# Patent

## Title
Methods for generating resistance against CGMMV in plants

## Abstract

The present invention relates to methods for generating resistance against Cucumber Green Mottle Mosaic Virus (CGMMV) in plants, in particular in plants susceptible to infection by CGMMV, such as Cucurbitaceae species, including melon, cucumber, watermelon and bottlegourd. The methods are based on the use of genetic constructs that induce post-transcriptional gene silencing and/or use a nucleotide sequence that encodes a defective variant of the replicase of CGMMV.

## Background
The present invention relates to a method for generating resistance against Cucumber Green Mottle Mosaic Virus (CGMMV) in plants, in particular in plants that are susceptible to infection by CGMMV, such as species of the Cucurbitaceae family.
The invention further relates to genetic constructs suitable or use in said method, and to CGMMV-resistant transgenic plants obtained via said method.
Methods of introducing DNA sequences into the genome of plants have been known for many years and have been widely used to alter the properties of plants varieties. Such methods are among others Agrobacterium-mediated transformation (Horsch et al., 1985; Rogers et al., 1986), protoplast transformation using electroporation or other techniques to introduce naked DNA molecules into the plant call (Shillito et al. 1985), and particle bombardment to introduce naked DNA molecules into plant cells or tissues (Christou et al., 1994).
Among the most important applications of plant genetic engineering are those aimed at introducing resistance genes to a wide variety of plant pests and plant pathogens, such as bacteria, fungi, nematodes, insects and viruses. Many examples of virus resistance in a wide variety of plant species have been described over the last decades (Wilson et al., 1993). The various methods to obtain virus resistance in plants through the introduction of gene sequences are either based on the use of genes of plant origin; on the use of sequences/genes derived from the viral pathogen itself (so-called pathogen-derived resistance (Wilson et al., 1993), or on the use of genes of yet different origin. Sequences originating from the viral genome can be either cloned or PCR-amplified DNA sequences obtain from the genome of DNA viruses, such as geminiviruses (Kunik et al., 1994) or the cDNA sequences obtained from the genomes of RNA viruses through the use of cDNA cloning or RT-PCR amplification.
Examples of sequences/genes of RNA viruses that have been successfully used in the engine of virus resistance in plants include:

    
    
        1. cost protein genes of tobamoviruses, cucumoviruses, potyviruses, potexviruses (Beachy et al., 1990);
        2. RNA dependent RNA polymerase genes (replicase genes) of tobamoviruses, cucumoviruses, potyviruses (Anderson et al., 1992; Donson et al., 1993; Audy et al., 1994);
        3. nucleoprotein genes of tospoviruses (Goldbach and De Haan, 1993; Prins et al., 1994; Vaira et al., 1995);
        4. movement protein genes of tobamoviruses and cucumviruses (Cooper et al, 1995).
    
    


Cucumber Green Mottle Mosaic Virus (CGMMV) is a member of the tobamovirus group and infects plant species of the Cucurbitaceae family: melon (Cucumis melo), cucumber (C. sativus), watermelon (Citrullus vulgaris) and bottlegourd (Lagenaria siceraria), but not apparently Cucurbita pepo (squash, pumpkin, courgette). The host range of the virus is basically restricted to members of the Cucurbitaceae and/or the diagnostic species Datura stramonium and Chenopodium amaranticolor (Hollings et al., 1975).
Several different strains can be distinguished serologically and by their response in C. amaranticolor and D. stramonium (Hollings et al., 1975) The “type strain” was originally identified in Europe and does not normally cause fruit symptoms in cucumber. Another European strain, called the cucumber aucuba mosaic strain, cucumber virus 4 or Cucumis virus 2A causes fruit symptoms in cucumber. A number of strains are known from Japan. In watermelon, the watermelon strain causes serious disease, whereas the Japanese cucumber strain (also called Kyuri Green Mottle Mosaic Virus) and the Yodo strain cause fruit distortions in cucumber. The CGMMV-C strain from India is a pathogen on bottlegourd and serious infectious can cause complete crop losses.
In cucumber, CGMMV causes vein clearing light and dark green leaf mottle, leaf blistering and malformation and stunted growth, seriously affecting fruit yield. The East European isolates of the aucuba mosaic strain produces bright yellow leaf mottling and fruit discoloration.
CGMMV is transmitted through seed, but mostly through mechanical infection via the roots in contaminated soil, and through foliage contact and handing of plants (Hollings et al., 1975). The virus particles are extremely stable and survive several months at normal temperatures. This stability combined with the very high infectivity through mechanical contact of the foliage is responsible for the economic importance of this virus as even one or a few infected plants in a cucumber greenhouse can eventually cause the infection and loss of the total crop. Also, infection may not only spread rapidly over a current crop, but also—due to the strong persistence of the virus—affect subsequent crops. Therefore, a CGMMV infection may require sterilization of an anti greenhouse, as well as the use of sterile tools and materials.
The complete sequence of only one isolate of CGMMV has been determined (Ugaki et al., 1991; Genbank accession numbers D12505 and D01188). This isolate “SH” had been found in infected watermelon plants in East Asia. Furthermore, the sequence of the coat protein gene of one other isolate (“W”) obtained from infected watermelon is known (Meshi et al., 1983; Genbank accession numbers V01551 and J02054), as well as the sequence of the 29 kD movement protein gene of a watermelon strain (Saito et al., 1988; Genbank accession number J04332). The nucleotide sequence of the CGMMV-SH isolate shows 55 to 56% identity with tobacco mosaic virus (TMV) and tobacco mild green mosaic (TMGMV), both other members of the tobamovirus group (Ugaki et al., 1991).
As described by Ugaki et al., the genome of CGMMV consists of a single-stranded RNA molecule coding for at least four open reading frames, encoding putative proteins of 186 kD, 129 kD, 29 kD and 17.3 kD, of which the 17.3 kD ORF is known to encode the coat protein. In this respect, Ugaki et al. state “No CGMMV-encoded proteins except for the coat protein have yet been identified in vivo”.
The CGMMV genome is schematically shown in FIG. 1. As can be seen therein, the ORF encoding the 186 kD protein starts at the same site as the ORF encoding the 129 kD protein, and adds a putative 57 kD polypeptide to the 129 kD ORF. The presence of this 57 kD protein alone has not been detected in infected plants. Instead, the 186 kD protein has been found, being the product of a read-through translation of the 129 kD and the 57 kD ORFs.
This 186 kD protein is thought to play a role in virus replication. Also, the 129 kD ORF is thought to encode a replicase function, whereas the 29 kD ORF is thought to encode a movement protein.
Hereinbelow, the nucleotide sequence corresponding to the ORF encoding the 129 kD protein will be referred to as “129 kD sequence”, the sequence corresponding to the 186 kD readthrough protein will be referred to as “186 kD sequence”, and the nucleotide sequence corresponding to the ORF encoding the 57 kD readthrough part will be referred to as “57 kD sequence”. These nucleotide sequences and the corresponding protein sequences are given in the sequence listings, as further described below.
Object of the invention was to provide a method for protecting plants, in particular plants susceptible to infection with CGMMV such as species of the Cucurbitaceae family, against infection with CGMMV, and in particular against infection with strains of CGMMV prevalent in Europe, such as the strains encountered in the cultivation of cucumbers in greenhouses.
Further objects were to provide means for use in said method, in particular a genetic construct that can be used for transforming plants or plant material so as to provide transgenic plants resistant against infection with CGMMV. Further objects of the invention will become clear from the description given hereinbelow.
For these purposes, applicant has investigated the symptomatology and the nucleotide sequence of the coat protein genes of 10 European strains of CGMMV, and compared these with the SH strain described by Ugaki et al. A list of these strains, with their geographical origin and symptoms on cucumber, is given in Table 1.







TABLE 1







List of collected CGMMV-isolates with their geographical


origin and symptoms on cucumber.









CGMMV




isolate
Geographical origin
Symptoms on cucumber





 1
Eastern Europe
vein clearing, mosaic


 2
Eastern Europe
vein clearing, mosaic


 3
IPO-DLO, the Netherlands
almost without symptoms


 4
The Netherlands
weak leaf chlorosis


 5
The Netherlands
weak leaf chlorosis


 6
Proefstation Naaldwijk,
Chlorosis



the Netherlands


 7
Rijk Zwaan, the Netherlands
Chlorosis


 8
Israel
Chlorosis


 9
Almeria, Spain
chlorotic leaf spots


10
Almeria, Spain
weak leaf chlorosis


CGMMV-SH
Japan
strong chlorotic leaf




mosaic









It was found that the sequences for the 10 European isolates are highly homologous (i.e. homology on the nucleotide level of 97%), and show about 90% homology (on the nucleotide level) with the SH-isolate. The nucleotide sequences encoding the coat proteins of each of the isolates 1-10, as well as strain SH, are given in the sequence listings, as further described below. The corresponding phytogenetic tree is shown in FIG. 2. This shows that the European isolates can be considered to constitute a subgroup of the CGMMV species.
In the sequence listings:

    
    
        SEQ ID no.1 gives the nucleotide sequence encoding the 129 kD replicase protein of CGMMV isolate 4, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.2 gives the amino acid sequence of the 129 kD replicase protein of CGMMV isolate 4; with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.3 gives the nucleotide sequence encoding the 57 kD protein of CGMMV isolate 4, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.4 gives the amino acid sequence of the 57 kD replicase protein of CGMMV isolate 4, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.5 gives the nucleotide sequence encoding the 186 kD readthrough protein of CGMMV isolate 4, with the ORF of the coat protein staring with the ATG codon at bp 523-525;
        SEQ ID no.6 gives the amino acid sequence of the 186 kD readthrough protein of CGMMV isolate 4, with the ORF of the coat protein staring with the ATG codon at bp 523-525;
        SEQ ID no.7 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 1, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.8 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 2, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.9 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 3, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.10 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 4, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.11 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 5, with the ORF of the coat protein stating with the ATG codon at bp 523-525;
        SEQ ID no.12 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 6, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.13 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 7, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.14 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 8, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.15 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 9, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.16 gives the nucleotide sequence encoding the coat protein of CGMMV isolate 10, with the ORF of the coat protein starting with the ATG codon at bp 523-525;
        SEQ ID no.17 gives the nucleotide sequence encoding the 129 kD replicase protein of CGMMV isolate SH;
        SEQ ID no.18 gives the amino acid sequence of the 129 kD replicase protein of CGMMV isolate SH;
        SEQ ID no. 19 gives the nucleotide sequence encoding the 57 kD protein of CGMMV isolate SH;
        SEQ ID no.20 gives tie amino acid sequence of the 57 kD replicase protein of CGMMV isolate SH,
        SEQ ID no.21 gives the nucleotide sequence encoding the 186 kD readthrough protein of CGMMV isolate SH;
        SEQ ID no.22 gives the amino acid sequence of the 186 kD readthrough protein of CGMMV isolate SH;
        SEQ ID no.23 gives the nucleotide sequence encoding the coat protein of CGMMV isolate SH;
        SEQ ID's nos. 24-40 give the nucleotide sequences of the primers used in the Examples;
        SEQ ID's nos. 41-44 give the nucleotide sequences used in assembling the leader sequences used in the constructs described in the Examples;
    
    


