others, and a codon preference table summarises these preferences. Accordingly, the transgene may be optimised so that the amino acids encoded in its open reading frame(s) are specified by the most preferred codons with respect to the host plant.

Plant transformation: once the transgene construct has been prepared, this is introduced into plant cells by a process of transformation. There are a variety of ways in which this can be accomplished. There are physical methods such as electroporation (Rhodes et al., (1988) Science 240:204-207; Shimamoto et al., (1989) Nature 338:274-276), or fibre-mediated transformation (Nagatani et al., (1997) Biotech. Techniques 11:471-473). There are also chemical methods such as PolyEthyleneGlycol (PEG) treatment (reviewed in (Davey et al., (1989) Plant Molecular Biology 13:273-285; Maliga et al., (1995) 'Methods in Plant Molecular Biology:A laboratory manual' Cold Spring Harbour Laboratory Press; Potrykus (1991) Ann. Rev. Plant Physiol.& Plant Mol. Biol. 42:205-225)), so-called 'ballistic' methods (reviewed by (Klein ef al., (1992) Biotechnology 10:286-291 ; Sanford et al., (1993) Methods In Enzymology 217:483-509))as well as the more widely used technique of Agrobacterium- mediated transformation.

Despite the apparent diversity of the methods available for transformation of plant cells, in practice the Agrobacterium -mediated techniques are used wherever possible, especially for experimentally tractable dicot. plants such as cucurbits.

DNA transfer from Agrobacterium tumefaciens into plant cells is a relatively well understood process, and relies on genetic determinants carried on the Agrobacterial Ti' plasmid. Naturally occurring Ti plasmids encode several large sets of bacterially expressed genes (reviewed in (Hooykaas and Schilperoort (1992) Plant Mol. Biol. 19:15-38) ). These include the virulence (vir ) genes which are variously necessary for different aspects of the DNA transfer process. There are also conjugative genes which are needed to bring the bacterial and plant cells into contact with one another immediately prior to the DNA transfer itself. There are also genes for opine catabolism. During tumourigenesis, the plant cells are directed to make opines (e.g. nopaline or octopine). These are catabolised by the bacteria, and the genes needed for such catabolism reside on the Ti plasmid.

/Agrobacter/^'u -mediated plant transformation makes use of the 'binary vector system' (Bevan (1984) NAR 12:8711-8721) . The binary system literally means having two plasmids. One is small (a few Kb), can be shuttled through E.coli for ease of handling, and carries the transfer-DNA or T-DNA. The second, 'helper', plasmid remains in A.tumefaciens and is essentially a Ti plasmid with all the relevant vir genes etc., but without a T-DNA region. When both plasmids are in the same bacterial cell, the helper plasmid specifies all the necessary components for transformation of the plant cell, and the T-DNA from the sister plasmid is mobilised and introduced into the host cell.

A transgene containing the CYSDV coat protein, which is the subject of the present invention, is placed into the T-DNA of an appropriate binary vector by in vitro DNA manipulation techniques known to those skilled in the art, and as described abo Binary vectors can be purchased from Clontech (http://www.clontech.com). They c widely available from researchers world-wide (e.g. (Bevan (1984) NAR 12:8711-871 Deblaere et al., (1987) Meths. Enzymol. 153:277-292; Rogers et al. (1987) Met Enzymol. 153:253-277)), or may be obtained from the vector collection of the ATC (http://vectordb.atcg.com) for a nominal fee.

In order to bring about transformation, cultured protoplasts are inoculated with Agrobacteria harbouring the required T-DNA/Ti plasmids. After a period of incubation (usually a few days) the Agrobacteria are removed by the addition of antibiotics. A selective agent is included in the culture medium to select for transformed plant cells which are expressing the marker gene (e.g. kanamycin resistance). Once the cultured cells have regenerated into shoots, these are moved to root-regeneration medium. The resulting transgenic plantiets are then gradually acclimatised, moved out of tissue culture into a greenhouse and analysed.

Particular instruction in the transformation of cucurbits such as Cucumis melo is provided in (Guis et al., (1998) Biotech. & Genet. Eng. Rev. 15:289-311), which is incorporated herein by reference.

The present invention will now be described by way of example, without limitation to the specific examples presented.

EXAMPLES

To produce an antiserum against the coat protein (CP) of cucurbit yellow stunting disorder virus (CYSDV), the sequence could be advantageously determined. To achieve this, sequence information was used from the only cloned parts of the CYSDV genome, and Rapid Amplification of 3' cDNA Ends (RACE) PCR was carried out with the double stranded (ds) RNA form of the virus as template. These prior art clones are referred as CYSDV5 (677bp long) and p410 (465 nucleotides long) (Tian et al., (1996) Phytopathology 86:1167-1173; Celix et al., (1996) Phytopathology 85:1370-1376, respectively). Both fragments represent parts of the heat shock protein 70 (HSP70) homolog gene of CYSDV, which is located close to the 5' end of CYSDV RNA2 of the virus.

Example 1 : EXTENSION OF THE SEQUENCE CYSDV RNA2 USING RAPID AMPLIFICATION OF cDNA ENDS (RACE) PCR

A specific oligonucleotide primer CYSDV1 (5'-AGAGACGGTAAGTATGTC-3') was designed on the coding (sense) strand of the p410 clone, in order to initiate a complementary DNA (cDNA) strand synthesis towards the 3'-end of the HSP70 homologue gene.

cDNA was made from dsRNA which was prepared as follows:

50ng of CYSDV dsRNA was isolated from plants according to (Coffin and Coutts

(1992) Intervirology 33:197-203) with minor modifications as set out below.

