The present invention relates to a modified RDR gene which is capable of conferring and/or increasing virus resistance to a plant. The invention further relates to the use of such a modified RDR gene for conferring and/or increasing virus resistance to a plant.

Viruses are one of the major groups of pathogens that attack plants, resulting in negative effects influencing relevant crop aspects such as plant growth, plant vigour, product quality, and yield potential. Like most eukaryotes, plants have established active defense mechanisms against invading pathogens, amongst which viruses.

Besides structural and physical barriers, like cell walls, that protect plants against pathogens, roughly three types of plant immune systems are currently recognized. The first type responds to molecules from many classes of microbes, using transmembrane pattern recognition receptors (PPRs) that can react to microbial associated molecular patterns (MAMPs) and pathogen- associated molecular patterns (PAMPs).

The second type of plant immune system uses the polymorphic NB-LRR

(nucleotide binding leucine rich repeat) protein products encoded by resistance (R) genes. The NB- LRR proteins are able to detect pathogen effector proteins or their activity from diverse sources. On the pathogen side, a specific avirulence (Avr) protein or effector, encoded by Avr genes, triggers the R-protein-mediated immune responses by the host plant. The R gene and Avr action/reaction causes the hypersensitive response (HR), one of the most common plant reactions to many types of pathogenic organisms. However, despite the availability of several cloned R genes and their corresponding Avr proteins, it is still not fully clear how pathogen Avr proteins are recognized by their host.

A third important defense mechanism that plants use to defend themselves against viruses or other pathogens is based on RNA silencing or RNA interference. RNA silencing refers to a class of gene silencing effects by which the expression of one or more genes is downregulated or entirely suppressed by small RNAs. A number of gene families and their related protein families have been identified as components of the RNA silencing system. Amongst those are Dicer (DCR) genes, Dicer-Like (DCL) genes, Argonaute (AGO) nuclease genes, and RNA-dependent RNA polymerase (RDR) genes. Each of those gene families contains several members, which can act in different combinations in different species, and have their own particular functionality and type of interaction against specific pathogens or groups of pathogens.

Upon viral infection of the host plant an RNA virus establishes viral replication, resulting in the presence of virally derived dsRNA. This dsRNA triggers the plant's RNA silencing system by functioning as a substrate for a specific DCL protein, an endoribonuclease, which cleaves the dsRNA into short fragments of typical lengths ranging from about 20-25 basepairs. After further processing, single-stranded forms of these short virus-derived siRNA fragments are loaded unto an AGO protein, which is part of the RNA-induced silencing complex (RISC) of the host plant. In the RISC, the siRNA is used by the AGO proteins as a guiding template which ultimately results in the degradation of viral RNA through a series of recognition and cleavage processes.

RDR genes are thought to play a role in this immunity system by the enhanced production of virus-specific dsRNA substrates for DCL proteins through de novo synthesis. The resulting siRNA accumulation then presumably increases the efficiency by which the recognition of the viral RNA occurs, which can consequently be more actively silenced or degraded.

As a counter measure many viruses have developed a system to circumvent or inhibit RNA silencing pathways, by activation of RNA silencing suppressors (RSS). The presence of a particular RSS can contribute to inhibition of the plant's defense, which in turn re -enables viral genome replication and spread in the targeted host plant, thereby facilitating further infection.

The balance between the silencing and suppressing pathways of a certain plant and a particular virus plays a large role in determining whether a virus will succeed in establishing an infection, or if the plant will prove strong enough to be completely or sufficiently resistant to withstand the attack.

Although the involvement of the various mentioned genes in plant immunity systems as such has been recognized, much about the regulatory mechanisms, the relationships between those genes, and the ways in which the different proteins function remains elusive.

Moreover, the contribution of the individual genes to viral silencing, while overcoming the antisilencing of the pathogen, has not been resolved. In addition, further research to unravel the structure of the relevant genes and the way their expression is regulated is still ongoing.

Consequently, it is unclear which gene that is involved in the RNA silencing pathway would be a preferred candidate to target for inducing resistance against pathogens, in particular against viruses, and in which way it should be modified to obtain the desired effect.

It is an object of the present invention to provide a means for conferring virus resistance to a plant.