In the above sequence listings, the nucleotide sequences given are DNA sequences, as the genetic constructs of the invention described below will usually contain or consist of a DNA. As CGMMV is an RNA virus, it will be clear to the skilled person that these sequences will occur in the virus as the corresponding RNA sequence (i.e. with U replacing T). Also, it will be clear to the skilled person that the nucleotide sequences given above may be followed —both in the virus as well as in a construct of the invention—with a suitable termination codon, i.e. TAA/UAA, TAG/UAG or TGA/UGA (not shown).
Furthermore, as will be clear to the skilled person, the nucleotide sequence encoding the coat protein win usually start with an ATG codon. For example, in SEQ ID NOs 1-16, the nucleotide sequence encoding the coat protein starts at the ATG codon at base positions 523-525. (In the nucleotide sequence of SEQ ID NOs 1-16, the nucleotide sequence encoding the coat protein is preceded by another nucleotide sequence, e.g. encoding a movement protein. Accordingly, when hereinbelow reference is made to any nucleotide sequence of SEQ ID NOs 1-16, this also explicitly includes the nucleotide sequence starting at the ATG codon at base positions 523-525 of these SEQ ID's).
A particular purpose of the invention is therefore to provide a method that can provide plants with resistance against all the strains simultaneously, and more in particular a type of resistance that is agronomically useful, i.e. that can be used to generate a resistance of an extreme nature and/or that can be used to protect (crops of) plants that are cultivated under circumstances wherein the high infectivity and persistence of CGMMV can be a major problem, such as the cultivation of cucumbers in greenhouses. When generating a resistance of an extreme nature it is preferred that not even low levels of accumulation of viral RNA in the resistant plants is tolerated.
In one aspect of the present invention, this problem is solved by transforming a plant with a polynucleotide sequence (e,g. as part of a genetic construct) that is capable of including resistance against CGMMV by a mechanism that triggers sequence-specific gene silencing. Induction of PTGS (Post-transcriptional gene silencing) is a method to obtain down-regulation of gene expression of genes homologous to the inducing sequence. It has previously been employed to down regulate endogenous genes or transgenes. The present invention employs this principle for the silencing of viral genes and more in particular CGMMV genes. The natural mechanism of PTGS is not entirely understood. Plant viruses however, have evolved to overcome or suppress PTGS in order to be infective. The efficacy of PTGS against viruses has therefor not yet proven to be a wide-spread or general mechanism. The efficacy of PTGS and similar concepts will therefore largely if not mainly depend an the evolutionary development of the plant in question as well as the virus concerned. PTGS is considered to be sequence specific and it has been theorized that induction occurs by aberrant forms of RNA homologous to the genes. Aberrant form of RNA are for example extremely high levels. Of specific RNA molecules such as appear after viral infection of plant cells. Hence, it appears that sequence-specific gene silencing is induced by either high levels of transgene transcription or by the production of aberrant RNA.
One of a number of ways of inducing sequence-specific gene silencing is by expressing in a cell sense and antisense RNA molecules. These sense and antisense RNA molecules comprise nucleotide sequences respectively homologous and complementary to at least part of the nucleotide sequence of the nucleic acid of interest. In the case the nucleic acid of interest derives from a virus, the nucleotide sequence is (art of) a viral gene, for instance a gene encoding for a coat protein, a movement gene or a replicase gene.
The sense and antisense RNA molecules may be provided as one RNA molecule, for instance in the form of one or more inverted repeat sequences. Alternatively the sense and antisense RNA molecules may be provided as (a part) of two or more RNA molecules. The sense and antisense RNA may be linked by a spacer nucleotide sequence.
Without be bound thereto, the theory is that the sense and antisense RNA are capable of forming a double stranded RNA molecule (dsRNA). The dsRNA subsequently triggers a sequence-specific RNA degradation mechanism. This phenomenon has been observed in a variety of organisms, such as C. elegans, Drosophila and Arabidopsis (see or example Chuang, Z, Marcowitz, Proc. Nat acad, Sci 2000, 97, 4985-4990). Alternatively the dsRNA causes hybrid arrest of translation of co-factors required for viral replication or the hybridization of the RNA affects intra-molecular base pairing required for viral replication. At present and for the purposes of the present invention there is no preference for either theoretical mechanism. The use of gene silencing in relation to inducing virus resistance has been described previously in a number of articles such as by Waterhouse et al. in Trends in Plant Science, 1999, 4, 452-457; Kooter et al. in Trends in Plant Science, 1999, 4, 340-347; Andrew Fire in Trends In Genetics 1999, 15, 358; Muskens et al. in Plant Molecular Biology 2000, 43, 243-260.
The present invention provides a method for generating resistance in a plant or in a plant cell or against infection with CGMMV, said method comprising at least each step of transforming said plant or plant cell with one or more polynucleotide sequence that upon (at least) transformation into a plant and transcription into RNA generates resistance against infection wit CGMMV in said plant, preferably upon (at least) transformation into a plant and transcription into RNA the polynucleotide sequence does not lead to generation of (any) replicase activity in said plant; wherein the one or more polynucleotide sequence(s) comprises a first and a second DNA sequence, wherein the first DNA sequence comprises a promoter operably linked to a first DNA region capable of being transcribed into a sense RNA molecule comprising a nucleotide sequence of at least 10 consecutive nucleotides having between 75 and 100% sequence identity with at least part of the nucleotide sequence of the genome of a CGMMV virus; and preferably a further DNA region capable of controlling transcription termination and/or polyadenylation in the plant or plant cells, whereby the further DNA region is operably linked to the first DNA region. The second DNA sequence comprises a promoter operably linked to a second DNA region capable of being transcribed into an antisense RNA molecule comprising an nucleotide sequence including at least 10 consecutive nucleotides, having between about 75% to about 100% sequence identity with the complement of at least 10 consecutive nucleotides of the sense nucleotide sequence; and preferably a further DNA region capable of controlling transcription termination and polyadenylation in the plant or plant cells, The sense and antisense RNA molecules are capable of forming a double stranded RNA region by base-pairing between the regions which are complementary. Preferably, transforming the plant with the nucleotide sequence according to the invention and transcription of the nucleotide sequence into RNA does not lead to generation of (any) replicase activity in said plant. The first and second DNA sequence are either integrated separately, for instance in different loci in the nuclear gene of the transformed cell or they are linked on one recombinant DNA (i.e. one locus) such that DNAs are integrated together in the nuclear genome of the transgenic plant cells.
In order to provide resistance in the present invention, the nucleotide sequence derived from the genome of a CGMMV virus may be from a strain of the virus that in itself is not capable of infecting the plant, but which sequence is suitable for the generation of resistance against tobamoviruses in general and CGMMV and in particular.
The polynucleotide sequence according to the invention or at least apart thereof is preferably capable of forming at least one double strained RNA molecule by complementary base pairing of at least part of the sense and antisense RNA sequences. The polynucleotide according to the present invention is in general capable of virus induced gene silencing or similar mechanisms as herein described, resulting in the generation of resistance, preferably extreme resistance of the plant cells against CGMMV.
Preferably, the first and second DNA regions, encoding the sense and antisense RNA molecule, are derived from the nucleotide sequence encoding the RNA dependent RNA polymerase of CGMMV. Other nucleotide sequences derived from CGMMV are also suitable for the generation the first and second DNA regions according to the invention, based on the presently provided nucleotide sequence of CGMMV. In a preferred embodiment, a fragment derived from a nucleotide sequence encoding a RNA dependent RNA polymerase, preferably from CGMMV, is cloned in inverted repeat orientation, separated by a stuffer fragment. Transcription of the fragment in this arrangement will produce an RNA molecule that is capable of framing a hairpin structure. These constructs are evaluated in cucumber as will be further explained in the examples below. The use of dsRNA in a method for inducing virus resistance has been previously described in WO 99/53050. In this particular case, tobacco was transformed to obtain transgenic tobacco resistant against Potato Virus Y (PVY). The experiments showed that transforming plants with specifically designed constructs that contain a PVY protease sequence in only a sense orientation or only an antisense orientation resulted in virus resistance in 4 to ca. 10% of Me total number of treated plants. Improved resistance was found when the construct contained said PVY protease sequence in both a sense orientation and an antisense orientations WO 99/53050 hence teaches that in tobacco plants that are already susceptible of being rendered resistant by either a selected sense or a selected antisense sequence of said PVY protease alone, resistance may be improved by modifying the constructs to such that they express both sense and antisense RNA sequences.
Little is known at present regarding the defense mechanism against viruses in the Cucurbitaceae family. Cucumber, as an example of the Cucurbitaceae family is known to be highly susceptible to a wide variety of viruses and has, due to this susceptibility in certain cases even been used as a diagnostic tool for the detection of viruses. It has been hypothesized that his may be due to the fact that the antiviral defense mechanisms in the Cucurbitaceae family are not well developed. In the art, hence, no knowledge is available that provides guidance to the skilled man that the mechanism for conferring resistance described in the case of PVY infections in tobacco can easily be modified or transferred to other plants, especially to the Cucurbitaceae family without undue experimentation and with a reasonable expectation of success. This holds especially in the case of the Cucumber Green Mottle Mosaic Virus, of which the nucleotide sequence has only now been made available by the present applicants.
Furthermore, WO 99/53050 provides no insight or set of teachings that cat guide the skilled man in the process of selecting the parts of the sequence of CGMMV that when transformed into a plant cell are capable of conferring resistance to other viruses than Potato Virus Y in general, and to members of the Tobamovirus group of viruses in particular. Potato Virus Y is a member of the Potyvirus group, whereas CGMMV is a member of the Tobamovirus group. Although both viral groups are characterized by viral genomes consisting of one single positive RNA strand (positive mug that the single strand RNA encodes the viral proteins directly, as opposed to viral proteins being encoded by a complementary RNA molecule synthesized from the genomic RNA stand), they employ completely different replication strategies. Potyviruses encode on their RNA one single Open Reading Frame, that upon infection in plant cells is being translated into a single large polyprotein. This polyprotein is subsequently cleaved and processed into the various functional viral proteins by protease activity provided by the polyprotein itself. WO 99/53050 teaches the use of sense and antisense nucleotide sequences derived from that part of the potyvirus genome, that encodes the protease domains. Thus, sequence-specific degradation directed toward this particular part of the potyvirus genome will at least prevent the transition of peptides with this protease activity.
Tobamoviruses in general, and CGMMV in particular, do not encode proteases or protease activity. Instead, upon infection of a plant cell with these types of viruses, tile most 5′ located Open Reading Frame of the viral genome will be translated into a functional RNA dependent RNA polymerase (RdRP, also termed ‘replicase’), that, in turn is capable of not only replicating the entire viral genomic RNA, but that more specifically will generate subgenomic RNA molecules from the 3′ part of the viral genome. These subgenomic RNA molecules encode the more 3′ located viral Open Riding Frames, from which the movement proteins and coat proteins are the translated. In view of this totally different replication strategy in Tobamoviruses, the choice of the nucleotide sequences to be employed in sense and antisense gene constructs of the present invention cannot be deduced from WO 99/53050.
In a preferred embodiment of the present invention, the sense and antisense RNA molecules may be provided as one single RNA molecule, wherein preferably but not necessarily, the sense and antisense RNA sequence may be linked together through a spacer nucleotide sequence and are capable of forming a double stranded RNA molecule, also referred to as a hairpin structure. Providing the sense and antisense RNAs in a single molecule has the advantage that the ability to form a double stranded RNA molecule will become independent from the concentrations of the sense and antisense RNAs.
The spacer nucleotide sequence is preferably located between the sense and antisense nucleotide sequence. The spacer sequence is preferred for stability of the gene constructs in the process of gene cloning. In the absence of such a spacer sequence, the RNA molecule will still be able to form a double-stranded RNA, particularly if the sense and antisense nucleotide sequence are larger than about 10 nucleotides and part of the sense and/or antisense nucleotide sequence will be used to form the loop allowing tie base-pairing between the regions with sense and antisense nucleotide sequence and formation of a double stranded RNA. There are no length limits or sequence requirements associated with the spacer region, as long as these parameters do not interfere with the capability of the RNA regions with the sense and antisense nucleotide sequence to form a double stranded RNA. Hence the spacer may comprise artificial sequences that preferably are designed to aid in formation of the loop. The spacer, in a preferred embodiment, comprises an intron. In a preferred embodiment, the spacer region varies in length from 4 to about 2000 bp, preferably from 50 to 1500 bp, more preferably from 100-1250 bp. However, as previously mentioned, may be absent in which case the sense and antisense RNAs will be directly linked to each other.
In the present invention of generating resistance, preferably extreme resistance against CGMMV, it is preferred that the genetic conduct that is used for triggering the RNA degradation mechanism is formed by a sequence that comprises a promoter, operably linked to a first DNA sequence in sense direction, optionally followed by a spacer, followed by a second DNA sequence in antisense direction, optionally followed by a DNA sequence capable of controlling transcription termination or polyadenylation.
The genetic construct of the invention encode RNA molecules capable of forming more than one secondary structures such as hairpins or stem-loop sutures. Preferably, the genetic constructs of the invention are designed that hey encode an RNA molecule capable of adopting a secondary structure of the RNA that has the lowest free energy, preferably under physiological conditions (as they may occur in the cell). In accordance with the invention, the RNA molecule to be produced in the cell is designed in such a way that at least in its lowest free energy state, which it can assume under physiological conditions (within the cell), it will comprise the desired hairpin.
As used herein “hairpin RNA” refers to any self-annealing double stranded RNA molecule. In its simplest representation, a hairpin RNA consists of a double studded stem made up by the annealing RNA strands, connected by a single stranded RNA loop, and is also referred to as a “pan-handle RNA”. However, the term “hairpin RNA” is also intended to encompass more complicated secondary RNA structures comprising self-annealing double stranded RNA sequences, but also internal bulges and loops. The specific secondary structure adapted will be determined by the fee energy of the RNA molecule, and can be predicted for different situations using appropriate software such as FOLDRNA (Zuker and Stiegler, 1981).
As used herein, the term “plant-expressible promoter” or “promoter” means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S, the subterranean clover virus promoter No 4 or No 7, or T-DNA gene promoters. It is preferred to use a promoter that has been reported active is cucumber for example, and preferred 35S.
The term “expression of a gene” refers to the process who a DNA region which is oprerably linked to appropriate regulatory regions, particularly to a promoter, is transcribed into an RNA which is biologically active i.e., which is either capable of interaction with another nucleic acid or which is capable of being translated into a polypeptide or protein. A gene is said to encode an RNA when the end product of the expression of the gene is biologically active RNA, such as e.g. an antisense RNA, a ribozyme or a replicative intermediate. A gene is said to encode a protein when the end product of the expression of the gone is a protein or polypeptide.
As used herein, “reduction of expression of the target nucleic acid” refers to the comparison of the expression of the nucleic acid of interest in the eucaryotic cell in the presence of the RNA or chimeric genes of the invention, to the expression of the nucleic acid of interest in the absence of the RNA or chimeric genes of the invention. The expansion in the presence of the chimeric RNA of the invention should thus be lower than the expression in absence thereof, preferably be only about 25%, particularly only about 10%, more particularly only about 5% of the expression of the target nucleic acid in absence of the chimeric RNA, especially the expression should be completely inhibited for all practical purposes by the presence of the chimeric RNA or the chimeric gene encoding such an RNA. The present invention preferably provides for sequence specific RNA degradation mechanism that leads to the essential annihilation of the viral genome.
A nucleic acid of interest is “capable of being expressed”, when said nucleic acid, when introduced in a suitable host cell, particularly in a plant cell, can be transcribed (or replicated) to yield an RNA, and/or translated to yield a polypeptide or protein in that host cell.
As used herein “a nucleic acid of interest” or a “target nucleic acid” refers to any particular RNA molecule or DNA sequence which may be present in a eucaryotic cell, particularly a plant cell. The term “gene” means any DNA fragment comprising a DNA region (the “transcribed DNA region”) that is transcribed into a RNA molecule (e. g., an mRNA) in a cell operably linked to suitable regulatory regions, e. g., a plant-expressible promoter. A gene may thus comprise several operably linked DNA fragments such as a promoter, a 5′ leader sequence, a coding region, and a 3′ region comprising a polyadenylation site. A plant gene endogenous to a particular plant species (endogenous plant gene) is a gene which is naturally found in that plant species or which can be introduced in that plant species by conventional breeding. A chimeric gene is any gene which is not normally found in a plant species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.
As used herein, “sequence identity” with regard to nucleotide sequences (DNA or RNA), refers to the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences. The alignment of the two nucleotide sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window-size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data, including sequence alignment as described above, can, a. g., be conveniently performed using the programs of the Intelligentics Suite (Intelligenetics Inc., C.A.). Sequences are indicated as “essentially similar” when such sequence have a sequence identity of at leas about 75%, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical. It is clear than when RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
It is an object of the invention to provide a virus resistant plant, comprising a first and second chimeric DNA integrated in the nuclear genome of at least some of its cells, wherein the first chimeric DNA comprises a plant-expressible promoter, operably liked to a first DNA region capable of being transcribed into a sense RNA molecule comprising a nucleotide sequence of at least 10 consecutive nucleotides having between 75 and 100% sequence identity with at least part of the nucleotide sequence of the genome of a virus capable of infecting the plant, and optionally a DNA region involved in transcription termination and polyadenylation functioning in plant cells. The second chimeric DNA comprises a plant-expressible promoter, operably linked to a second DNA region capable of being transcribed into an antisense RNA molecule comprising an antisense nucleotide sequence including at least 10 consecutive nucleotides, having between about 75% to about 100% sequence identity with the complement of the at least 10 consecutive nucleotides of the sense nucleotide sequence, and optionally a DNA region involved in transcription termination and polyadenylation functioning in plant cells. Preferably the at least 10 nucleotides share sequence identity with part of the vial genome that encodes a replicase function, and more preferably the virus is a CGMMV.
The sense and antisense RNA molecules are capable of forming a double stranded RNA region by base-pairing between the regions which are complementary. The first and second chimeric DNA are integrated either in one locus or in different loci in the nuclear genome.
In a preferred embodiment of the invention, the RNA molecule transcribed from the chimeric gene, consists essentially of the hairpin RNA.
In a preferred embodiment, the order of the sense and antisense nucleotide sequence in the RNA molecule is not critical.
Thus, in other words, the chimeric DNA ha a transcribed DNA region, which when transcribed, yields a RNA molecule comprising an RNA region cable of forming an stem-loop structure, wherein one of the annealing RNA sequences of the stem-loop structure comprises a sequence, essentially similar to at least part of the nucleotide sequence of the nucleic acid of interest, and wherein the second of the annealing RNA sequences comprises a sequence essentially similar to at least part of the complement of at least part of be nucleotide sequence of the nucleic acid of interest. The RNA molecule may comprise more than one hairpin structures, which may be designed to reduce the expression of different nucleic acids of interest.
In a preferred embodiment, the nucleic acid of interest, whose expression is targeted to be reduced or whose degradation is desired, is a viral nucleic acid, particularly a viral RNA molecule, more in particular a tobamovirus, most in particular a CGMMV RNA molecule capable of infecting a eulcaryotic cell, particularly a plant cell. In a preferred embodiment, the expression to be reduced is the replication of the virus and/or the degradation of the viral DNA. It is also preferred to reduce or to remove the disease symptoms caused by the infecting virus. The reduction of expression or the degradation of other genes from CGMMV such as the genes encoding for movement proteins or coat proteins or the degradation of other viral nucleic acid sequences or the degradation of subgenomic RNAs is also explicitly included within the scope of the present invention.
Preferably, the nucleotide sequence of the target nucleic acid corresponding to the sense nucleotide sequence is part of a DNA region which is transcribed, particularly a DNA region which is transcribed and translated (in other words a coding region). It is particularly preferred that the target sequence corresponds to one or more consecutive exons, more particularly is located within a single exon of a coding region.
The length of the sense nucleotide sequence may vary from about 10 nucleotides (nt) up to a length equaling the length (in nucleotides) of the target nucleic acid. Preferably the total length of the sense nucleotide sequence is at least 10 nt, preferably 15 nt, particularly at least about 50 nt, more particularly at least about 100 nt, especially at least about 150 nt, more especially at least about 200 nt, quite especially at least about 550 nt. In principle there is no upper limit for the total length of the sense nucleotide sequence, other than the total length of the target nucleic acid. However for purely practical reason (such as e. g. stability of the chimeric genes, ease of manipulating the genetic constructs) the length of the sense nucleotide sequence should preferably not exceed 5000 nt, more preferably should not exceed 2500 nt and may preferably be limited to about 1000 nt.
It will be appreciated that the longer the total length of the sense nucleotide sequence is, the less stringent the requirements for sequence identity between the total sense nucleotide sequence and the corresponding sequence in the target gene become. Preferably, the total sense nucleotide sequence should have a sequence identity of at least about 75% with the corresponding target sequence, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially be identical to the corresponding part of the target nucleic acid. However, it is preferred that the sense nucleotide sequence always includes a sequence of about 10 consecutive nucleotides, particularly about 20 nt, more particularly about 50 nt, especially about 100 nt, quite especially about 150 nt with 100% sequence identity to the corresponding part of the target nucleic acid. Preferably, for calculating the sequence identity and designing the corresponding sense sequence, the number of gaps should be minimized, particularly for the shorter sense sequences.
The length of the antisense nucleotide sequence is largely determined by the lengths of the sense nucleotide sequence, and will preferably correspond to the length of the latter sequence. However, it is possible to use an antisense sequence which differs in length by about 10%. Similarly, the nucleotide sequence of the antisense region is largely determined by the nucleotide sequence of the sense region, and preferably is identical to the complement of the nucleotide sequence of the sense region. Particularly with longer antisense regions, it is however possible to use antisense sequences with lower sequence identity to the complement of the sense nucleotide sequence, preferably with at least about 75% sequence identity, more preferably with at least about 80%, particularly with at least about 85%, more particularly with at least about 90% sequence identity, especially with at least about 95% sequence to the complement of the sense nucleotide sequence. Nevertheless, it is preferred that the antisense nucleotide sequences always includes a sequence of about 10, preferably 15 consecutive nucleotides, particularly about 20 nt more particularly about 50 nt, especially about 100 nt, quite especially about 150 nt with at least 80%, preferably at leas 90% more preferably at least 95% and most preferred 100% sequence identity to the complement of a corresponding part of the sense nucleotide sequence. Again, preferably the number of gaps should be minimized, particularly for the shorter antisense sequences. Further, it is also preferred that the antisense sequence has between about 75% to 100% sequence identity with the complement of the target sequence.
In a preferred embodiment the hairpin RNA formed by the sense and antisense region and if appropriate the spacer region, is an hairpin RNA.
By “artificial hairpin RNA” or “artificial stem-loop RNA structure”, is meant that such hairpin RNA is not naturally occurring in nature, because the sense and antisense regions as defined are not naturally occurring simultaneously in one RNA molecule, or the sense and antisense regions are separated by a spacer region which is heterologous with respect to the target gene, particularly, the nucleotide sequence of the spacer has a sequence identity of less than 75% with the nucleotide sequence of the target sequence, at the corresponding location 5′ or 3′ of the endpoints of the sense nucleotide sequence. A hairpin RNA can also be indicated as artificial, if it is not coded within the RNA molecule it is nay associated with. It is conceivable to use in accordance with the invention a chimeric DNA whose transcription results in a hairpin RNA structure with a naturally occurring nucleotide sequence (which otherwise meets he limits as set for i this specification) provided this hairpin RNA is devoid of the subsiding RNA sequences (not involved in the hairpin structure formation).
Although it is preferred that the RNA molecule comprising the hairpin RNA does not further comprise an intron sequence, it is clear that the chimeric DNA genes encoding such RNAs may comprise in their transcribed region one or more introns.
The transformed plant cells are preferably used for the generation of transformed plants that can be fisher used in conventional breeding schemes to provide for more plants or to introduce the desired transformation, in the present invention resistance against CGMMV, to other varieties of the same or related plant species or in hybrid plants. Seeds obtained from the transformed plants containing the chimeric genes of the invention are also encompassed within the presently claimed scope.
As herein defined, with “inverted repeat sequence” is meant a DNA or RNA sequence that contains two identical nucleotide sequences in opposite directions (i.e. sense and anti-sense). The identical nucleotide sequences may be divided by a spacer. Identical in this respect is to be seen in the terms of sequence identity as herein defined.
The RNA sequence of the viral genome that may be used in the design of a suitable construct for use in the present invention preferably comprises nucleotides sequences that are derived from nucleotides sequences of the virus of interest, in the present case and preferably CGMMV, encoding (part(s) of) the movement, coat and/or replicase proteins, of which nucleotide sequences coding for the replicase protein are most preferred. However, other nucleotide sequences that can be expressed such that resistance is conferred by virus-derived transgenes are included within the present invention. Such nucleotide sequences are sequences that are homologous, preferably functionally homologous, to the sequences of the present invention. The term homologous in terns of the present invention indicates a certain amount of sequence identity on the nucleotide level. 100% homology indicates that the sequences are 100% identical. Sequences are also considered homologous if one or more nucleotides from the sequence are deleted, added or replaced as long as a certain percentage of sequence identity remains, for instance with a most preferred limit of 99%, more preferably 95, 85, 80, preferably 75, 70 or 65%. Also percentages as low as 50 or 60% may very well be considered as homologous. Whether or not a sequence can be regarded as homologous also depend on the function of that sequence. For instance a nucleotide e sequence encoding for a protein will still be considered as homologous if the protein it encodes for is able to perform its function. Hence homology is present if the functionality is maintained, thereby allowing for well known principles as degeneracy. By the term “functionally homologous” is meant the following. A sequence (for instance a gene) is considered functionally homologous if that sequence (gene) is homologous to another sequence, hence at least one nucleotide is deleted, inserted, replaced such as inversed (in case of more than one nucleotide) or transversion or transition while the function of said sequence (gene) is substantially maintained. This may also apply to chemically modified sequences. When a sequence is functionally homologous, there may very well be a low percentage of homology, but the functionality of that sequence is substantially maintained. Such sequences, whether DNA or RNA are also included within the scope of the present invention.
In a preferred embodiment, the sequence used to design the construct of the present invention is the “nucleotide sequence encoding a defective variant of the replicase gene of CGMMV” as herein defined.
In another aspect of the present invention, this problem is solved by transforming a plant with a polynucleotide sequence (e.g. as part of a genetic construct) that can provide the plant with so-called “replicase-mediated” resistance against CGMMV. In particular, this will be a polynucleotide sequence that