The concentration of nucleic acid preparations was determined by ultraviolet (UV) adsorption spectroscopy using a Beckman Model 35 spectrophotometer with quartz cells of path length 1cm. The concentration of nucleic acids was calculated from the reading at 260 nm according to the formula:

1 O.D.260= 50mg/ml ds DNA or ds RNA 1 O.D.260= 40mg/ml ss DNA or ss RNA

The ratio between the readings at 260nm and 280 nm (O.D.₂₆o/ .D.₂₈o) provided an estimation of the purity of the nucleic acid preparation. Pure preparations of DNA or RNA were assumed to have a ratio of 1.8-2.0.

10Og of plant tissue (leaves) was ground to fine powder in liquid N₂. The resulting fine powder was directly added to a mixture containing 150 ml GPS (200 mM glycine, 100 mM Na₂HPO₄, 600 mM NaCl, [pH 9.5]), 150 ml GPS-saturated phenol, 150 ml chloroform/isoamyl alcohol (24:1), 15 ml 10% SDS, and 1.5 ml b-mercaptoethanol, and stirred at 4°C for 1 h. After centrifugation for 10 min at 10,000g, the aqueous phase was carefully separated and ethanol was added to a final concentration of 16.5% (v/v) together with 3g of Whatman CF-11 cellulose (Sigma). The mixture was gently stirred at room temperature for 1h followed by centrifugation at 10,000g for 10min. The pelleted cellulose was washed four times by resuspension in STE buffer (100 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, [pH: 7.5]) containing 16% ethanol. DsRNAs were eluted four times in STE buffer in a final volume of 80ml. After ethanol precipitation, concentrated crude dsRNA was obtained by centrifugation for 30min at 11 ,500g.

In order to remove contaminating DNA and ssRNA from the crude dsRNA preparation, digestions were performed using deoxyribonuclease (DNase I) and S1 nuclease respectively. 10 units of DNase I and 100 units of S1 nuclease (both Pharmacia, USA) were applied in a total volume of 100ml of S1 nuclease buffer (200mM NaCl, 50mM NaOAc [pH: 4.5], 1mM ZnSO₄, 0.5% glycerol) and the reactions incubated for 1h at 37°C. The reactions were then phenol/chloroform extracted: Phenol was melted at 68°C and then equilibrated five times with an equal volume of 1 M Tris-HCl (pH: 8.0) and 0.1% 2-hydroxy-quinoline, before overlaying with 0J M Tris-HCl (pH: 8.0). An equal volume of phenol/chloroform/iso-amyl alcohol (24.5:24.5:1) was added to each reaction and vigorously mixed until an emulsion was formed. These rections were then centrifugated for 3min to separate the two phases. The aqueous phase was removed and extracted with an equal volume of chloroform/iso-amyl alcohol (24:1) (SEVAG), in order to remove residual phenol. The dsRNA was then precipitated by the addition of 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH: 5.5). The tube was inverted and nucleic acids were precipitated at -20°C overnight or -70°C for at least 1h and centrifugation at 12,000 rpm for 20min. The supernatant was aspirated and the pellet was washed in 70% ethanol, air dried and resuspended in 10mM Tris-HCl (pH 7.5) or sterile distilled water.

The resulting dsRNAs were separated in a 1% agarose gel: A non-denaturing agarose gel was prepared by melting molecular grade agarose (Bio-Probe) to a final concentration of 0.8-2.0% (w/v) in 1xTAE buffer (10x: 400mM Tris-base, 11.4 ml/l glacial acetic acid, 10 mM EDTA, [pH 8.0]). Ethidium bromide was added to a final concentration of 0.5mg/ml when the temperature had cooled to approximately 55°C. The samples were mixed with 5xloading buffer (δxloading buffer: 50% glycerol, 0.2% bromophenol blue, 0.2% xylene cyanol) before loading. The samples were run at 40-80V submerged in 1xTAE buffer and their progress was monitored by the migration of the bromophenol blue and xylene cyanol dyes.

The dsRNA2 species was then recovered using the BIO 101 kit (Anachem, Germany). The band of interest was excised from the gel with a minimal exposure to UV light, and after its weight was roughly estimated, three volumes of RNA binding salt was added. The suspension was incubated at 50°C for 5-10 min until the agarose was completely melted. 5ml of RNA matrix solution (able to bind 5mg of dsRNA) was added and the mixture was vigorously mixed in order to create a homogenous suspension. This suspension was left to stand at room temperature for 5-10 min. After two washing steps with washing buffer, the pellet was resuspended in 20ml of nuclease-free water. After incubation at 50°C for 5 min and centrifugation, the clear supernatant was collected.

This supematent was then phenol-chloroform extracted: An equal volume of phenol/chloroform/iso-amyl alcohol (24.5:24.5:1) was added to each of the recovered dsRNA samples, and vigorously mixed until an emulsion was formed. These samples were then centrifugated for 3min to separate the two phases. The aqueous phase was removed and extracted with an equal volume of chloroform/iso-amyl alcohol (24:1) (SEVAG), in order to remove residual phenol. The dsRNA was then precipitated by the addition of 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH: 5.5). The tube was inverted and nucleic acids were precipitated at -20°C overnight or -70°C for at least 1h and centrifugation at 12,000 rpm for 20min. The supernatant was aspirated and the pellet was washed in 70% ethanol, air dried and resuspended in 10mM Tris-HCl (pH 7.5) or sterile distilled water.