During research that led to the present invention, a modification in an RDR gene was detected. This modification resulted in the enhanced expression of the RDR gene as compared to the wild type RDR gene. The enhanced expression can be detected as an increase in the mRNA accumulation level of the RDR gene; as an increase in the level of the RDR protein; and/or as an increase in the activity of the RDR protein.

The RDR gene family, encoding RNA dependent RNA polymerases, comprises several members. In plants, the RDR genes are commonly grouped into four clusters, indicated as clusters I-IV. In many plants a combination of RDR genes is present in the genome. The Arabidopsis thaliana genome encodes six RDR genes, AtRDRl to AtRDR6, while for example the Oryza sativa genome contains 5 RDR genes, including OsRDRl to OsRDR4, as well as OsSHL2 which is clustered with RDR6. A certain species may also comprise several RDR gene copies of the same cluster, for example soybean (Glycine max), which has two RDRl genes and two RDR2 genes, or sorghum (Sorghum bicolor), which has four RDR6 gene copies in its genome.

AtRDR3, AtRDR4, and AtRDR5 are grouped together in cluster III, while AtRDRl is in cluster I, AtRDR2 in cluster II, and AtRDR6 in cluster IV. Orthologous RDR genes of other species are grouped accordingly.

Plant genes belonging to the RDRl cluster (cluster I), the RDR2 cluster (cluster II), and the RDR6 cluster (cluster IV) can be identified by a highly conserved common DLDGD motif in the putative catalytic domain of the encoded protein, which distinguishes them from RDR genes of cluster III, which contain a common DFDGD motif. The RDR genes with a DLDGD motif are grouped together as the RDRa class, while the RDR genes with the DFDGD motif belong to the RDRy class. Next to the DLDGD motif, RDRa class genes share several other common conserved motifs, by which they can easily be grouped to the RDRa class. The further categorization into cluster I, II, or IV is more difficult because in spite of the common motifs a rather broad range of sequences is represented in the RDR genes, even if only RDRa genes are considered. The RDR gene of the invention was however categorized as an RDRl gene.

The present invention thus provides a modified RDRl gene capable of conferring resistance to a plant and/or increasing virus resistance in a plant, which modification results in enhanced expression of the RDRl gene, and wherein the modification is selected from a modification that increases the mRNA level of the RDRl gene; and/or a modification that increases the level of the RDRl protein; and/or a modification that increases the activity of the RDRl protein. As used herein, the increase in a certain level or the increase in activity is as compared to a non-modified wild-type RDRl gene.

To obtain a relevant effect, the increase in mRNA level is an increase in steady state mRNA level. The increase in steady state mRNA level can be caused by an increase in the mRNA synthesis; and/or a decrease in the breakdown of the mRNA; and/or an increase in the stability of the mRNA. The mRNA level as mentioned herein comprises the mRNA accumulation level.

Surprisingly, the enhanced expression of the gene resulted in virus resistance and/or increased virus resistance of a plant comprising the modified gene. The mechanism through which genes confer resistance against pathogens is frequently a mechanism in which the resistance gene is silenced or reduced in expression. Examples of resistance genes that work through reduction of their expression are an elf4E gene, a DMR6 gene, a homoserine kinase gene, MLO genes, and others. It was therefore unexpected that in the present invention the activity of the RDRl gene was actually enhanced, and thereby resistance against viruses induced and/or increased.

In one embodiment the modification that increases the mRNA level of the RDRl gene and/or the level of the RDRl protein and/or the activity of the RDRl protein comprises a modification upstream of the coding sequence of the RDRl gene and/or a modification in the coding sequence of the RDRl gene and/or a modification downstream of the coding sequence of the RDRl gene.

Enhancing the expression of an RDRl gene can be achieved in various ways. One option is modification of the region upstream of the start of the gene coding sequence, which region comprises the promoter and the 5 '-untranslated region (5'-UTR), also called the leader sequence. Since these regions are involved in the regulation of the gene transcription to mRNA and the subsequent translation, and therefore in gene expression, suitable modification can lead to an increase of the expression through an increase of the mRNA level and/or an increase in the level of the protein.

Expression of the RDRl gene can likewise be boosted by creation of a

modification in the coding sequence (CDS) of the RDRl gene, whereby the CDS is defined as comprising the whole region between the 5'-UTR and the 3'-UTR, including exons as well as introns. Polymorphisms in the CDS resulting in allelic variants of the wild-type RDRl gene are potentially able to cause an enhancement in the expression of the gene. Combinations of individual modifications in promoter, leader, and/or CDS can further enhance the expression and/or activity of the RDRl gene.