    
    
        i) has been derived from the 129 kD sequence, the 57 kD sequence, or the 186 kD readthrough sequence of native CGMMV;
        ii) upon (at least) transformation into the plant and transcription into RNA —and usually also translation into the corresponding encoded protein—can provide the plant with resistance against CGMMV; but
        iii) does not encode any replicase activity.
    
    


In one aspect of the invention, in case of the “replicase-mediated” resistance, a polynucleotide sequence according to the invention can encode a polypeptide or protein that is capable of providing a plant with resistance against GCMMV, but that by itself has no replicase activity, for resistance due to one or more alterations in its amino acid sequence, compared to the amino acid sequence encoded by the 129 kD sequence, 57 kD sequence, and/or 186 kD readthrough sequence of native CGMMV.
However, according to one specific embodiment of the invention, the polynucleotide sequence may also comprise, or even consist of the native 57 kD sequence.
In one aspect, the invention therefore relates to a method for generating resistance in a plant against CGMMV, said method comprising at least the step of transforming said plant with a polynucleotide encoding a defective variant of the replicase gene of CGMMV.
In a further aspect, the invention also relates to a method for providing a transgenic plant and/or plant cell that is resistant against infection with CGMMV, comprising at least the step of transforming said plant or plant cell with a polynucleotide sequence encoding a defective variant of the replicase gene of CGMMV.
In another aspect, the invention also relates to a genetic construct suitable for transforming a plant, said construct at least comprising a polynucleotide sequence encoding a defective variant of the replicase gene of CGMMV, and optionally further elements of genetic constructs known per se. The invention also relates to a plant, plant cell and/or plant material that has been transformed with a genetic construct of the invention.
The invention also relates to transgenic, plants that contain a polynucleotide sequence encoding a defective variant of the replicase gene of CGMMV, and/or that have been provided with resistance against infection with CGMMV by the method of the invention.
In the context of the invention, by the “replicase gene of CGMMV” is meant the native 129 kD sequence, the native 57 kD sequence, and/or the combined native 186 kD “readthrough” product of the native 129 kD and native 57 kD sequences.
By a “native” sequence is meant any RNA sequence that naturally occurs in CGMMV, including all isolates and strains thereof, as well as any DNA sequence that corresponds to these naturally occurring RNA sequences. Examples of such native sequences are the 129 kD nucleotide sequences given in SEQ. ID no.1 and SEQ. ID no.17, the 57 kD nucleotide sequences given in SEQ. ID no.3 and SEQ. ID no.19 and the 186 kD nucleotide sequences given in SEQ. ID no.5 and SEQ. ID no.21. It will be clear to the skilled person that there may be (further) naturally occurring variants of the RNA sequence from which the DNA sequences in the sequence listings were derived, and these (and the DNA sequences corresponding thereto) are also included within the term “native sequence”.
By “a polynucleotide sequence encoding a defective variant of the replicase gene of CGMMV” in its broadest sense is meant a polynucleotide sequence that

    
    
        i) upon (at least) transformation into a plant and transcription into RNA generates resistance against infection with CGMMV in said plant; and
        ii) upon (at least) formation into a plant and transcription into RNA does not lead to generation of (any) replicase activity in said plant (or at least—when it does lead to expression of some replicase activity—leads to expression of a replicase activity that is severely reduced compared to expression of the native gene encoding CGMMV replicase).
    
    


Herein, the terms “plant”, “transformed plant” and/or “transgenic plant” include all parts or tissues of such a plant, including but not limited to individual cells of such a plant. These terms also includes material of or for such a plant, such as material that can be regenerated into a (mature) plant, including but not limited to protoplasts and/or callus tissue, or material that can be cultivated into a mature plant, such as cultivation material.
The plant is preferably a pit that is susceptible to infection with CGMMV, more preferably a plant belonging to the Cucurbiteceae family, such as melon (Cucumis melo), cucumber (C. sativus), watermelon (Citrullus vulgaris) and bottlegourd (Lagenaria siceraria).
Included within the term “CGMMV” are all known strains thereof, including those prevalent in Europe and Asia. In particular, the method of the invention can be used to protect plants against strains of CCMMV prevalent in Europe (including Israel), such as those which are a problem in the cultivation of melons and in particular cucumbers in greenhouses, although the invention is not limited thereto.
In doing so, a major advantage of the invention is that it can provide protection against several and preferably all, (such) strains of CGMMV simultaneously. Another advantage of the invention is that it provides “absolute” protection against CGMMV, which means that— upon expression of a polynucleotide sequence encoding a defective replicase in a plant— essentially no viral particles can be detected in the transformed plant (material). The method of the invention therefore does not lead to a deferral or slowing down of the onset of symptoms, as may occur when so-called “coat protein-mediated” resistance is used. Also, the method of the invention leads to a high level of resistance, and may also have the advantage of a favorable temperature effect. Usually, the “nucleotide sequence encoding a defective variant of the replicase gene of CGMVV” will be a nucleotide sequence in which—compared to a nucleotide sequence encoding the corresponding native replicase of CGMMV—one or more nucleotides have been added, replaced and/or removed. In particular, the “nucleotide sequence encoding a defective variant of the replicase gene of CGMMV may be a nucleotide sequence that comprises, and preferably consists of:

    
    
        a nucleotide sequence corresponding to the native 129 kD sequence in which—compared to said native sequence—one or more nucleotides have been added, replaced and/or removed;
        a nucleotide sequence corresponding to the native 186 kD sequence in which—compared to said native sequence—one or more nucleotides have been added, replaced and/or removed, e.g. in the part of the native 186 kD sequence corresponding to the 129 kD sequence, to the 57 kD sequence, or both;
        a nucleotide sequence corresponding to the native 57 kD sequence;
        a nucleotide sequence corresponding to the native 57 kD sequence in which—compared to said native nucleotide—one or more nucleotides have been added, replaced and/or removed;
        such that said nucleotide sequence is capable—upon (at least) transformation into a plant and transcription into RNA—to confer to said plant resistance against infection with CGMMV, and such that said nucleotide sequence—upon (at least) transformation into a plant and transcription into RNA—is not capable of generating of (any) replicase activity in said plant.
    
    


Usually, the “nucleotide sequence encoding the defective variant of the replicase gene of CGMWV” will encode a protein or polypeptide, more specifically a protein or polypeptide that:

    
    
        1) upon being expressed in a plant is capable of generating resistance against CGMMV in said plant; and
        2) upon being expressed in a plant has no replicase activity (or —when it has some replicase activity—has severely reduced replicase activity compared to the native CGMMV replicase).
    