The resulting recovered dsRNA was denatured for 15min at room temperature, in 10ml of 20mM methyl mercuric hydroxide solution containing lOOpmol of CYSDV1 primer. The denatured dsRNA was added to a first-strand cDNA synthesis mixture to create a total volume of 50ml, and immediately incubated at 42°C for 50min. First-strand cDNA reaction mixture:

The entire reaction was then diluted in 2ml of TE buffer (10mM Tris [pH 7.4], 1mM EDTA [pH 8.0]) before excess oligonucleotide primers and low molecular weight contaminants were removed by two passages through a Centricon 100 (Amicon) column, by centrifugation at 25000 rpm for 10min each. cDNA was concentrated in a total volume of 50ml and one fifth was added to a PCR mixture (50mM KCI, 10mM Tris-HCl, [pH 9.0], 0.1% Triton X-100, 4mM MgCI₂, lOOpmol CYSDV4 primer, lOOpmol anchor primer [5'-ATGCGT-3'], 2.5units Taq polymerase [Promega]) to a total volume of 100ml. The reactions were overlaid with 40ml of mineral oil and PCR amplification was carried out in a Hybaid thermocycler, using the following cycling regime:

96°C for 5 min 1 cycle

96°C for 30 sec/ 42°C for 45 sec/ 72°C for 2 min 3 cycles 96°C for 30 sec/ 55°C for 30 sec/ 72°C for 2 min 32 cycles 72°C for 10 min 1 cycle

One tenth of the PCR mixture was separated on a 1% agarose gel: A non-denaturing agarose gel was prepared by melting molecular grade agarose (Bio-Probe) to a final concentration of 0.8-2.0% (w/v) in 1xTAE buffer (10x: 400mM Tris-base, 11.4 ml/I glacial acetic acid, 10 mM EDTA, [pH 8.0]). Ethidium bromide was added to a final concentration of 0.5mg/ml when the temperature had cooled to approximately 55°C. The samples were mixed with δxloading buffer (δxloading buffer: 50% glycerol, 0.2% bromophenol blue, 0.2% xylene cyanol) before loading. The samples were run at 40-80V submerged in 1xTAE buffer and their progress was monitored by the migration of the bromophenol blue and xylene cyanol dyes.

The amplified DNA product of approximately 2.2kbp was recovered using the QIAEX II kit (QIAGEN, Germany). After electrophoresis, the desired band was excised from the gel, its weight was roughly estimated and three volumes of DNA binding salt buffer (QX1 ) were added. The solution was incubated at 50°C for 5 min in order to melt the agarose and 5-10ml of DNA matrix (QIAEX II) capable of binding 2mg of DNA were added. After washing twice with QX1 solution and twice with PE buffer, the pellet was air-dried. The pellet was resuspended in 20-60ml of nuclease free water and after incubation at room temperature for 5 min and centrigugation for 30sec the clear supernatant was collected.

This recovered DNA was then cloned into the 'pGEM-T Easy' vector (Promega) as follows:

DNA was prepared for ligation:

First, the DNA supernatent was phenol-chloroform extracted: An equal volume of phenol/chloroform/iso-amyl alcohol (24.5:24.5:1) was added to each of the recovered DNA samples, and vigorously mixed until an emulsion was formed. These samples were then centrifugated for 3min to separate the two phases. The aqueous phase was removed and extracted with an equal volume of chloroform/iso-amyl alcohol (24:1) (SEVAG), in order to remove residual phenol. The DNA was then precipitated by the addition of 2.5 volumes of ethanol and 1/10 volume of 3 M sodium acetate (pH: 5.5). The tube was inverted and nucleic acids were precipitated at -20°C overnight or -70°C for at least 1h and centrifugation at 12,000 rpm for 20min. The supernatant was aspirated and the DNA pellet was washed in 70% ethanol, air dried and resuspended in 10mM Tris-HCl (pH 7.5) or sterile distilled water.

The ligation was as follows: 200ng of this DNA were ligated with 50ng of pGEM-T Easy vector (Promega) in a total volume of 10 ml containing T4 ligation buffer (δxligation buffer: 250mM Tris-HCl [pH: 7.6], 50mM MgCI₂, 5mM ATP, 5mM DTT, 25% [w/v] PEG-8000) and 3.0 units of T4 ligase enzyme (Gibco BRL). Ligation reactions were incubated at 12-16°C for 16h.

Sucessfully ligated products were recovered by transformation of competent E.coli cells.

Electro-Competent E.coli cells were prepared as follows:

A single colony of E.coli (strain DH5a, genotype: f80dlacZDM15, recA1 , endendAI , endA1 , gyrA96, thi-1 , hsdR17(r_k ^"m_k ⁺), supE44, relA1 , deoR, D(iacZYA-argF)U169) was grown overnight in 10ml of Luria-Bertani medium (LB medium: 1 litre contains 5g bacto-tryptone (Difco), 10g bacto-yeast extract(Difco), 5g NaCl (Sigma), pH 7), supplemented to 2mM MgSO₄ and 2mM MgCI₂ in the absence of any antibiotic at 37°C and 225 rotations per minute (rpm). This 10ml culture was transferred to a 2- litre sterile flask containing 500ml of fresh LB medium, and was further incubated at 37°C, 225rpm until an OD₅₅₀ of 0.5 had been achieved. This culture was then chilled on ice for 30min, before being centrifuged at 2000g at between 0 and 4°C. The cell pellets were resuspended in 500ml ice cold sterile H₂O and centrifuged at 2000g for 15min. The supernatant was carefully removed by aspiration, and the pellets were resuspended in 250ml of ice cold H₂O before centrifugation at 4000g for 20 min. The supernatant was removed by aspiration and the pellets were resuspended in a total volume of 18ml ice cold 10% glycerol. The suspension was centrifuged at 3000g for 10min and the supernatant was again discarded. The cells were resuspended in a total volume of 1.5ml of ice cold 10% glycerol, distributed into 60ml aliquots and stored at -70°C. Electro-transformation of competent cells:

In order to transform electro-competent cells, an aliquot of 60ml was removed from - 70°C, and placed on ice until completely thawed. One tenth of the ligation reaction mixture was added and mixed by flicking the tube gently. The cell/ligated DNA mixture was placed into an electroporation chamber cuvette with 0.2cm electrode gap (Bio-Rad), which was kept on ice wherever possible during the electroporation procedure. The cells were transformed (electroporated) by applying 2.5KV per cm with a pulse duration (time constant) of approximately 9ms, using a pulse controller (Bio-Rad). After the pulse, 940ml of LB liquid medium was immediately added to the electroporated cells. The mixture was removed from the electroporation chamber, transferred to a sterile eppendorf tube, and recovered at 37°C for one hour. One quarter volume of the cells was plated on LB-agarose plates (LB medium plus 1.5% w/v bacto-agar or agarose (Difco/Sigma), containing 100mg/ml ampicillin, and incubated at 37°C overnight.

Individual ampicillin-resistant colonies were grown in 10ml LB cultures as above, supplemented with 100mg/ml ampicillin. Putative recombinant plasmids were prepared from 1-1.5ml of these bacterial cultures as follows: Cells were pelleted in an eppendorf tube by centrifugation at 14000rpm for 2min. After discarding the supernatant, the bacterial cells were resuspended in 250ml of Wizard Plus SV (Promega, USA) cell resuspension solution (50mM Tris-HCl [pH 7.5], 10mM EDTA, 100mg/ml RNase A). The cells were lysed by the addition of 250ml of cell lysis solution (0.2M NaOH, 1% SDS) and inversion of the tube. When the suspension was clear, 250ml of neutralization solution (4.09 M guanidine hydrochioride, 0.759 M potassium acetate, 2J2 M glacial acetic acid) was added and gently mixed. After centrifugation at 14000rpm for 10 min, the supernatant was collected avoiding the pellet, and passed through a Wizard Plus SV spin column. The columns were washed twice with 750ml and 250ml of DNA washing solution (60% ethanol, 60mM potassium acetate, 10mM Tris-HCl) respectively, and the DNA was eluted with 100 ml of nuclease free water by centrifugation at 14000rpm for 30sec.

Purified plasmids were then digested with £coRI DNA restriction enzyme, according to the manufacturers' instructions, at a concentration of approximately 1 unit of restriction enzyme per 100 mg of DNA, with incubation at the optimal temperature for 2-3 hours.

DNA restriction fragments were then size separated in a 1% non-denaturing agarose gel as described above. One of the recombinant plasmids examined ('clone 2') was sequenced automatically:

Approximately 10mg of DNA plasmid was purified and ethanol/sodium acetate precipitated. The DNA pellets were air-dried and sent in eppendorf tubes in a closed envelope to be sequenced automatically by MWG (West Germany).

Clone 2 was proven to include a 2295bp long insert which provided new sequence knowledge towards the 3' end of the HSP70 gene homologue, and a part of the heat shock protein 90 (HSP90) gene homologue of CYSDV.

In order to extend the new sequence knowledge of the CYSDV RNA2 towards the CP gene, another specific oligonucleotide primer was designed: CYSDV 101 (5'-CTT CCT GAA CAC ATA GAT GAA-3') based on the sequence data obtained with the method described above. CYSDV101 specific primer was used in combination with the same random hexamer anchor primer (δ'-ATGCGT-3') in a new RACE-PCR experiment as described above (without modification except for the omission of CYSDV1 primer, and the inclusion of lOOpmol of CYSDV 101 primer instead). The result of this experiment was a DNA product of approximately 2kbp that was gel extracted and ligated into a PGEM-T Easy vector (Promega) as described above. DH5a cells were electro-transformed (as above) and plated on LB-agar plates containing 100mg/ml ampicillin, incubated at 37°C (as above).

The existence of recombinant plasmids was again verified by digestion of purified DNA plasmids with EcoR\ restriction enzyme and agarose gel electrophoresis (as above). One DNA clone ('clone e') was sequenced automatically (by MWG (West Germany) as above) and the length of the insert in this plasmid was proven to be 2148bp. Detailed sequence analysis of clone e showed that 892 nucleotides after the 5' end of the CYSDV101 primer, and immediately downstream of the stop codon of an ORF homologous to the p9 ORF of LIYV (Klaassen ef al., (1995) Virology 208:99-110), there is an ATG start codon encoding methionine (Met). The total length of this new ORF is 756 nucleotides which encodes a 252 amino acids predicted protein product. This predicted protein product shares a high level of homology with the CP gene of the Crinivirus genus lettuce infectious yellows virus (LIYV). This new sequence also contains recognisable motifs of closterovirus CP genes.

Example 2: ISOLATION OF THE CYSDV COAT PROTIEN GENE: EXPRESSION AND PURIFICATION OF CYSDV COAT PROTEIN

Based on sequence information derived from clone e, a specific set of specific primers CYSDV CP1 (5'-CATCCCGGGAATGGCGAGTTCGAGTGAGAA-3'. and CYSDV CP2 (5'-GTAG____ICTCAATTACCACAGCCACCTG-3') were designed which corresponded to the actual 5' and 3' ends of the CYSDV CP gene, respectively. CYSDV CP1 primer was designed on the coding strand (sense) of the RNA and included a Smal restriction site (nucleotides underlined) upstream of the specific sequence. CYSDV CP2 was designed to be complementary to the coding strand (sense) of the RNA and included a Sad restriction site (nucleotides underlined) immediately downstream of the termination codon (5'-TCA-3'). Both primers included three extra nucleotides at their 5' ends, for cloning purposes. An extra adenosine (marked in bold) between the Smal restriction site and the ATG starting codon of the CYSDV CP1 oligo was included in order to maintain the correct reading frame of the CP gene with respect to the expression vector.