In one embodiment the modification that increases the mRNA level of the RDRl gene and/or the level of the RDRl protein and/or the activity of the RDRl protein is a modification of a cis-acting regulatory element and/or the modification of a trans-acting regulatory element.

Regulatory elements are essential for the transcription of the gene to mRNA, and regulate both the spatial and temporal rate of transcription, thereby affecting the expression of a gene.

Cis-acting regulatory elements, also shortened as cis-acting elements or cis- regulatory elements, are usually located in the vicinity of the actual gene sequence that is to be transcribed, and are for example commonly present in the promoter sequence and the leader sequence of a gene, although they may also be located at a great distance either upstream or downstream from the transcription start of the gene. In addition, also the 3'-UTR region which immediately follows the CDS can harbour cis-acting elements, which are often, although not solely, involved in the regulation of post-transcriptional gene expression. Cis-regulatory elements include binding sites for transcription factors ('TF binding sites'), both for transcriptional repressors as well as activators, and microRNA target sites. Trans-acting regulatory elements, also called trans-acting factors, include transcription factors and small RNA's, for example microRNA's. Transcription factors are proteins that bind to specific binding sites, i.e. their corresponding cis -regulatory elements, on the DNA and thereby control the transcription of a gene. Transcription factors perform this function alone or with other proteins in a complex by promoting, as a transcriptional activator, or blocking, as a transcriptional repressor, the recruitment of RNA polymerase to specific genes.

MicroRNA's are small non-coding RNA molecules which function in transcriptional and post-transcriptional regulation of gene expression via base-pairing with complementary sequences within their rriRNA target molecules and inducing gene repression through for example degradation of their target transcripts or prevention of translation.

MicroRNA's form an important class of small RNA's; siRNA's are another class of small RNA's. Usually small RNA's are generated by distinct genes that do not code for proteins, and are therefore also called non-coding RNA's. In a few cases small RNA's have been described that originate from intronic sequences of protein coding genes, and very rarely also from other elements.

Transcriptional repressors negatively influence transcription and reduce gene expression; transcriptional activators in turn have a positive effect on transcription and therefore enhance expression of the gene.

Modification of a transcriptional repressor or its cis-regulatory binding element, which modification results in reduction or absence of the repression, can result in increase of transcription and a higher level of mRNA. A modification that for example results in the deletion or partial deletion of a transcriptional repressor or its binding element is expected to have the effect of an increase in mRNA level. In addition, a modification that for example results in the disruption of the function of a transcription factor that acts as a transcriptional repressor is expected to have the effect of an increase in mRNA level.

Modification, for example duplication, of a transcriptional activator or its cis- regulatory binding element can likewise result in further increase of transcription. A modification leading to a higher rate of activation or more efficient activation could lead to a higher level of mRNA.

Modification of a small RNA, for example a microRNA, of the sequence encoding or generating the small RNA, or of its complementary target site(s) in the targeted gene, can correspondingly result in reduced repression of gene expression of the targeted gene, through an increase in a higher level of steady state mRNA.

The invention relates to an RDR1 gene comprising a modified regulatory element, wherein the regulatory element is selected from a transcription factor binding site for a transcriptional repressor, the modification of which leads to reduction or absence of transcriptional repression; and/or a transcription factor binding site for a transcriptional activator, the modification of which leads to induction or enhancement of transcription; and/or a microRNA binding site, whereby the modification leads to reduction or absence of gene repression; and/or a small RNA sequence, for example a microRNA sequence, whereby the modification leads to reduction or absence of gene repression.

In one embodiment the modification that increases the mRNA level of the RDRl gene and/or the level of the RDRl protein and/or the activity of the RDRl protein comprises the deletion or the partial deletion of a cis-acting element, preferably the deletion or the partial deletion of a transcription factor binding site for a transcriptional repressor.

In one embodiment the modification that increases the mRNA level of the RDRl gene and/or the level of the RDRl protein and/or the activity of the RDRl protein comprises a modification in the promoter or in the 5'-UTR region of the RDRl gene, preferably of a cis-acting element in the promoter or in the 5'-UTR region.