    


Such a protein or polypeptide will be generally referred to hereinbelow as “defective replicase”; and a polynucleotide sequence encoding such a protein or polypeptide will be referred to as a “polynucleotide sequence encoding a defective replicase”.
Usually, the defective replicase will be a derivative—such as an analog, homolog, variant, mutant, part fragment or combination of two or more such parts or fragments, etc.—of the amino acid sequence encoded by the native 129 kD sequence, the native 186 kD sequence and/or the native 57 kD sequence, in which—compared to the amino acid sequence encoded by the corresponding native sequence—one or more amino acids have been added, replaced or removed, preferably replaced or removed, more preferably removed, leading to loss of replicase activity (or at least an inability to generate replicase activity when expressed in the plant).
In particular, the defective replicase may be a protein or polypeptide that comprises, and preferably consists of:

    
    
        an amino acid sequence corresponding to the amino acid sequence encoded by the native 129 kD sequence, in which—compared to said native sequence —one or more amino acids have been added, replaced or removed, preferably replaced or removed, more preferably removed;
        an amino acid sequence corresponding to the amino acid sequence encoded by the native 186 D sequence, in which—compared to said native sequence —one or more amino acids have been added, replaced or removed, preferably replaced or removed, more preferably removed, leading to loss of replicase activity;
        an amino acid sequence corresponding to the ammo acid sequence encoded by native 57 kD sequence;
        an amino acid sequence corresponding to the amino acid sequence encoded by the native 57 kD sequence, in which—compared to said native sequence—one or more amino acids have been added, replaced or removed, preferably replaced or removed, more preferably removed;
    
    


or any combination thereof, provided that the resulting protein or polypeptide shows no replicase activity, but is still capable—upon expression in a plant—to generate resistance against CGMMV in said plant.
More in particular, the defective replicase may be a protein or polypeptide that comprises, and preferably consists of:

    
    
        an amino acid sequence corresponding to a part or fragment of the amino acid sequence encoded by the native 129 kD sequence, or to a combination of two or more such parts or fragments;
        an amino acid sequence corresponding to a part or fragment of the amino acid sequence encoded by the native 186 kD sequence, or to a combination of two or more such parts or fragments; or
        an amino acid sequence corresponding to the amino acid sequence encoded by the native 57 kD sequence.
    
    


such that the resulting protein or polypeptide shows no replicase activity, but is still capable—upon expression in a plant—to generate resistance against CGMMV in said plant.
An amino acid sequence “corresponding to apart or fragment of the amino acid sequence encoded by the native 186 kD sequence, or to a combination of two or more such parts or fragments” may for instance comprise: i) at least one part or fragment of the amino acid sequence encoded by the native 129 kD sequence combined with at least one part or fragment of the amino acid sequence encoded by the native 57 kD sequence (which combination of parts or fragments may or may not correspond to a contiguous amino acid sequence encoded by the native 186 kD sequence); ii) at least one part or fragment of the amino acid sequence encoded by the native 129 kD sequence combined with the full amino acid sequence encoded by the native 57 kD sequence, and/or iii) at least one part or fragment of the amino acid sequence encoded by the fill native 129 kD sequence combined with at least one part or fragment of the amino acid sequence encoded by he native 57 kD sequences.
It is know however, that expression in a plant of a nucleotide sequence encoding the full 129 kD sequence of the native replicase usually does not provide resistance against infection with CGMMV, but may even—upon infection of the plant—promote or facilitate multiplication of the virus. Therefore, in one embodiment the invention does not comprise the expression in a plant of said replicase, nor the use of a polynucleotide sequence encoding such a replicase.
Even more preferably, the defective replicase is a protein or polypeptide that consists of;

    
    
        an amino acid sequence corresponding to a part or fragment of the amino acid sequence encoded by the native 129 kD sequence, or a combination of two or more such parts or fragments; such that the resulting protein or polypeptide shows no replicase activity, but is still capable, upon expression in a plant, to generate resistance against CGMMV in said plant, or
        an amino acid sequence corresponding to the amino acid sequence encoded by the native 57 kD sequence.
    
    


Any such parts or fragments may also contain one or more further amino acid substitutions, insertions or deletions compared to the native sequence, but this is not preferred.
Most preferably, the defective replicase is a so-called “truncated replicase”, i.e. an amino add sequence corresponding to the amino acid sequence encoded either by the 129 kD sequence and/or by the 186 kD sequence, from which—compared to the native amino acid sequence—one or more amino acid residues are lacing at the carboxyl-terminus, such that the resulting protein or polypeptide shows no replicase activity, but is still capable—upon expression in a plant—to generate resistance against CGMMV in said plant. (In case of a truncated replicase based upon the 186 kD sequence, this usually means that the resulting protein will contain the fill acid sequence of the 129 kD sequence, as well as part of the amino acid sequence of the 57 kD sequence (i.e. that is contiguous to the 129 kD sequence in the amino acid sequence encoded by native 186 kD sequence), with one or more amino acids lacking at the carboxy-terminus of the 57 kD part, although the invention in its broadest sense is not limited thereto).
The polynucleotide sequence that encode such a truncated replicase may either comprise, or preferably consist of, the full native 129 kD sequence or 186 kD sequence, respectively, in which a stopcodon has been introduced at a desired site, or a polynucleotide sequence from which—compared to the full native 129 kD sequence or 186 kD sequence, respectively—one or more codons coding for the carboxy-terminal amino acid residues have been removed, i.e. starting from the 3′ end of the native sequence(s).
As mentioned below, preferably a stopcodon is introduced in to the native sequence, in particular in the so-called GDD motive or in the P-loop. Examples thereof are the polynucleotide sequences comprised in the vectors shown in FIGS. 3-8, and as described in the Experimental Part.
Again, any such truncated replicase may also contain one or more amino acid substitutions, insertions or deletions compared to the native sequence, but this is not preferred.
As mentioned above, (the polynucleotide sequence encoding) the defective replicase is such that—after expression in a plant or plant cell—it is still capable of generating resistance against CGMMV in said plant. Usually, this means that the defective replicase will have at least one biological function that allows the defective replicase to protect the plant against CGMMV infection, such as for example down-regulation of viral replication or interference with the replication of the wild-type CGMMV, for instance by competing with wild-type virus for the replication machinery in the plant (cell). It will be clear that in order to achieve such a biological function, the defective replicase must usually have a certain minimal level of amino acid similarity wit the amino acid sequences encoded by the native 129 kD, 186 kD and/or 57 kD sequences. In so far as the defective replicase is similar to the corresponding native amino acid sequence, this may be because it contains—on the corresponding amino acid positions—the same amino acid residues as the native amino acid sequence, or amino acid residues comparable thereto. The latter will usually comprise so-called “conservative” amino acid substitutions, for instance involving replacing a given acidic or basic amino acid residue by another acidic or basic amino acid residue.
However, there will also be dies in amino acid sequence between the defective replicase and the native replicase (i.e. the 129 kD, 186 kD or 57 kD protein), such it the defective replicase will no longer provide replicase activity. The skilled person will be able to select appropriate alterations to the amino acid sequence of the native replicase. As will be clear to the skilled person, a single (amino acid or nucleotide) alteration may be sufficient, or two or more such alterations may be required, dependant upon the position and nature of the alteration(s) compared to the ammo acid sequence of the native replicase.
Whether a given polynucleotide sequence encodes a defective replicase according to the invention—or at least is capable of protecting a plant against infection with CGMMV—can simply be tested by transforming a plant, plant cell or plant material with a construct containing said polynucleotide sequence, and then exposing the plant, plant cell, plant material, and/or a mature plant generated therefore, to CGMMV under conditions such that infection may occur. It can then be easily determined whether the polynucleotide sequence/construct is capable of protecting the plant, i.e. by suitably determining the presence of the virus, or simply by the presence or absence of symptoms of CGMMV-infection.
In general, as a minimum, when the defective replicase contains my amino acid substitutions or insertions, it will have amino acid homology (i.e. identity on corresponding position) with the corresponding native replicase protein of at least 80%, preferably at least 90%, more preferably at least 95%, with amino acid deletions not being taken into account, and a single amino acid insertion being counted as a single alteration.
In general, as a minimum, when the defective replicase contains one or more amino acid deletions, it will usually contain at least 30%, preferably at least 50%, more preferably at least 70%, and usually 80-90%, and may even contain as much as 95-99%, of the amino acid sequence of the corresponding native replicase protein, with any amino acid insertions or substitutions not being taken into account.
A truncated replicase based upon the 129 kD sequence will usually contain at least 50%, preferably at least 70%, and may contain as much as 80-95%, of the amino acid sequence of the native replicase. A truncated replicase based upon the 186 kD sequence may contain the full 129 kD protein followed by one or more amino acids from the 57 kD sequence, and usually contains the full 129 kD sequence followed by 1-95%, preferably 5-50%, of the 57 kD sequence.
The differences in acid sequence mentioned above can be differences compared to any of tie amino acid sequences given in SEQ ID's 2, 4, 6 and/or 18, 20, 22, and/or compared to any naturally occurring variant of these amino acid sequences. These differences are at least such that the resulting protein does not correspond to a naturally occurring/native protein (including those given in SEQ ID's 2, 4, 6 and/or 18, 20, 22).
The polynucleotide sequences used in the invention am such that they encode the above defective replicases. For this purpose, they may contain the same codons as in the corresponding positions on the native 129 kD, 186 kD and/or 57 kD sequence, or codons equivalent thereto due to the degeneracy of the genetic code.
The polynucleotide sequence encoding the defective replicase can be provided in a manner known per se, for instance starting from the known sequence of the native 129 kD, 57 kD and/or 186 kD sequences, and/or from a nucleic acid that encodes said sequences. Usually, this will involve introducing one or more deletions, substitutions and/or insertions of one or more nucleotides, or even of one or more codons into, or compared to, the native sequence. Such deletions, substitutions and/or insertions will be collectively referred to hereinbelow as “alterations”.
Accordingly, the polynucleotide sequence encoding the defective replicase may be a sequence that contain one or more such alterations compared to any of the nucleotide sequences given in SEQ ID's 1, 3, 5 and/or 17, 19, 21, and/or compared to any naturally occurring variant of these nucleotide sequences (including DNA sequences corresponding to the RNA sequences as present in the virus). These differences are at least such that the protein encoded by the polynucleotide sequence does not correspond to a naturally occurring/native protein (including those given in SEQ ID's 2, 4, 6 and/or 16, 18, 22).
Furthermore, besides the alterations mentioned above, and compared to nucleotide sequences given in SEQ ID's 1, 3, 5 and/or 17, 19, 21 and/or compared to any naturally occurring variant of these nucleic acid sequences (including DNA sequences corresponding to the RNA sequences as present in the virus), the polynucleotide sequences may ether contain one or more alterations that lead to a codon that encodes the same amino acid as the codon given for the corresponding position in SEQ ID's 1, 3, 5 and/or 17, 19, 21, and this may even lead to a fully or totally artificial and/or synthetic sequence. Also, compared to nucleotide sequences given in SEQ ID's 1, 3, 5 and/or 17, 19, 21 and/or compared to any naturally occurring variant of these nucleic acid sequences (include DNA sequences corresponding to the RNA sequences as present in the virus), the polynucleotide sequences may further contain one or more alterations that lead to a conservative amino acid substitution, i.e. as mentioned above.
Providing a polynucleotide sequence that contain the desired alterations will be within the skill of the artisan and can involve techniques such as nucleic acid synthesis using an automated nucleic acid synthesis technique; introduction of (point)mutations into a nucleic acid that comprises the native 129 kD, 57 kD, and/or 186 kD sequences; and/or using or suitably combining parts or gents of the 129 kD, 57 kD and/or 186 kD sequences, or any combination thereof. Also, in providing such a polynucleotide sequence, the skilled person may take into account the degeneracy of the genetic code and/or conservative amino acid substitutions, as mentioned above.
In order to provide a polynucleotide sequence that encodes a truncated replicase as defined above, a technique involving the introduction of a stopcodon into the native sequence is particularly preferred.
A particularly preferred technique of introducing the above alterations—including stopcodons—involves the use of a PCR reaction, in which the desired alterations are introduced into the amplified sequence(s) by the use of modified primers, i.e. primers that contain a suitable “mismatch” compared to the template sequence, leading to the desired alteration in the amplified sequence. This PCR-based technique may also be used to introduce one or more restriction sites into the amplified sequence in order to facilitate the cloning of the amplification products into the desired transformation on vectors.
As further described in the Experimental Part, this may involve a single PCR-reaction, but may also involve two or more PCR, reactions, each leading to a pat of intended final sequence encoding the defective replicase in which the priers (e.g. with the desired alteration) form the ends of the fragments. These fragments may then be combined, for instance to provide a polynucleotide sequence that comprises a combination of such fragments, and/or to reconstitute the full 129 kD, 57 kD and/or 186 kD sequence, now containing the desired alteration compared to the native sequence, such as a stopcodon.
The PCR-reactions and the further steps following amplification, such as combining/joining the amplified sequences, can be carried out in a manner known per se, for instance as described in the Experimental Part and/or using the techniques described in U.S. Pat. No. 4,683,202; Saiki et al., Science 239 (1988), 487-491 or PCR Protocols, 1990, Academic Press, San Diego, Calif., USA.
As the template for the PCR-reaction a nucleic sequence encoding the native 129 kD, 57 kD and/or 186 kD sequence can be used, such as a cDNA derived from the native RNA sequence, or a plasmid containing such a sequence, including those described in the Experimental Part. The template used may itself already contain one or more alterations, compared to the corresponding native sequence.
As mentioned above, a preferred alteration involves the introduction of a stopcodon into the native 129 kD or 186 kD sequence, such that—upon transformation into a plant—the polynucleotide sequence thus obtained causes expression of a truncated replicase. In particular, such a stopcodon may be introduced into a sequence corresponding to the native 129 kD sequence, more in particular to that part of the native sequence that corresponds to the so-called GDD-motivee or to the so-called P-loop.
The polynucleotide sequence encoding the defective replicase is preferably in the form of—e.g. forms part of and/or is incorporated within—a genetic construct. The genetic construct is preferably a construct suitable for the transformation of a plant, plant cell and/or plant material, such as a plasmid, cosmid or vector, including co-integration vectors or binary vectors. The genetic construct may be DNA or RNA, and is preferably dsDNA.
Preferably the genetic construct comprising the polynucleotide sequence encoding for the defective replicase is combined with the genetic construct comprising the polynucleotide sequence encoding for the hairpin sequence. By providing plants with these omnipotent constructs resistance can be generated against different strains of a virus, preferably the CGMMV virus, depending on the vulnerability of a strain for a particular method of generating resistance.
Such a construct may further contain all known elements for genetic constructs, and in particular for genetic constructs intended for the transformation of plants, as long as the presence thereof does rot interfere with the CGMMV resistance to be provided by the polynucleotide sequence encoding the defective replicase. Some non-limiting examples of such elements include leader sequences, terminators, enhancers, integration factors, selection markers, reporter genes, etc., and suitable elements will be clear to the skilled person.
These further elements may or may not be derived from plants, and may or may not be homologous to the plant that is to be transformed with the conduct of the invention (hereinbelow referred to as the “target plant”). For instance, the after elements may also have been derived from micro-organisms, viruses, etc., and may also be elements that are natively associated with the CGMMV sequence, such as the native CGMMV leader sequence (5′-UTR sequence).
The nucleotide sequences encoding these further elements may have been isolated and/or derived from a naturally occurring source—for instance as cDNA—and/or from known available sources (such as available plasmids, etc.), and/or may have been provided synthetically using known DNA synthesis techniques.
For instance, a construct of the invention will usually contain a suitable promoter operatively linked to the polynucleotide sequence encoding the defective replicase or the hairpin, e.g. such that it is capable of directing the expression of the polynucleotide sequence. Suitable promoters can be chosen from all known constitutive, inducible, tissue specific or other promoters that can direct expression of a desired nucleotide sequence in a plant and/or in part of a plant, including specific tissues and/or individual cells of the plant. In particular, promoters are used that are suitable for use in species of the Cucurbitaceae family, such as cucumber.
A specifically preferred promoter is the plastocyanine-promoter. Use of the 35S promoter is less preferred, as it may be less reliable in cucumber.
The terminator can be any terminator that is effective in plants. A particularly preferred terminator is the nos-3′ terminator.
The selection marker can be any gene that can be used to select—under suitable conditions such as the use of a suitable selection medium—plants, plant material and plant cells that contain—e.g., as the result of a successful transformation—the genetic construct containing the marker. A particularly premed selection marker is the nptII-gene, which can be selected for using kanamycin.
The construct of the invention further preferably contains a leader sequence. Any suitable leader sequence, including those of viral origin, can be used. Preferably, a leader sequence essentially identical to the 5′ untranslated (5′-UTR) region of the CGMMV genome is used. This may be derived from the viral RNA, or may be provided synthetically, e.g. as described in the Experimental Part.
Although not preferred, the invention also encompasses constructs that encode a fusion of a defective replicase as mentioned above, and at least one further amino acid sequence, such as a protein or polypeptide, or a part or fragment thereof, Preferably, expression of a defective replicase as (part of) such a fusion does not detract from the desired biological activity (i.e. protection against infection with CGMMV).
The construct of the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989) of F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987).
According to one embodiment, the genetic construct is preferably (also) in a form that can be maintained stable or inherited in a micro-organism, in particular a bacterium, more in particular a bacterium that can be used to transform a plant or plant material, such as Agrobacterium. In a further aspect, the invention also relates to such a micro-organism, in particular a bacterium, more in particular a bacterium that can be used to transform a plant, such as Agrobacterium, that contains a genetic construct according to the invention.
The genetic construct can be transformed into the target plant, plant cell or plant material by any suitable transformation technique known per se, including transformation with Agrobacterium, transformation with “denuded” DNA, for instance through particle bombardment or transformation of protoplast through electroporation or treatment with PEG.
Examples of suitable vectors systems for use with Agrobacterium are for instance binary vectors such as pBI121 and derivatives thereof co-integration vectors such as pGV1500 and derivatives of pB322. Suitable systems for transformation with denuded DNA include E. coli-vectors with high copy number, such as pUC-vectors and pBluescript II (SK+) vectors.
Upon transformation, the construct may for instance be incorporated into the genomic DNA of the plant, or it may be maintained/inherited independently in the plant (cell).
In a further aspect, the invention therefore comprises a method in which a plant, plant cell or plant material is transformed with a genetic construct as described above.
This method may also comprise cultivating the transformed plant cell or plant material into a mature plant, and may also comprise sexually or asexually reproducing or multiplying the transformed plant (and/or the mature plant obtained from the transformed plant cell or plant material).
The invention therefore also relates to a plant, plant cell or plant material, that has been transformed with—or more generally contains—a genetic construct as described above. Preferably, such a plant, plant cell or such plant material is resistant against infection with CGMMV as described herein.
The invention furthermore relates to cultivation material such as seed, tubers, roots, stalks, seedlings etc. for such a plant, as well as descendants of such a plant, obtained through sexual or asexual reproduction techniques. Such cultivation material and/or descendants most preferably still contain or have inherited the genetic construct of the invention, and more preferably also are resistant against infection with CGMMV as described herein.