CYSDV CP1 and CP2 primers were used in RT-PCR experiments before using dsRNA as template for the amplification of the intact CYSDV CP genes. First, cDNA was prepared as follows. Approximately δOng of CYSDV RNA2 dsRNA species was recovered from a 1% agarose gel as described in Example 1. After phenol/chloroform extraction and ethanol precipitation, dsRNA was denatured in 10ml of 20mM methyl mercuric hydroxide for 1δmin at room temperature, in the presence of lOOpmol of CYSDV CP2 primer. The denatured dsRNA was added to first-strand cDNA synthesis mixture in a total volume of 50ml that was further incubated at 37°C for 50min, as in Example 1.

First-strand cDNA reaction mixture:

The entire reaction was diluted in 2ml of TE buffer before excess primer and low molecular weight contaminants were removed by two passages through a Centricon 100 (Amicon) column (as in Example 1). The first-strand cDNA was then concentrated in a total volume of 50ml and 10ml was added into a total volume of 100 ml of PCR mixture (50mM KCI, 10mM Tris-HCl, [pH 9.0], 0.1% Triton X-100, 4mM MgCI₂, lOOpmol CYSDV CP1 and lOOpmol CYSDV CP2 primer, 2.5units of Taq polymerase). The reactions were overlaid with 40ml of mineral oil and PCR amplification was carried out in a Hybaid thermocycler using the following cycling regime:

96°C for 5 min 1 cycle

96°C for 30 sec/ 55°C for 30 sec/ 72°C for 2 min 32 cycles 72°C for 10 min 1 cycle The expected amplified DNA PCR product of 756 bp was gel purified (recovered) using the QiaEX II kit (Qiagen, UK) as described in Example 1.

δOOng of this recovered PCR product was digested using Smal and Sad restriction enzymes. 200ng of digested PCR product was ligated with δOng of similarly digested pGEX-PG expression vector (Pharmacia). The ligation procedure, electro- transformation and recovery of E.coli (strain DHδa), selection of putative recombinants and purification and restriction analysis of the plasmid DNAs was conducted as described in Example 1 , with the exception that Smal and Sac\ restriction enzymes were used to restrict the DNAs, (instead of £coRI being used as in Example 1), and the expected size of the DNA insert was approximately 7δ0 bp (instead of approximately 2.2kbp as in Example 1).

The resulting pGEX-PG-CYSDV CP clone was used to transform E.coli BL21 (DE3) competent cells (BL21 (DE3) genotype: F^'.ompT, hsdS_B, (r_B ^", m_B ^"), dcm, gal, l(DE3)). Transformed cells were plated on LB-agar plates containing ampicillin and incubated at 37°C overnight.

Single colonies containing the correct insert cloned in frame with the pGEX-PG expression vector were grown in LB medium containing ampicillin overnight at 37°C, 225 rpm. The culture was then diluted 1 :100 into fresh LB medium and grown at 37°C, 225 rpm until the optical density (O.D.) at 5δ0nm reached 0.6. Bacterial cells were induced to express the protein product by adding isopropy! b-D- thiogalactopyranoside (IPTG) to a final concentration of 1mM. CYSDV CP fusion protein was isolated from induced bacterial cells in form of inclusion bodies.

To isolate inclusion bodies containing the CYSDV CP fusion protein from the cells, the following protocol was adapted from (Kleid et al., (1981) Science 214:1125-1129): 1. Bacterial cells expressing the protein were centrifuged at 7000g for δmin.

2. The cell pellet was resuspended in 100mM NaCl, 1 mM EDTA, δOmM Tris (pH 8.0) to a final concentration of 10% (vol/vol). Lysozyme was added to 1 mg/ml concentration and incubated at room temperature for 20min.

3. The suspension was centrifuged at δOOOg for 10min and the supernatant removed. The pellets were transferred to ice.

4. The spheroplasts were resuspended in ice-cold 100mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, δOmM Tris (pH 8.0) and incubated on ice with occasional mixing for 10min. δ. MgCI₂ and DNasei were added to a final concentration 8mM and lOmg/ml, respectively, and the mixture was incubated at 4°C for at least 15 min.

6. The inclusion bodies were removed from the suspension by centrifugation (10000g for 10min). The pellets were washed in 1% Nonidet (NP-40, Sigma), 100mM NaCl, 1mM EDTA, 50mM Tris-HCl (pH 8.0), and concentrated by centrifugation.

7. Finally, the pellets were resuspended in 100mM NaCl, 1mM EDTA, δOmM Tris- HCl (pH 8.0) and were ready for SDS-PAGE.

The CYSDV CP fusion protein was separated by 10% SDS PAGE:

10% Mini-Protean II (Bio-Rad) dual slab gels were used for gel electrophoresis of protein samples. The gels were consisted of a separation and a stacking gel. The separation gel was prepared by mixing the following reagents:

Separation gel

After pipetting a few drops of water onto the surface the gel, the system was incubated at room temperature for 30 min to polymerize. After polymerization, the water was removed a comb was inserted, and the stacking gel (4% acrylamide) was poured using a pasteur pipette.