The invention will now be illustrate by means of the following non-limiting Experimental Part and by means of the Figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the genome of CGMMV;
FIG. 2 gives a phylogenetic tree of CGMMV-coat protein (cp) for CGMMV-SH and the ten European isolates, using the method of J. Hein with weighed residue table.
FIG. 3 shows the genetic construct pKG433 1.
FIG. 4 shows the genetic construct pKG4332.
FIG. 5 shows the genetic construct pKG43 3 3.
FIG. 6 shows the genetic construct pKG4334.
FIG. 7 shows the genetic construct pKG4335.
FIG. 8 shows the genetic construct pKG4336.
FIG. 9a shows the construct pKG4322.
FIG. 9b shows the construct pKG1572.
FIG. 9c shows the construct pCG3.
FIG. 10a shows the construct pCG11.
FIG. 10b shows the construct pCG13.
FIG. 11 shows the plasmid construct pKG4359.
FIG. 12 shows the plasmid construct pKG4358.
FIG. 13 shows the plasmid construct pKG4375.
FIG. 14 shows the plasmid construct pKG4377.
FIG. 15 shows the plasmid construct pKG4374.
FIG. 16 shows the plasmid construct pKG4376.



Also, in the Experimental Part hereinbelow, enzymes, kits, etc. were usually used according to the instructions of the manufacturer and/or using well-established protocols, unless indicated otherwise.
Experimental Part
EXAMPLE I
Cloning of the Coat Protein Genes of 10 CGMMV-Isolates
1. Collecting CGMMV Isolate
To make use of coat protein-mediated protection (CPMP) strategy against CGMMV, it is necessary to clone the coat protein cistrons of the isolates, that are economically important As the only sequence information available for CGMMV is derived from watermelon strains from the Far East, it was first decided to collect CGMMV isolates of important cucumber culture areas in Europe and the Mediterranean area. Table 1 lists the isolates collected from various geographical areas. All isolates were propagated on cucumber, and infected leaf material was stored at −80° C. The symptoms obtained after infection of cucumber cv. Hokus are listed in Table 1.
2. Design of PCR Primers
The possibility of sequence divergence among the various collected isolates, and between the isolates and the published sequences of CGMMV-SH and CGMMV-W exists. In order to identify nucleotide regions with a high degree of sequence conservation, that could serve as a basis of PCR primer design, an alignment study was carried out on corresponding sequences of CGMMV-SH, CGMMV-W and of some other related members of the tobamovirus group: Sunn-Hemp Mosaic Virus (SHMV, a variant of TMV) and Pepper Mild Mottle Virus (PMMV). For this purposes a region of 800 nucleotides just 5′ of the coat protein cistron and a region of 170 nucleotides forming the far 3′ of the viral genome were compared. In this sequence alignment, region with sufficient sequence homology among all compared viruses were identified. Based on these sequences, sets of PCR primers were designed, which are listed in Table 2.








TABLE 2







Design of primers for the RT-PCR



amplification of coat protein sequences of


CGMMV-isolates.










position




on CGMMV-









Primers
Sequence
SH sequence













5′ primers





 


97G01
AGGTGTCAGTGGAGAACTCATTGA
5004



SEQ ID NO:24


 


97G02
GGCGTTGTGGTTTGTGG
5210



SEQ ID NO:25


 


97G03
CTGTAGGGGTGGTGCTACTGT
5248



SEQ ID NO:26


 


3′ primer


 


97G18
GCCCATAGAAACTTCAACGTC
6370



SEQ ID NO:27









3. Amplification of the Coat Protein Regions
From leaf meal of cucumber plants infected with each of the 11 isolate described in Table 1, a total RNA extraction was prepared. Using each of the 5′ primers listed in Table 2 in combination with 3′ primer 97G18, reverse transcription of RNA and PCR amplification of cDNA with an annealing temperature of 55° C. was established using a kit manufactured by Perkin Elmer Cetus. Especially in the reactions with the 5′ primer 97G03 amplification products of the correct size were obtained for each of the 11 RNA samples The PCR amplification products were directly cloned in T/A cloning vector pCR2.1 and introduced in E. coli stain INVαF′. For each of the RNA samples, the correct size of the cloned product (1.12 kb) was verified, and the clones were stored at −80° C. The amplification products of CGMMV-isolates 1 to 10 cloned in pCR2.1 were designated pKG4301 to pKG4301, and the one of CGMMV-SH cloned in pCR2.1 was designated pKG4311.
4. Nucleotide Sequence Analysis of the Coat Protein Cistrons
The sequences of the complete inserts of the plasmids pKG4301 to pKG4310 were determined by reading in both directions using m13 forward en m13 reverse sequencing primers. The sequence of the insert of pKG4311 was already known, as this plasmid contains a cDNA fragment of CGMMV-SH.
Sequence analysis confirmed, that in each case indeed the correct cDNA fragment of CGMMV had been obtained and cloned. With one exception, each amplified and cloned cDNA fragment consisted of 1123 base pairs, containing the CGMMV coat protein in and a large part of the CGMMV movement protein cistron.
The cloned sequences of all collected European isolates (isolates 1 to 10) are approximately 97% homologous among each other, but differ on average by 10% from the published sequence of CGMMV-SH. Comparison of each individual sequence revealed, that isolates 1 and 2, both from Eastern Europe are extremely alike. The same very high degree of identity was found between both isolates from cucumber greenhouses in the Netherlands (isolates 4 and 5) and between both isolates obtained from the Almeria area in Spain (isolates 9 and 10). None of the cDNA sequences was 100% identical to any of the other ones, but the differences in sequence are no more than a few nucleotides, and sometimes only one nucleotide in the coding region of the coat protein cistron. The Japanese isolate CGMMV-SH is clearly different from any of the European isolates.
5. Coat Protein Amino Acid Sequence Analysis
Based on the nucleotide sequences of the Open Reading Frames (ORF) of the coat protein cistrons of the 10 isolates, the to acid sequence could be deduced. In each of the analyzed sequences, the ORF consisted of a region of 486 nucleotides, coding for a protein of 161 amino acid residues. The predicted molecular mass of this proton is 17.3 kD, corresponding to earlier published results. The homology among the predicted protein sequences of the various isolates is as high as 98.1%. The only deviations are found for amino acid residue 19 (usually valine), residue 65 (mostly serine) and residue 84 (mostly leucine).
The sequence of the coat protein of the Japanese isolate CGMMV-SH only differs by 1 amino acid (residue 65) from the consensus sequence.
EXAMPLE II
Cloning of the Replicase Gene of CGMMV
1. Strategy for Replicase-Mediated Protection
By way of example, two approaches to replicase-mediated protection (RMP) against virus infections in plants were investigated.
One approach makes use of defective replicase genes in the form of truncated Open Reading Frames (ORF), in which the sequence downstream from the GDD motif had been truncated or altered through mutation.
The other approach makes use of the expression of the ‘read-through’ part of the replicase gene, i,e. the 57 kD sequence. It is thought that this ORF is not translated in the plant cell, but forms part of a larger ‘read-through’ ORF combining the coding regions of both the 129 kD replicase gene and the putative 57 kD protein gene, resulting a protein of 189 kD. However, expressing merely the 57 kD protein ORF in plant cells may result in a extremely strong resistance to infection by both virus particles and viral RNA which also would be capable of resisting high temperatures, as well as high inoculum concentrations.
For either of these approaches, either the full-length CGMMV replicase gene must be cloned, or both constituting parts must be cloned separately.
2. Design of Primers
Because of the high sequence homology of the coat protein genes of 11 CGMMV isolates it was assumed, that the sequences of the replicase genes of the various isolates would be also highly conserved. Based on the complete sequence of the CGMMV-SH isolate, primers were designed for the PCR amplification of the 57 kD ORF and of the 129 kD ORF (Tables 3 and 4). The primers were designed such, that they contain restriction sites for the future cloning of the amplification products. The 5′ primers contain an NcoI-site positioned such, that it will coincide with the ATG start codon of the amplified ORF. The 3′ primers contain a SacI-site downstream from the stop codon.








TABLE 3







Design of primers for the LR-RT-PCR



amplification of the 57 kD replicase


sequence of CGMMV.










position on




CGMMV-SH









Primers
sequence
sequence













5′ primer





 


98A88
CCATGGAGAATTCGCTGTATGTCC
3497



SEQ ID NO:28


 


3′ primer


 


98A86
CGAGCTCTCGACTGACACCTTAC
5001



SEQ ID NO:29

















TABLE 4







Design of primers for the LR-RT-PCR



amplification of the 129 kD replicase


gene sequence of CGMMV.










position on




CGMMV-SH









Primers
sequence
sequence













5′ primers





 


98A84
CCATGGCAAACATTAATGAAC
59



SEQ ID NO:30


 


98A85
CAACCATGGCAAACATTAATG
56



SEQ ID NO:31


 


3′ primer


 


98G63
TAACAGGGAGGAAAATATTACG



SEQ ID NO:32









3. Long-Range Reverse Transcriptase Polymerase Chain Reactions
From the known sequence of CGMMV-SH it was derived that the size of the 57 kD protein gene is 1.5 kb. Such a size is at the limit of the size image that can be amplified in a PCR with standard Taq polymerase. For the amplification of this cDNA fragment, and certainly for the amplification of the cDNA fragment for the 129 kD replicase gene, a different polymerase suitable for long range amplifications must be used. In these experiments, rTth DNA polymerase was used.
For direct amplification of cDNA fragments from total RNA extractions a RT-PCR kit is normally employed, combining in one reaction the activity of the Reverse Transcriptase (RT), producing a single cDNA strand complementary to the RNA template strand beginning at one primer annealed to the 3′ end of the RNA molecules, and the activity of the Polymerase, amplifying the thus produced single stranded cDNA molecule in a normal PCR fashion.
Because of the need to use long range polymerase, it was attempted to combine the RT with the long range polymerase to produce in one reaction large-size amplification products directly from total RNA extracts. This type of reaction was called a Long Range Reverse Transcriptase Polymerase Chain Reaction (LR-RT-PCR).
4. LR-RT-PCR Amplification and Cloning of the 57 kD Protein Gene
Using the primers listed in Table 3 and the LR-RT-PCR described above, a specific 1.5 kb amplification product was obtained from total RNA extracts of cucumber leaves infected with CGMMV-4. This isolate was chosen, as it originated from the Dutch cucumber greenhouse cultures, and would thus represent an economically important isolate. Because long range polymerases contain a ‘proof reading’ activity and do not leave A-additions on the amplification products, as does the Taq polymerase normally employed in PCR, direct cloning of the amplification products in a TA vector accommodating the A-additions was not possible. Therefore, the amplification products were briefly treated with Taq polymerase, resulting in the addition of A-overhangs on the amplified DNA molecules. These molecules could then sly be cloned in the TA vector pCR2.1, and transformed to E. coli MC1061. Clones with the correct insert size of 1.5 kb were stored at −80° C. and are known as pKG4321.
5. Sequence Analysis of the 57 kD Protein Gene
The nucleotide sequence of the cloned insert of pKG4321 was determined by double-stranded sequencing using m13 forward en m13 reverse primers and subsequent primer walking steps. The ORF coding for a putative 57 kD protein gene (SEQ ID no 3) showed 90% homology at the nucleotide level to the corresponding sequence of the Japanese isolate CGMMV-SH (SEQ ID no 19). The predicted amino acid sequence (SEQ ED no 4) shows a 98.2% homology to the one predicted by the CGMMV-SH sequence (SEQ ID no 20).
The GDD motif characteristic for viral replicase genes resides at amino acid residues 364-366.
6. LR-RT-PCR Amplification and Cloning of the 129 kD Replicase Gene
Using the primers listed in Table 4 in a Long Rage Reverse Transcriptase Polymerase Chain Reaction as described under 3, one specific amplification product of 3.5 kb representing the viral 129 kD replicase gene was obtained from total RNA of cucumber leaves infected with CGMMV isolate 4 (Table 1). Because long range polymerases contain a ‘proof reading’ activity and do not leave A-additions on the amplification products, as does the Taq polymerase normally employed in PCR, direct cloning of the amplification products in a TA vector accommodating the A-additions was not possible. Therefore, the amplification products were briefly treated with Taq polymerase, resulting in the addition of A-overhangs on the amplified DNA molecules. These molecules could easily be cloned in the TA vector pCR2.1, and transformed to E. coli MC1061. Clones with the correct insert size of 3.5 kb were stored at −80° C. and are known as pKG4322.
7. Sequence Analysis of the 129 kD Protein Gene
The nucleotide sequence of the amplification product cloned in pKG4322 was determined by double-sided sequencing using m13 forward en m13 reverse primers, and a primer walking strategy. The ORF coding for the 129 kD replicase gene (SEQ ID no 1) showed 88% homology at the nucleotide level to the corresponding sequence of the Japanese isolate CGMMV-SH (SEQ ID no 17). The ORF of the Dutch cucumber greenhouse isolate codes for a replicase protein of 1144 amino acids, which is one amino acid in extra in comparison to the CGMMV-SH strain. The predicted amino acid sequence (SEQ ID no. 2) shows a 97.1% homology to the one predicted by the CGMMV-SH sequence (SEQ ID no 18).
Two GDD motifs are found at amino acid residues 256258 and 540-542.
8. Site-Directed Mutagenesis of the 129 kD ORF
As explained above, one approach to obtain RMP in plant cells was to make use of replicase genes truncated either in the GDD motif, or truncated in the P-loop of the helicase domain. In order to create gene compression cassettes carrying such truncated genes, a site-directed mutagenesis approach was followed to introduce stop codons at the required positions in the ORF. To this end, several parts of the 129 kD replicase ORF were re-amplified from pKG4322 as a template using specifically designed primers that included unique restriction sites for future re-assembling of the thus amplified products, as well as the required mutations in the form of stop codons (Table 5). These stop codons should ensure the proper truncation of the translation of the protein. Several stop codons were designed one after the other in the three reading frames in these primers, thus ensuring an effective translation-deficient mutation.








TABLE 5







Design of primers for the site-directed



mutagenesis of the 129 kD replicase


gene of CGMMV.










Primers
sequence















98L99
GAGCTCGGATCCACTAGTAACGGC





SEQ ID NO:33



 



98L107
TAGAGCTCTTGAAGCTAAGCAAATTCCG




SEQ ID NO:34



 



98L108
TTCAAGAGCTCTAATCACCGAAGACAAAGGC




SEQ ID NO:35



 



98L102
GAATTATATCGATTATCTATCGGC




SEQ ID NO:36



 



98L103
GATAATCGATATAATTCTTCATCTGCC




SEQ ID NO:37



 



98L104
AACTAGTAATTGATGATCTGTTCAAGAAG




SEQ ID NO:38



 



98L105
AATTACTAGTTTCCGGAAGCAAGCAGCTCAG




SEQ ID NO:39



 



98L106
GCCCTCTAGATGCATGCTCGAG




SEQ ID NO:40










Using primers 98L103 and 98L104, a fragment from the downstream half of the 129 kD gene from the GDD motif up to the ClaI-site was amplified, while simultaneously stop codons were introduced at the site of the GDD motif. This fragment cloned in TA vector pCR2.1 was called pKG4325.
Using primers 98L105 and 98L106, a fragment corresponding to the 5′ half of the 129 kD gene up to the GDD motif was amplified, while simultaneously a stop codon was introduced at the site of the GDD motif. This fragment cloned in T/A vector pCR2.1 was called pKG4326.
Replacing an XbaI-ClaI fragment of pKG4322 with the combined amplified products of pKG4325 and pKG4326 reconstitutes the full-length 129 kD replicase ORF of pKG4322 with stop codons introduced at the site of the GDD motif. This construct is named pKG 4329.
Using primers 98L99 and 98L107, a fragment at the far downstream end of the 129 kD gene from the P-loop to the end of the ORF was amplified, while simultaneously stop codons were introduced at the site of the P-loop. This fragment cloned in T/A vector pCR2.1 was called pKG4327.
Using priers 98L108 and 98L102, a fragment corresponding to a central part of the 129 kD gene from the GDD motif up to the P-loop was amplified, while simultaneously a stop codon was introduced at the site of the P-loop. This fragment cloned in T/A vector pCR2.1 was called pKG4328.
Replacing an BamHI-ClaI fragment of pKG4322 with the combined amplified products of pKG4327 and pKG4328 reconstitutes the full-length 129 kD replicase ORF of pKG4322 with stop codons introduced at the site of the P-loop. This construct is named pKG4330.
EXAMPLE III
Transformation of Cucumber
1. Construction of a CGMMV-Leader Sequence
For optimal expression and stability of the replicase gene transcripts in plant cells, it was thought necessary to add a sequence identical to the 5′ untranslated (5′ UTR) region of the CGMMV genome upstream from the ORF sequence in the plant expression cassette. Because the 5′ UTR of viral genomes contain highly repetitive RNA, this sequence could not be obtained by RT-PCR amplification, as no specific primers could be designed. Instead, a synthetic region identical to the 5′ UTR of CGMMV-SH was assembled from the four oligonucleotide sequences:









97G40
(CTAGAGTTTTAATTTTTATAATTAAACAAA),




SEQ ID NO:41,


 


97G41
(TCAAAATTAAAAATATTAATTTGTTTGTTGTTGTTG),



SEQ ID NO:42,


 


97G42
(CAACAACAACAACAACAAACAATTTTAAAACAACAC)



SEQ ID NO:43


 


and


 


97G43
(TTGTTGTTTGTTAAAATTTTGTTGTGGTAC)



SEQ ID NO:44.






These oligonucleotides were designed such, that outside the sequence corresponding to the 5′ UTR they contain restriction sites for XbaI and NcoI, thus facilitating further cloning. Adding the four oligonucleotides together will cause spontaneous assembling due to the design of extensive regions of overhang. Using these restriction sites, the assembled mixture was cloned in a plant expression vector containing an Arabidopsis thaliana plastocyanine promoter (Vorst et al, 1993) and a Agrobacterium tumefaciens nopaline synthase terminator sequence (Depicker et al., 1982) in a pUC19-derived plasmid. This expression cassette was called pKG1315. The complete expression cassette consisting of the plastocyanine promoter, the CGMMV leader sequence and the nos terminator was subsequently removed from pKG1315 using HindIII and EcoRI as restriction enzymes, and recloned in the corresponding restriction sites of:

    
    
        1) an intermediate type Agrobacterium transformation vector for cointegrate type vector systems containing an nptII selectable marker gene cassette to create pKG1575, and
        2) an intermediate type Agrobacterium transformation vector for cointegrate type vector systems containing no selectable marker gene to create pKG1110.
    
    


2. Construction of Transformation Vectors
The three cloned and modified replicase constructs of pKG4321, pKG4329 and pKG4330 were isolated from the plasmids by restriction with BamHI (filled in with Klenow) and NcoI and ligated into the SacI (filled in with Klenow) and NcoI sites of each of the two transformation vectors pKG1575 and pKG1110, resulting in a total of six transformation vectors, listed in Table 6.







TABLE 6







List of six transformation vectors for the expression in plants of parts of the


CGMMV-replicase gene.









Vector
vector type
modified CGMMV-replicase gene





PKG4331
Intermediate type with nptII
 57 kD ORF


PKG4332
intermediate type with nptII
129 kD ORF with stopcodon in GDD motif


PKG4333
intermedinte type with nptII
129 kD ORF with stopcodon in P-loop


PKG4334
intermediate type
 57 kD ORF


PKG4335
intermediate type
129 kD ORF with stopcodon in GDD motif


PKG4336
intermediate type
129 kD ORF with stopcodon in P-loop









3. Transformation of Cucumber
The intermediate type transformation vectors pKG4331 and pKG4333 were introduced into Agrobacterium tumefaciens strain GV2260 by tri-parental mating. Transconjugants which had incorporated the intermediate type vector into their Ti-plasmids through homologous recombination were selected on the basis of streptomycin and spectinomycin resistance and analyzed for the correct insertion of the vector.
Cucumber plants were transformed with these two strains of Agrobacterium, as well as wit an Agrobacterium strain harbouring only the nptII selection marker, using published procedures. A number of transgenic cucumber plant were obtained. The plants were transferred to a greenhouse to flower and set seed, The seedlings germinating from these R1 seed were mechanically infected with CGMMV isolate 1-3 weeks post-inoculation, the plants were scored for symptoms of virus infection, as described in the assay for tolerance to virus infection set out in under 4, below.
4. Assay for Tolerance to Virus Infection
The seedlings of transgenic cucumber germinating from these R1 seed were mechanically infected with CGMMV isolate 1. Fresh inoculum was prepared from a crude leaf extract of susceptible non-transgenic cucumber plants cv. Hokus pre-infected with this same isolate 3 weeks previously. Seedlings of non-transformed cucumber plants were used as controls in the assay. During 21 days post-inoculation the appearance of viral symptoms was scored visually every 2 days. In this assay, individual plants are scored as being tolerant when they remain free of visible symptoms for at least 7 days, and preferably more than 14 days, and more preferably more than 21 days post-inoculation.
Sixty-four independent transgenic lines were assayed, with 14 to 20 seedlings for each line. Control seedlings all became diseased within 9 days post-inoculation. A number of seedlings in seventeen of the transgenic lines showed clear absence of symptoms for a prolonged period of time, and remained free of symptoms after 21 days post-inoculation. Of some transgenic lines, the number of symptom-fee plants corresponded to Mendelian segregation of a transgene present in a single locus. In one particular transgenic cucumber line 4 out of 14 seedlings remained symptom-free during the assay period, which may indicate that the tolerant phenotype corresponds to the homozygous state of a transgene present in one single locus, although, as mentioned above, the invention is not limited to a specific mechanism.
EXAMPLE IV
1. Construction of Hairpin RNA Construct
1.1. Genome Organization of CGMMV
The genome of CGMMV consists of a single-stranded RNA molecule coding for a 129 kD protein with replicase function (RNA dependent RNA polymerase), a putative 54 kD protein, a 29 kD movement protein and a 17.3 kD coat protein The presence of the 54 kD protein has not been detected in infected plants. However, a 186 kD protein has been found instead, being the product of a read-through translation of the 129 kD and the 54 kD Open Reading Frames. The 186 kD protein is also thought to play a role in virus replication. The genome structure of CGMMV is thus very similar to those of other members of the tobamovirus group.
The complete sequence of only one isolate of CGMMV has been determined (Ugaki et al., 1991; Genbank accession numbers D12505 and D01188). This isolate “SH” had been found infected watermelon plants in East Asia. Furthermore, the sequence of the coat protein gene of one other isolate (“W”) obtained from infected watermelon is known (Meshi et al., 1983; Genbank accession numbers V01551 and J02054), as well as the sequence of the 29 kD movement protein gene of a watermelon strain (Saito et al, 1988; Genbank accession number J04332). The nucleotide sequence of the CGMMV-SH isolate shows 55 to 56% identity with tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV), both other members of the tobamovirus group (Ugaki et al., 1991).
1.2. Cloning of the RdRp Gene of CGMMV
In the present example of the invention, the sequence elected for constructing the constructs is the RNA dependent RNA polymerase of CGMMV.
a. Primer Design
Because of the high sequence homology of the coat protein genes of 11 CGMMV isolates it was assumed, that the sequences of the replicase genes of the various isolates would be also highly conserved. Based on the complete sequence of the CGMMV-SH isolate, primers were designed for the PCR amplification of the 54 kD ORF and of the 129 kD ORF (Tables 7 and 8). The primers were designed such, that they contain restriction sites for the future cloning of the amplification products. The 5′ primers contain an NcoI-site positioned such, that it will coincide with the ATG start codon of the amplified ORF. The 3′ primers contain a SacI-site downstream from the stop codon.