Stacking gel

Samples were mixed in 2xloading buffer (100mM Tris-HCl [pH 6.8], 2% b- mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) and placed in a boiling water bath for 5 min. After polymerization of the stacking gel, the comb was carefully removed and samples were loaded into the slots using a 20ml micropipette. SDS-PAGE gels were run for 1-1. δh at 100V until the bromophenol blue dye reached the bottom of the gel.

Proteins were visualised using copper chloride staining:

Rapid visualization of proteins in polyacrylamide gels was achieved using the copper chloride staining procedure described by (Lee ef al., (1987) Anal. Biochem. 166:308- 312): 1. After electrophoresis, the gels were washed 3x10min with a large volume (eg. 2δ0mls) of distilled water.

2. The gels were placed in a plastic tray and δ gel volumes of 0.3M CuCI₂ were added. The gels were incubated at room temperature with agitation for δmin.

3. Gels were washed for several minutes with distilled water and observed against a dark background.

Gel slices containing the protein of interest were excised using a scalpel. CYSDV CP fusion protein was purified by electroelution:

Proteins were electroeluted as described by (Leppard ef al., (1983) EMBO J. 2:1993- 1999):

1. Gel slices containing the protein of interest were transferred into a clean dialysis tube containing 1.0ml of 0.2M Tris/acetate (pH 7.4), 1% SDS, 100mM dithiothreitol per 0.1 g of wet polyacrylamide gel.

2. The dialysis tube was placed in a horizontal electrophoresis chamber. Running buffer (δOmM Tris/acetate (pH 7.4), 0.1% SDS, O.δmM sodium thioglycolate) was added to cover the tube and a voltage of 100 V was applied. 3. After 3h the dialysis tube was opened and the gel slices were removed. The dialysis tube was reclosed and dialyzed against several changes of 0.2M sodium bicarbonate, 0.02% SDS.

4. The remaining buffer in the dialysis tubing, containing the electroeluted protein, was passed through an 'Amicon 100' column, in order to concentrate the protein.

This CYSDV CP fusion protein was then ready for use, for example in immunisation.

Example 3: PRODUCTION ANTI-CYSDV CP ANTISERUM

Three aliquots of IδOmg, 2δ0mg and 2δ0mg of purified CYSDV CP antigen were emulsified with equal volumes of Freuds adjuvant and injected intramuscularly into a rabbit over a period of three weeks at intervals of 1 , 7 and 21 days. Blood was collected two weeks after the final injection and incubated at room temperature for 1 h and at 4°C overnight to allow for clotting. The serum was centrifuged twice at δOOOg for 10min before an equal volume of glycerol was added to the serum. Sodium azide was added to a final concentration of 0.02% (w/v). This antiserum was stored at - 20°C.

Example 4: DETECTION OF CYSDV CP IN INFECTED PLANT TISSUE

In order to detect the CYSDV CP in infected plant tissue the following protocol was used:

All plant material was grown under natural conditions. The plant/CYSDV samples used herein were supplied by Dr Kraakman (De Ruiter Research, Almeria, Spain) in (Cucumis melo L.) melon leaves (cv. MA 109, De Ruiter) following Bemisia tabaci- transmission of the virus; CYSDV-infected {Cucumis sativus L.) cucumber leaves (cv. Camaron, Enza Zuiden) were supplied by Dr Lambalk (Enza Zaden, The Netherlands). Healthy, insect-free cucumber and melon plants of the same cultivars were collected from plants maintained under similar growth conditions.

1. ELISA-plates were coated with δO ml of coating buffer (14mM Na₂CO₃, 3δmM NaHCO₃, 3mM NaN₃, [pH 9.6]). An equal volume of infected crude plant extract diluted in PBS-Tween (140mM NaCl, 20mM KH₂PO₄, 80mM Na₂HPO₄.2H₂O, 20mM KCL, [pH 7.4], 0.05% Tween) was added and incubated at 4°C overnight.

2. The plates were washed three times with PBS-Tween.

3. 100ml of antibody in PBS-Tween, raised against the CYSDV CP, was added. The plates were incubated 2h at 37°C.

4. The plates were washed three times with PBS-Tween.

5. 100ml of alkaline phospatase labelled goat-anti-rabbit IgG (diluted 1:5000 in PBS- Tween) was added. Plates were incubated for 2h at 37°C.

6. The plates were washed three times with PBS-Tween. 7. 100ml of freshly prepared p-nitrophenyl phosphate (PNPP) at 1.0 mg/ml in substrate buffer (96ml diethanolamine, 800ml H₂O, 0.2g NaN₃) was. The plates were incubated at room temperature until the yellow colour became visible. The plates were read in an ELISA-plate reader at 405nm. Example δ: PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST THE CUCURBIT YELLOW STUNTING DISORDER VIRUS (CYSDV) COAT PROTEIN (CP), AND ASSAY OF PLANT TISSUE FOR CYSDV CP

Cloning of the CYSDV CP gene.

Agarose gel-extracted CYSDV dsRNA (δO ng) is added to 100 pM CYSDV4 specific primer (see Table 1 below) and the mixture is denatured in 20 mM methyl mercuric hydroxide for 20 minutes at room temperature. This primed RNA is used for first- strand cDNA synthesis with 200 U of Superscript II reverse transcriptase (Gibco BRL) which is performed at 42°C for δO minutes. The reaction mixture is diluted in TE buffer and passed twice through a Centricon 100 column (Amicon). Several dilutions of the purified cDNA are used as template for PCR amplification, including 1pg, 10pg, 100pg, 1ng, 10ng and δOng, with a combination of the CYSDV4 primer and a random hexamer primer (5'-ATGCGT-3'; each 100 pM). The template cDNA is denatured at 96°C for δ minutes and amplification carried out for 3 cycles of 30 seconds at 96°C, 4δ seconds at 42°C and 2 minutes at 72°C, followed by 32 cycles of 30 seconds at 96°C, 4δ seconds at δδ°C and 2 minutes at 72°C, with a final extension step at 72°C for δ minutes. PCR amplified products are subsequently ligated into pGEM-T Easy vector (Promega) according to the manufacturer's instructions, and transformed into DHδa Escherichia coli cells. Sequence information of the above cloned DNA product is used to design another CYSDV-specific primer (CYSDV101 ; Table 1) for the amplification of the CYSDV CP gene, in RACE-PCR experiments as described above, and in Example 1.

Sequences of cDNA clones are determined by the Sanger chain termination method using Dye-Terminator Cycle Sequencing with AmpliTaq DNA polymerase FS and an ABI PRISM 377 automatic sequencer. Sequence analysis is performed using the UWGCG (University of Wisconsin Genetics Computer Group) programs (version 8J) (Deveraux ef al., (1984) Nucleic Acids Res. 12: 387-396; Gish (1993) Nat. Genet. 3: 266-272) and BLAST programs (see above). Transformation of E.coli, Southern blotting analysis and DNA manipulation are carried out as described in (Sambrook ef al (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Construction of a CYSDV CP expression vector.

To express the CYSDV CP in £ coli, a 77δ bp long DNA fragment containing the entire putative CP open reading frame (ORF) is amplified by RT-PCR from viral dsRNA. This amplification is performed as described above, except that two virus- specific 30-mer oligonucleotide primers are used (CP1 , CP2; Table 1), and the three PCR cycles of low annealing temperature (42°C) are omitted.

CP1 comprises the first 20 nucleotides of the CP ORF and 10 additional nucleotides including an extra A nucleotide, and a Smal site immediately upstream of the start codon to facilitate cloning and expression of the CP ORF. CP2 comprises the complement of the last 20 nucleotides of the CP ORF including the termination codon, and immediately downstream are included extra nucleotides which form an £coRI cleavage site, in order to facilitate cloning of the ORF. The PCR-amplified product is cloned into the pGEM-T Easy vector (Promega) according to the manufacturer's instructions, and transformed into DHδa Escherichia coli cells. The resulting plasmid is termed pGEM-CP. Following digestion of pGEM-CP with Smal and Sac\, a 819 bp fragment (including the entire CP ORF) is gel-purified from agarose using a QIAEX kit (Qiagen) and ligated to similarly digested pGEX-KG expression vector (Pharmacia) to produce pGEX-CYSDVCP. The correct ORF for expression and the CP ORF sequence in the recombinant plasmid pGEX-CYSDVCP is verified by DNA sequencing.

Expression of the CYSDV CP in Escherichia coli, purification of the fusion protein, and antiserum production.

The pGEX-CYSDVCP recombinant plasmid is transformed into £ coli BL21 cells, which are grown in Luria Broth under appropriate selective conditions (100mg/ml Ampicillin) at 37°C until they reach A₆₀₀nm 0.5-0.6. Expression of CYSDVCP is then induced by the addition of isopropyl-thio-β-D-galactopyranoside (IPTG) to a final concentration of 0J mM for 3 hours at 25°C. The soluble recombinant protein, which consists of the full-length CYSDV CP fused to a glutathione-S-transferase (GST) tag, is affinity purified using glutathione-Sepharose 4B (Pharmacia), following the manufacturer's instructions. The fusion protein is further purified to near homogeneity following 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) (Laemmli (1970) Nature 227: 680-685), by staining with 0.3 M copper chloride (Lee (1987) Anal. Biochem. 166: 308-312) and electro-elution of the GST-CYSDVCP antigen (Leppard (1983) EMBO J. 2: 1993-1999). The recombinant protein is concentrated using a Centricon column 30 (Amicon) according to the manufacturer's instructions. The recovery of the purified antigen is assessed via 10% SDS-PAGE, and Coomassie blue staining of the gels. Protein concentration is determined by a BCA protein assay (Pierce) according to the manufacturer's instructions.

Polyclonal antibodies are raised in rabbits by administering three intramuscular injections over a three week period on days 1 , 8 and 20 using 108, 210 and 240 μg respectively of purified GST-CYSDVCP, emulsified with an equal volume of Freund's adjuvant. Ten days after the last injection, serum is collected from the rabbit, and immunoglobulins (IgG) are purified according to (Harlow and Lane (Eds) 'Antibodies: A Laboratory Manual.' Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.;Clark and Adams (1977) J. Gen. Virol. 34: 476-483).

Assay Of Plant Tissue For CYSDVCP By Immunoblotting And Indirect ELISA.