TABLE 7







Design of primers for the LR-RT-PCR



amplification of the 54 kD replicase


sequence of CGMMV.










position on




CGMMV-SH









primers
sequence
sequence













5′ primer





 


98A88
CCATGGAGAATTCGCTGTATGTCC
3497



SEQ ID NO:28


 


3′ primer


 


98A86
CGAGCTCTCGACTGACACCTTAC
5001



SEQ ID NO:29

















TABLE 8







Design of primers for the LR-RT-PCR



amplification of the 129 kD replicase


gene sequence of CGMMV.










position on




CGMMV-SH









primers
sequence
sequence













5′ primers





 


98A84
CCATGGCAAACATTAATGAAC
59



SEQ ID NO:30


 


98A85
CAACCATGGCAAACATTAATG
56



SEQ ID NO:31


 


3′ primer


 


98G63
TAACAGGGAGGAAAATATTAC



SEQ ID NO:32









b. Long-Range Reverse Transcriptase Polymerase Chain Reactions
From the known sequence of CGMMV-SH it was clear, that the size of the 54 kD protein gene is 1.5 kb. Such a size is at the limit of the size range that can be amplified in a PCR with standard Taq polymerase. For the amplification of this cDNA fragment, and certainly for the amplification of the cDNA fragment for the 129 kD replicase gene, a different polymerase suitable for long range amplifications must be used. In these experiments, a long-range polymerase was used.
For direct amplification of cDNA fragments from total RNA extractions a RT-PCR kit is normally employed, combining one reaction the activity of the Reverse Transcriptase (RT), producing a single cDNA strand complementary to the RNA template strand beginning at one primer annealed to the 3′ end of the RNA molecules, and the activity of the Polymerase, amplifying the thus produced single stranded cDNA molecule in a normal PCR fashion.
Because of the need to use long range polymerase, it was attempted to combine the RT with the long range polymerase to produce in one reaction large-size amplification products directly from total RNA extracts. This type of reaction was called a Long Range Reverse Transcriptase Polymerase Chain Reaction LR-RT-PCR).
c. LR-RT-PCR Amplification and Cloning of the 54 kD Protein Gene
Using the primers listed in Table 1 and the LR-RT-PCR described above, a specific 1.5 kb amplification product was obtained from total RNA extracts of cucumber leaves infected with CGMMV-4. This isolate was chosen, as it originated from the Dutch cucumber greenhouse cultures, and would thus represent an economically important isolate. Because long range polymerases contain a ‘proof reading’ activity and do not leave A-additions on the amplification products, as does the Taq polymerase normally employed in PCR, direct cloning of the amplification products in a T/A vector accommodating the A-additions was not possible. Therefore, the amplification products were briefly treated with Taq polymerase, resulting in the addition of A-overhangs on the amplified DNA molecules. These molecules could then easily be cloned in the T/A vector pCR2.1, and transformed to E. coli MC1061. Clones with the correct insert size of 1.5 kb were stored at −80° C. and are known as pKG4321.
d. Sequence Analysis of the 54 kD Protein Gene
The nucleotide sequence of the cloned insert of pKG4321 was determined by double-stranded sequencing using m13 forward en m13 reverse primers. The ORF coding for a putative 54 kD protein gene showed 90% homology at the nucleotide level to the corresponding sequence of the Japanese isolate CGMMV-SH. The predicted amino acid sequence shows a 98.2% homology to the one predicted by the CGMMV-SH sequence.
The GDD motif characteristic for viral replicase genes resides at amino acid residues 364-366.
e. LR-RT-PCR Amplification and Cloning of the 129 kD Replicase Gene
Using the primers listed in Table 9 in a Long Range Reverse Transcriptase Polymerase Chain Reaction as described in 6.2.3, one specific amplification product of 3.5 kb representing the viral 129 kD replicase gene was obtained from total RNA of cucumber leaves infected with CGMMV isolate 4 (Table 1). Because long range polymerases contain a ‘proof reading’ activity and do not leave A-additions on the amplification products, as does the Taq polymerase normally employed in PCR, direct cloning of the amplification products in a T/A vector accommodating the A-additions was not possible. Therefore, the amplification products were briefly treated with Taq polymerase, resulting in the addition of A-overhangs on the amplified DNA molecules. These molecules could easily be cloned in the T/A vector pCR2.1, and transformed to E. coli MC1061. Clones with the correct insert size of 3.5 kb were stored at −80° C. and are known as pKG4322.








TABLE 9







Design of primers for the PCR amplification



of CGMMV target sequences


and plant intron sequences, to be assembled


in hairpin encoding gene constructs.










restriction site











Primer
target
added
sequence














primer 1
5′ RdRp
SacI
CGAGCTCATCTCGTTAGTCAGC






SEQ ID NO:45


 


primer 2
3′ RdRp
BamHI
GGGATCCACGTCTGGACAGG





SEQ ID NO:46


 


primer 3
5′ RdRp
XbaI
CTCTAGAATCTCGTTAGTCAGC





SEQ ID NO:47


 


primer 4
3′ RdRp
BamHI
AGGATCCTACACGAACCTATC





SEQ ID NO:48


 


primer 5
5′ AO3
BamHI
AGGATCCATTGCGGTAACACAAC





SEQ ID NO:49


 


primer 6
5′ AO3
BglII
TAGATCTATTGCGGTAACACAAC





SEQ ID NO:50


 


primer 7
3′ AO3
BglII
TAGATCTGTGTGATTCTGG





SEQ ID NO:51


 


primer 8
3′ AO3
BamHI
AGGATCCGTGTGATTCTGG





SEQ ID NO:52


 


primer 9
5′ IV2
BamHI
AGGATCCGTGTACGTAAGTTTC





SEQ ID NO:53


 


primer 10
5′ IV2
BglII
TAGATCTGTGTACGTAAGTTTC





SEQ ID NO:54


 


primer 11
3′ IV2
BglII
TAGATCTGTGATACCTGCAG





SEQ ID NO:55


 


primer 12
3′ IV2
BamHI
AGGATCCGTGATACCTGCAG





SEQ ID NO:56


 


primer 13
5′ RdRp
SacI
CGAGCTCATCTCGTTAGTCAGCTAGC





SEQ ID NO:57


 


primer 14
3′ RdRp
BamHI
AGGATCCTTTGTGCCTCTGTACATG





SEQ ID NO:58


 


primer 15
5′ RdRp
XbaI
CTCTAGAATCTCGTTAGTCAGCTAGC





SEQ ID NO:59


 


primer 16
3′ RdRp
BamHI
AGGATCCATCAACCCTAAATTGAGCC





SEQ ID NO:60


 


primer 17
5′ RdRp
BamHI
AGGATCCAGCAGGGAAATAAGTACGC





SEQ ID NO:61


 


primer 18
3′ RdRp
BamHI
AGGATCCGGTATGGACAAAATCAGC





SEQ ID NO:62


 


primer 19
5′ AO3
BamHI
AGGATCCATTGCGGTAACACAACCTCTC





SEQ ID NO:63


 


primer 20
3′ AO3
BglII
TAGATCTGTGTGATTCTGGAAAAG





SEQ ID NO:64


 


primer 21
3′ IV2
BglII
TAGATCTGTGATACCTGCACATCAAC





SEQ ID NO:65


 


primer 22
5′ IV2
BamHI
AGGATCCGTGTACGTAAGTTTCTGCTTC





SEQ ID NO:66


 


primer 23
5′ RdRp
XbaI
CTCTAGAATCTCGTTAGTCAGCTAGC





SEQ ID NO:67


 