For immunoblotting, 1 g of fresh leaf tissue is homogenized in liquid nitrogen, and extracted in 1 ml of buffer (100 mM Tris-HCl [pH 8.0], 10 mM EDTA, δ mM DDT). Samples of 30 ml are mixed with Laemmli's sample buffer (Laemmli (1970) Nature 227:680-686) and analyzed by 12% SDS-PAGE. Electroblotting of the separated proteins onto Immobilon N membrane (MiUipore) is performed according to the method of (Towbin (1979) Proc. Nat. Acad. Sci. USA 76:4360-4364), using an LKB transfer electroblot unit. The immunoblots are blocked for 1 hour in 1% (wt/vol) skimmed milk in 1x phosphate-buffered saline (PBS) containing 0.1% Tween 20. After blocking, the immunoblots are probed with purified anti-GST-CYSDVCP rabbit IgG (prepared as described above), followed by alkaline phosphatase (AP) conjugated goat anti-rabbit IgG (Sigma) at a dilution of 1 :20,000. To visualise antibody staining, membranes are exposed to nitro blue tetrazolium chloride (0.33 mg/ml) and δ-bromo-4-chloro-3-indolyl phosphate, toluidine salt (0J 6δ mg/ml) in AP substrate buffer (0.1 M Tris-HCl [pH 9.5], 0.1 NaCl, 50 mM MgCI₂). The membranes are washed thoroughly in PBS-Tween between each step.

For indirect ELISA, 1 g of leaf tissue is homogenized in 10 ml of coating buffer (14 mM Na₂CO₃, 35 mM NaHCO₃, 3mM NaN₃ [pH 9.6]). ELISA plates are coated with 200 ml of plant extracts and incubated at 4°C overnight. ELISA plates are then washed three times with PBS-Tween. Immunoglobulins (1 mg/ml) raised against the GST-CYSDVCP fusion protein are added at a 1 :500 dilution and incubated at 37°C for 2 hours. ELISA plates are then washed three times with PBS-Tween. Then 200 ml of 1 :5,000 dilution AP-labelled goat anti-rabbit IgGs are applied. After incubation for 2 hours at 37°C, ELISA plates are washed three times with PBS-Tween, and then 200 ml of freshly prepared p-nitrophenyl phosphate (1.0 mg/ml) in 0J M diethylamine substrate buffer (pH 9.8) is added to each well. After 1 hour the absorbance is measured at 40δ nm using a u-max microplate ELISA reader (Mol. Devices, USA).

TABLE 1. Specific oligonucleotide primers used for RACE-PCR amplification of cucumber yellow stunting disorder (CYSDV) RNA2 and the CYSDV coat protein gene.

Primer Primer sequence³ SEQ ID No

CYSDV4 GATGTGATGAATTACTGTGCTA SEQ ID No : 4

CYSDV2 TTGGGCATGTGACATAGAG SEQ ID No : 5

CYSDV101 CπCCTGAACACATAGATGAA SEQ ID No : 6

CP1 CAT_QC3£2AATGGCGAGπCGAGTGAGAA SEQ ID No : 7

CP2 GTAG_ .mTCMTTACCACAGCCACCTG SEQ ID No: 8

The primer sequences are shown in a δ'->3' orientation. ^a Restriction sites for Smal in CP1 and £coRI in CP2 are underlined. An additional adenine nucleotide in CP1 (shown in bold) maintains the correct reading frame of CYSDVCP with that of the expression vector. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. Isolated CYSDV coat protein, or a fragment, homologue or derivative thereof.

2. A protein comprising the sequence presented as SEQ. ID. No. 2, or a fragment, homologue, or derivative thereof.

3. A protein comprising the sequence presented as SEQ. ID. No. 2.

4. An antibody raised against a polypeptide of any of claims 1 to 3.

δ. An antibody which has immunoreactivity with a polypeptide of any of claims 1 to 3.

6. A polypeptide capable of having immunoreactivity with an antibody according to claims 4 or δ.

7. A nucleotide sequence encoding a polypeptide according to any of claims 1 to 3.

8. A nucleotide sequence selected from:

(a) a nucleotide sequence comprising a nucleotide sequence presented as SEQ ID No. 1 , or a fragment, homologue or derivative thereof; (b) a nucleotide sequence comprising a nucleotide sequence presented as

SEQ ID No. 1 , or the complement thereof;

(c) a nucleotide sequence capable of hybridising a nucleotide sequence presented as SEQ ID No. 1 , or a fragment thereof;

(d) a nucleotide sequence capable of hybridising to the complement of a nucleotide sequence presented as SEQ ID No. 1, or a fragment thereof;

(e) a nucleotide sequence which is degenerate to the nucleotide sequences defined in (a), (b), (c) or (d) with respect to the genetic code, such as SEQ ID No. 3.

9. A nucleotide sequence according to claim 7, comprising the nucleotide sequence as shown in SEQ ID No. 1.

10. A nucleotide sequence according to any of claims 7 to 9, operably linked to a promoter.

11. A construct capable of causing the expression of a polypeptide according to any of claims 1 to 3 in a host cell.

12. A vector comprising a nucleotide sequence according to any one of claims 7 to 11.

13. A transformed cell comprising a nucleotide sequence according to any one of claims 7 to 12.

14. A host cell comprising the nucleotide sequence according to any one of claims 7 to 12, wherein the nucleotide sequence is heterologous to the genome of the cell.

1δ. A process for preparing a polypeptide according to claims 1 to 3, comprising expressing a nucleic acid according to any of claims 7 to 12, and optionally isolating the polypeptide therefrom.

16. A kit for detecting CYSDV, comprising nucleic acid according to any of claims 7 to 12.

17. A kit for detecting CYSDV, comprising an antibody according to claim 4 or claim δ.

18. A method for detecting the prescence of CYSDV in a biological sample, comprising testing for the presence of CYSDV coat protein in said sample using an antibody according to claim 4 or claim 5.

19. A method for detecting the prescence of CYSDV in a biological sample, comprising testing this sample with a nucleic acid according to any of claims 7 to 12.

20. A transgenic organism comprising a transgene which expresses a polypeptide according to any of claims 1 to 3.

21. A transgenic organism comprising a transgene nucleotide sequence according to any of claims 7 to 12.