primer 24
3′ RdRp
BamHI
AGGATCCAGCAGGGAAATAAGTACGC





SEQ ID NO:68









f. Sequence Analysis of the 129 kD Protein Gene
The nucleotide sequence of the amplification product cloned in pKG4322 was determined by double-stranded sequencing using m13 forward en m13 reverse primers, and a primer walking strategy. The ORF coding for the 129 kD replicase gene showed 88% homology at the nucleotide level to the corresponding sequence of the Japanese isolate CGMMV-SH. The ORF of the Dutch cucumber greenhouse isolate codes for a replicase protein of 1144 amino acids, which is one amino acid in extra in comparison to the CGMMV-SH strain The predicted amino acid sequence shows a 97.1% homology to the one predicted by the CGMMV-SH sequence.
1.3. Cloning of Target Sequences
In one particular example of the invention, a fragment of 489 nt of the 5′ end of RdRP gene of CGMMV was chosen as a target sequence for the construction of sense and antisense sequences separated by a stuufer fragment. These fragments were isolated from the cloned 129 kD ORF of pKG4322 (described above) by PCR amplification PCR primers were designed a corresponded to the 5′ and 3′ parts of the chosen target sequence, and included in the 5′ part of the primer sequences, an additional restriction site to facilitate the cloning of the amplification products.
One primer set (primer 1 and primer 2, Table 9) was designed to amplified the chosen target fragment of 489 bp from pKG4322, whereby restriction sites for SacI (primer 1) and BamHI (primer 2) were introduced by the PCR process at either end of the fragment.
A second primer set (prier 3 and primer 4, Table 9) was designed to amplify from pKG4322 the same target sequence of 489 bp from pKG4322 plus an additional sequence of 332 bp downstream of the target sequence in the CGMMV RdRP gene. Primer 4 and primer 5 added restriction sites for XbaI and BamHI, respectively, at either end of the amplified fragment. Details of the priers are given in Table 9. The PCR products obtained by amplification of the target sequences using the respective primers were cloned in T/A cloning vector pCR2.1, resulting in pCG1 (489 bp target sequence) and pCG2 (821 bp fragment). The ligation product was transformed to E. coli MC1061 and stored at −80° C. The sequences of the cloned PCR products in pCG1 and pCG2 were verified by sequence analysis and found to correspond exactly to the sequence of the template DNA of pKG4322.
In a similar way, one fragment of the target sequence was obtained by PCR on template pKG4322 DNA using primers 13 and 14, which resulted in a 398 bp amplification product with restriction sites for BamHI and SacI on either end. The second fragment of the target sequence was obtained on pKG4322 DNA as template with primers 15 and 16, resulting in an amplification product of 698 bp, of which the first 398 bp were identical to the fragment obtained with primers 13 and 14 and which extended another 300 bp in the 3′ direction. This product contained restriction sites for BamHI and XbaI on either end. Both fragments were cloned in T/A cloning vector pCR2.1 to create plasmids pKG4347 and pKG4349, respectively.
Yet another set of amplification reactions was designed to obtain larger fragments of the target sequence. In a similar way as described above, one 805 bp PCR product of the target sequence was obtained with primers 13 and 17 with restriction sites fob SacI and BamHI on either end, and a second 1102 bp product was obtained with primers 15 and 18 and contained restriction sites for BamHI and XbaI on either end. The sequence of the first 805 bp of the second PCR product was identical to the sequence of the first PCR product, while the second product extended for another 297 bp in the 3′ direction. Both fragments were cloned in T/A cloning vector pCR2.1 to create plasmids pKG4351 and pKG4346, respectively.
1.4. Construction of Hairpin RNA Encoding Transformation Vectors
The restriction sites on the ends of the amplified target sequences allowed the simultaneous cloning of both fragments by a three-way ligation in a suitable transformation vector such as pKG1572. This information vector is a cointegrate type T-DNA vector for Agrobacterium—mediated transformation of plants, carrying between the T-DNA borders a) the plant selectable marker gene nptII driven by a nos promoter, b) a CaMV 35S promoter for constitutive expression in plants, c) a multiple cloning site, and d) the nos polyadenylation sequence (FIG. 9). Furthermore, this vector contain a backbone sequence homologous to pBR322, including tie ColE1 origin of replication for maintenance in E. coli, and the aadA selectable marker gene for bacterial resistance to streptomycin and spectinomycin.
The presence of the restriction sites for BamHI at both 3′ ends of the PCR products allowed the insertion of both fragments in reverse orientation to each other in the cloning vector. Thus, a construct was created, that included the target sequence of 489 bp in reverse orientation, separated by a ‘stuffer’ fragment of 332 bp, that was included in the amplification product generated with primers 3 and 4, This ‘stuffer’ fragment is included to guarantee stability of the inverted repeat sequences in E. coli. The construct obtained by the three-way ligation was named pCG3 and was transformed to E. coli MC1061 and stored at −80° C. The pCG3 can construct was verified by sequence analysis.
In a similar way, the cloned PCR products of pKG4347 and pKG4349 were inserted in transformation vector pKG1572 in a 3-way ligation, resulting in inverted repeat orientation of the 398 bp identical parts of the products, separated by a 300 bp ‘stuffer’ sequence. The resulting transformation vector was named pKG4359 (FIG. 11).
Similarly, the PCR products of pKG4351 and pKG4346 were inserted in a 3-way ligation in transformation vector pKG1572 to create pKG4358 (FIG. 12), consisting of 805 bp inverted repeats of the CGMMV target sequence, separated by a 297 bp ‘stuffer’. All constructs were transformed to E. coli MC1061 and stored at −80° C.
1.5. Transformation of Cucumber
Transformation vector pCG3 was subsequently transferred to the disarmed Agrobacterium tumefaciens strain GV2260 by tri-parental mating. Strain GV2260 carries in its Ti-plasmid pGV2260 a 3.8 kb sequence of pBR322, homologous to a similar fragment of pBR322 residing in the backbone of the cointegrate transformation vectors such as pKG1572 and pCG3. This homologous sequence allows the stable integration of the transformation vector into the Ti-plasmid by homologous recombination.
Agrobactrium colonies were grown and subcultured on streptomycin end spectinomycin to select for the presence of the integrated transformation vector. Selected colonies were subjected to Southern blot analysis with the aadA selectable marker gene present on the cointegrate vector as a probe to verify single integration events in the Ti-plasmid. Furthermore, the Agrobacterium colonies were subjected to PCR analysis using primer sets capable of amplifying overlapping fragments covering the entire T-DNA of the integrated transformation vector to verify the correct integration in the Ti-plasmid of the complete T-DNA. A number of Agrobacterium colonies verified in this way were named GV2260 (pGV2260::pCG3) and were stored at −80° C.
In a similar way, the transformation vectors pKG4358 and pKG4359 were transferred to Agrobacterium GV2260. These were named GV2260 (pGV2260::pKG4358) and GV2260 (pGV2260::pKG4359), respectively.
The hairpin RNA encoding constructs are introduced into the genomes of cucumber plants using Agrobacterium-mediated transformation procedures known i the art. Briefly, cotyledon explants of young cucumber seedlings germinated in vitro are inoculated with a suspension of an Agrobacterium strain containing any one of the previously described transformation constructs integrated on their Ti-plasmids. The explants, after 1 to 5 days of cocultivation with Agrobacterium, are transferred to Petri dishes with regeneration medium containing, in addition to minerals, vitamins, sugars and plant growth regulators, kanamycin sulphate in concentrations of 50 to 300 mg/l as a selective aged, and incubated in growth chambers under the appropriate temperature and light conditions for the specific cucumber cultivar under study. The cotyledon explants will, in the course of the following weeks, produce primordia, that grow out to shoots. When the shoots have grown sufficiently long, the are transferred to glass jars with rooting medium containing the selective agent kanamycin sulphate. Truly transformed shoots will remain green and form roots on this medium, are ultimately hardened off transplanted to soil and transferred to a greenhouse. Viral resistance assays are preferably performed on young seedlings originating from crosses between transformed maternal cucumber plants and a pollinator line. Virus resistance assays can simply be carried out by mechanical inoculation of the seedlings with a crude extract in phosphate buffer of leaves of a severely diseased cucumber plant previously infected. The resistance phenotype is observed 21 days post-inoculation by absence of leaf chlorosis and stunted growth, which has become apparent in non-transgenic control sets. Depending on the number of independently integrated copies of the gene construct in the plant genome, the number of resistant seedlings versus the number of susceptible seedlings will correspond to a Mendelian segregation.
The resistance against virus infection obtained may be expressed as the degree of tolerance, by scoring the period in number of days post-infection which it takes for 50% of transformed seedlings in the infected population to show symptoms of virus infection. In many cases, however, the resistance to CGMMV infection obtained by hairpin RNA constructs is sufficiently effective that a score of 50% of transformed seedlings showing symptoms will not be observed within a period of several months. In such case, all seedlings remaining free of symptoms 21 days post-inoculation are scored as being resistant, and the number of resistant seedlings out of the total number of infected transformed seedlings is expressed as a percentage of effectiveness of resistance. In this way, differences in the effectiveness of the various described intron-spliced hairpin RNA constructs in conferring virus resistance are evaluated.
The virus resistance assays described above can be performed using inoculations of viral isolates of different origin. In this way, the intron-spliced hairpin RNA constructs targeted again CGMMV are shown to be effective against all isolates of CGMMV described in Table 1, including the Japanese isolate CGMMV-SH, as well as to isolates of the related cucurbit-infecting tobamoviruses Kyuri Green Mottle Mosaic Virus (KGMMV) and Cucumber Fruit Mottle Mosaic Virus (CFMMV).
EXAMPLE V
2.1. Cloning of Plant Intron Sequences
In a second example, an alternative ‘stuffer’ fragment necessary for stable maintenance of the inverted repeat structure in E. coli is chose in this case, use is made of a plant intron sequence capable of being spliced after transcription of the inverted repeat sequence in plant cells. The publication of Smith et al. (Nature 407: 319-320, 2000) describes the use of intron 2 of the PdK gene Flaveria as a ‘stuffer’ fragment in gene silencing constructs to obtain a high degree of resistance to infections with PVY. However, this Flaveria intron is very large (1.8 kb) and the correct splicing of this intron in Cucumis plant cells is uncertain. In this example, two types of plant introns are employed, of which the correct splicing in Cucumis has been verified. The intron is the 188 bp IV2 intron of the potato LS-1 gene, that is frequently encountered in gusA reporter gene constructs to obtain expression of beta-glucuronidase in plant cells with simultaneous absence of beta-glucuronidase expression in bacterial cells such as Agrobacterium tumefaciens. From experience it is known that cucumber and melon plants correctly express beta-glucuronidase from introduced gene constructs containing the gusA gene with the potato IV2 intron.
The second intron employed is the 532 bp Cucumis melo ascorbate oxidase intron AO3, which by its very nature is known to be spliced correctly in Cucumis melo (melon plants and is expected to function properly in the related species Cucumis sativus (cucumber).
Both these intron sequences were obtained by PCR amplification using primers, which, in the 5′ part of the primer sequences, include an additional restriction site to facilitate the cloning of the amplification products. Thus, melon intron AO3 was amplified from total genomic DNA of young melon seedlings using primers 5 and 7 (see Table 9), which each contains a restriction site fox BamHI or BglII, respectively, at their 5′ ends.
An alternative PCR reaction to obtain gene melon AO3 intron employed primers 19 and 20, and yielded a PCR product with 546 bp of amplified intron sequence, which corresponded to the known AO3 intron sequence, and which contained restriction sites for BamHI and BglII on either end. This PCR product was cloned in the T/A cloning vector pCR2.1 to yield plasmid pKG4355.
In order to test the effect of the cloned intron sequences in the hairpin RNA encoding gene constructs, control gene constructs were anticipate in which the intron sequences were placed in reverse orientation. To this end, a similar primer set was designed, consisting of primers 6 and 8, in which the restriction sites for BamHI and BglII were reversed as compared to primers 5 and 7 (Table 9). The PCR products obtained by amplification of the AO3 intron sequence using said primer sets were cloned in T/A cloning vector pCR2.1, resulting in pCG4 (BamHI-AO3 intron-BglII) and pCG5 (BglII-AO3 intron-BamHI). The sequences of the cloned PCR products were verified by sequence analysis and found to correspond exactly to the known sequences of intron.
The potato IV2 intron was amplified from the vector construct pKGT-3 carrying a gusA gene containing this intron with primes 9 and 11 (see Table 9), each carrying an additional restriction site for BamHI and BglII, respectively. Also for this intron, an additional PCR product with BamHI and BglII at the removed positions on either side of the amplification product was using PCR primers 10 and 12 (Table 9). The PCR products thus obtained were cloned in cloning vector pCR2.1, and named pCG6 (BamHI-potato IV2 intron-BglII) and pCG7 (BglII-potato IV2 intron-BamHI).
An alternative reaction to obtain the potato IV2 intron for cloning in the correct orientation employed primers 21 and 22 in a PCR reaction on template DNA of pKG1600, a plasmid vector containing the gusA gene of this intron. The reaction yielded an amplification product with 202 bp of IV2 intron sequence, and which contained restriction sites for BamHI and BglII on either end. This PCR product was cloned in the T/A cloning vector pCR2.1 to yield plasmid pKCG4353.
2.2. Cloning of Introns in the Hairpin RNA Encoding Expression Cassettes
The target sequence of 489 bp of the CGMMV RdRP gene was reamplified from the cloned CGMMV RdRp gene in vector pKG4322 by PCR using primer 2 and primer 3, as described in Example IV. This PCR reaction produced a fragment containing the target sequence, that was identical to the insert of pCG1 of Example IV) except that the 5′ restriction site generated at the 5′ end of the PCR product is a recognition site XbaI instead of for SacI. The PCR product was cloned in T/A vector pCR2.1 to produce pCG8. The ligation product was transformed to E. coli MC1061 and stored at −80° C. The sequences of the cloned PCR product in pCG8 was verified by sequence analysis and found to correspond exactly to the sequence of the template DNA of pKG4322.
In a similar way, the 398 bp CGMMV target sequence was reamplified from pKG4322 template DNA using primers 23 and 14 to create restriction sites for BamHI and XbaI on either end, to facilitate the cloning in intron-containing repeat constructs. This PCR fragment, after cloning in the T/A cloning vector pCR2.1, was named pKG4348.
Yet in another, similar, PCR reaction, an 806 bp CGMMV target sequence was reamplified from pKG4322 template DNA using primers 23 and 24 to create restriction sites for BamHI and XbaI on either end, to facilitate the cloning in intron-containing repeat constructs. This PCR fragment, after cloning in the T/A cling vector pCR2.1, was named pKG4350.
The vector pCG1 of Example IV was digested with restriction enzymes SacI and BamHI and the rent containing the 489 bp target sequence was isolated from gel and ligated into the transformation vector pKG1572 (described in Example IV) digested with the same two restriction enzymes. This ligation product, named pCG9, was transformed to E. coli MC1061. The correct structure of pCG9 was then verified by restriction analysis.
Next, both plant intron sequences in sense and in antisense orientations, cloned in vectors pCG4 to pCG7 were isolated from their vectors by digestion with BamHI and BglII and ligated into pCG9 digested with BamHI. The ligation products were transformed to E. coli MC1061. This cloning step placed the plant intron sequences next to the 489 bp CGMMV target sequence in the expression cassette of the transformation vector. Since restriction enzymes BamHI and BglII are isoschizomers and produce identical ‘sticky ends’, two orientations of the intron sequences were obtained in the ligation products. Colonies of all four cloning reactions were analysed by restriction enzyme digestion, and only those colonies of all four reactions were retained for further cloning, that contained the single BamHI site at a position between the in sequence and the CaMV 355 promoter. The cloning intermediates were named pCG10 (sense AO3 intron), pCG12 (sense IV2 intron), pCG14 (antisense AO3 intron) and pCG16 (antisense IV2 intron).
Subsequently, the 489 bp target sequence of pCG8 was isolated from the vector by digestion with XbaI and BamHI and ligated into the vectors pCG10, pCG12, pCG14 and pCG16, each digested with XbaI and BamHI. This Ligation step produced the final transformation vectors containing two copies of the 489 bp target sequence in reverse orientation to each other, thus encoding a hairpin RNA structure, and separated from each other by plant intron sequences in sense and antisense orientation The ligation products were named pCG11 (sense AO3 intron), pCG13 (sense IV2 intron), pCG15 (antisense AO3 intron) and pCG17 (antisense IV2 intron), and were transformed to E. coli MC1061 and stored at −80° C. The correct structure of the vectors was verified by sequence analysis,
The other cloned amplification products of target and intron sequences described in this example were assemble in the follow manner. The melon AO3 intron of pKG4355, as a BamHI-BglII fragment, and the 398 bp CGMMV RdRp target sequence of pKG4347, as a BamHI-SacI fragment, were simultaneously ligated in the transformation vector pKG1572. Subsequent insertion of a BamHI-XbaI fragment of pKG4348 into the ligation product yielded transformation vector pKG4375 (FIG. 13), which carried inverted repeats of the 398 bp CGMMV RdRp target sequence, separated by the melon AO3 intron.
To create a similar construct with the longer CGMMV target sequences, tie melon AO3 intron of pKG4355, as a BamHI-BglII fragment, and the 805 bp CGMMV RdRp target sequence of pKG4351, as a BamHI-SacI fragment were simultaneously ligated in the transformation vector pKG1572. Subsequent insertion of a BamHI-XbaI fragment of pKG4350 into the ligation product yielded transformational vector pKG4377 (FIG. 14), which carried inverted repeats of the 805 bp CGMMV RdRp target sequence, separated by the melon AO3 intron.
Likewise, transformation vectors with CGMMV RdRp inverted repeats separated by the potato IV2 intron were created. The potato IV2 intron of pKG4353, as a BamHI-BglII fragment, and the 398 bp CGMMV RdRp target sequence of pKG4347, as a BamHI-SacI fragment, were simultaneously ligated in the transformation vector pKG1572. Subsequent insertion of a BamHI-XbaI fragment of pKG4348 into the ligation product yielded transformation vector pKG4374 (FIG. 15), which carried inverted repeats of the 398 bp CGMMV RdRp target sequence, separated by the potato IV2 intron.
Also, the potato IV2 intron of pKG4353, as a BamHI-BglII fragment, and the 805 bp CGMMV RdRp target sequence of pKG4351, as a BamHI-SacI fragment, were simultaneously ligated in the transformation vector pKG1572. Subsequent insertion of a BamHI-XbaI fragment of pKG4350 into the ligation product yielded transformation vector pKG4376 (FIG. 16), which carried inverted repeats of the 805 bp CGMMV RdRp target sequence, separated by the potato IV2 intron.
All constructs were transformed to E. coli MC1061 and stored at −80° C.
2.3. Transformation of Cucumber
After transferred the transformation vectors to Agrobacterium tumefaciens strain GV2260 as described in Example IV, cucumber plants transformed with these Agrobacterium strains will be resistant to CGMMV infection. The preferred manner to assay virus resistance is described in Example IV. With all four gene constructs resistance to CGMMV infection is obtained. The efficacy of the intron sequences in sense orientation as opposed to constructs with introns in antisense orientation is apparent from the high percentage of cucumber lines showing extreme resistance to CGMMV infection.
Hereinabove, the invention has been described under the assumption that resistance against CGMMV is generated “at the protein level”, i.e. that the “nucleotide sequence encoding a defective variant of the replicase gene of CGMMV” codes for a “defective replicase”, the expression of which at cellular level generates the desired resistance against CGMMV. Hereinabove, the invention has been described under the assumption, that the resistance to CGMMV can also be generated at the RNA-level, e.g. down-regulation of gene expression due to RNA sequence homology. However, the invention is not limited to any explanation or mechanism, and is not particularly limited to the use of a particular type of nucleotide sequence (i.e. encoding a “defective replicase” or a “hairpin”).
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## Claims

1. A method for generating resistance in a plant or in a plant cell against infection with Cucumber Green Mottle Mosaic Virus (CGMMV), comprising generating a transformed plant or plant cell by transforming a plant or plant cell with one or more polynucleotide sequences that
upon transcription into RNA generate resistance against infection with CGMMV in said plant and produce a sense and an antisense RNA molecule capable of forming a double stranded RNA region by base-pairing between the regions which are complementary, said polynucleotide sequences comprising a first and a second DNA sequence, wherein:
said first DNA sequence comprises a promoter, operably linked to a first DNA region capable of being transcribed into a sense RNA molecule with a nucleotide sequence comprising a sense nucleotide sequence of at least 100 consecutive nucleotides having 100% sequence identity with the replicase encoding nucleotide sequence of the genome of the CGMMV virus capable of infecting the plant or the plant cell;
said first DNA sequence optionally further comprises a DNA region involved in transcription termination and polyadenylation functioning in plant cells operably linked to said first DNA region; and
said second DNA sequence comprises a second DNA region capable of being transcribed into an antisense RNA molecule with a nucleotide sequence comprising an antisense nucleotide sequence including at least 100 consecutive nucleotides, having 100% sequence identity with the complement of said at least 100 consecutive nucleotides of the sense nucleotide sequence;
said second DNA sequence optionally further comprises a promoter operably linked to said second DNA region and optionally a DNA region involved in transcription termination and polyadenylation functioning in plant cells operably linked to said second DNA region,

and wherein said replicase encoding nucleotide sequence of the genome of the CGMMV virus is:
(A) the nucleotide sequence of SEQ ID NO:1;
(B) the nucleotide sequence of SEQ ID NO:5; or
(C) the nucleotide sequence of SEQ ID NO:3.

2. The method according to claim 1, wherein the transformed plant is generated by crossing transformed parent plants comprising either the first or the second DNA sequence.

3. The method according to claim 1, wherein the transformed plant is generated by transforming a plant cell with the first and second DNA sequence and regenerating a plant from the transformed plant cell.

4. The method according to claim 1, wherein the first and second DNA sequence are integrated separately in the nuclear genome of the plant cell.

5. The method according to claim 1, wherein the polynucleotide sequence is transcribed into an inverted repeat RNA sequence, optionally linked by a spacer and wherein the spacer is preferably an intron.

6. The method according to claim 1, further comprising at least one step of cultivating the transformed plant cell into a mature plant.

7. The method according to claim 1, further comprising at least one step of sexually or asexually reproducing or multiplying the transformed plant and/or a mature plant obtained from the transformed plant cell.

8. The method according to claim 1, in which the plant is a plant that is susceptible to infection with CGMMV, more preferably a plant belonging to the Cucurbitaceae family, such as melon (Cucumis melo), cucumber (C. sativus), watermelon (Citrullus vulgaris) and bottlegourd (Lagenaria siceraria).

9. A transgenic plant or plant cell, obtainable or obtained by a method according to claim 1, or a descendant of such a plant; wherein the plant, plant cell, or descendant comprises said polynucleotide sequences.

10. The transgenic plant or plant cell or descendent according to claim 9, wherein said plant or plant cell or descendent is resistant against infection with strains of CGMMV prevalent in Europe.

11. The plant according to claim 9, being a plant that is susceptible to infection with CGMMV, more preferably a plant belonging to the Cucurbitaceae family, such as melon (Cucumis melo), cucumber (C. sativus), watermelon (Citrullus vulgaris) and bottlegourd (Lagenaria siceraria).

12. A cultivation material such as seed, tubers, roots, stalks, seedlings of a plant according to claim 9; wherein said cultivation material comprises said polynucleotide sequences.

13. The method according to claim 1, wherein the sense RNA molecule comprises a sense nucleotide sequence of at least 150 consecutive nucleotides having 100% identity with the replicase encoding nucleotide sequence of the CGMMV virus genome.

14. The method according to claim 1, wherein said transformed plant or plant cell is resistant against infection with strains of CGMMV prevalent in Europe.

15. The method according to claim 14, wherein said CGMMV strains prevalent in Europe are strains encountered in the cultivation of cucumbers in greenhouses.

16. The method according to claim 1, wherein the sense RNA molecule comprises a sense nucleotide sequence of at least 200 consecutive nucleotides having 100% identity with the replicase encoding nucleotide sequence of the CGMMV virus genome.

17. The method according to claim 1, wherein the sense RNA molecule with a nucleotide sequence comprises a sense nucleotide sequence of at least 550 consecutive nucleotides having 100% sequence identity with the replicase encoding nucleotide sequence of the CGMMV virus genome.