In one embodiment the modification in the RDRl gene is a modification in an exon of the 5'-UTR region of the RDRl gene, preferably a modification of a cis-acting element in an exon of the 5'-UTR region.

In a further preferred embodiment the modification comprises the deletion or the partial deletion of a transcription factor binding site for a transcriptional repressor in an exon, optionally in the 1 ^st exon, of the 5'-UTR region of an RDRl gene.

In one embodiment the modified RDRl gene is an endogenous RDRl gene.

During the research that led to the present invention, a Cucumis sativus plant comprising a mutation in an RDR gene was identified. The mutation resulted in enhanced expression, which was detected as a significant increase of the steady state mRNA level of the RDR gene. This particular RDR gene that was modified was categorized as belonging to the RDRa class, as it comprised the typical DLDGD motif, and in particular to the RDRl cluster. This gene is designated hereafter as CsRDRl_II. The wild-type CsRDRl_II sequence can be found in Figure 1 (SEQ ID No. 4 (DNA) and SEQ ID No. 5 (protein)).

Further research identified that the mutation in the CsRDRl_II gene was a 46 bp deletion at around 1000 bp upstream of the ATG start codon of the CDS of the CsRDRl_II gene. More specifically, the deletion was determined to be in the 5'-UTR, and particularly in the 1 ^st exon of the 5'-UTR region of the gene (Fig. 2, SEQ ID No. 6).

The region upstream of the CDS of a protein coding gene commonly comprises a large number of cis-regulatory elements. In some cases it can also harbour sequences potentially resulting in small RNA's, in particular microRNA's. Modification in this upstream region can therefore have an effect on the expression of the gene. The wild-type sequence of the CsRDRl_II gene comprises a number of sequences coding for cis-regulatory elements in the modified area, specifically in the 5'-UTR's 1 ^st exon, which were affected by the deletion. The identified cis-regulatory elements in the 5'-UTR include at least two short sequences coding for PY-boxes or pyrimidine boxes, which function as transcription factor binding sites. The deletion included part of one of the PY-boxes, leading to the functional absence of this particular PY-box. In addition, a transcription factor (TF) binding site with a WAAAG sequence, whereby W can be A or T, was located in the modified region and partly deleted (EXAMPLE 1). Optionally, other TF binding sites were also located in and affected by the modification.

Besides TF binding sites, the 5'-UTR region of the CsRDRl_II gene comprised a stretch of about 23 nt which was identified to potentially behave in a microRNA-like manner. Part of the sequence coding for this miRNA-like element was deleted, leaving the miRNA-like element to be non-functional (EXAMPLE 1).

MicroRNA's typically need a target mRNA sequence for pairing, which for plants is usually located in the CDS of a protein coding gene. It was remarkably determined that the 3'- end of the CDS of the Cucumis sativus CsRDRl_II gene itself comprises a plausible target sequence for the detected miRNA-like element, as indicated by the high level of sequence complementarity with the detected miRNA-like element in the 5'-UTR of the same gene.

Subsequent detailed research was performed, and it was very surprisingly and highly interestingly detected that other RDR1 genes in the Cucumis sativus genome comprised an almost fully complementary target sequence to the microRNA-like element from CsRDRl_II in the 3'-ends of their respective CDS's. (EXAMPLE 2, Fig. 3).

It was concluded that the deletion or partial deletion of a regulatory element upstream of the start codon, particularly in the 5'-UTR of the Cucumis sativus CsRDRl_II gene, resulted in an increase in the level of mRNA of the CsRDRl_II gene. The deletion or partial deletion renders the regulatory element non-functional. The non-functional regulatory element results in an increase of the mRNA level. The increase in mRNA leads to enhanced expression of the CsRDRlJI gene.

In one embodiment the invention relates to a modified RDR1 gene comprising a deletion in the 1 ^st exon of the 5'-UTR. In a preferred embodiment the deletion in the 1 ^st exon of the 5'-UTR of the RDR1 gene comprises the deletion or partial deletion of a cis-acting element, which cis-acting element is preferably a transcription factor binding site, more preferably a transcription repressor binding site, and/or the deletion or partial deletion of a miRNA or miRNA-like element.

In a certain embodiment the modified cis-acting element is a transcription factor binding site with SEQ ID No. 1 and/or SEQ ID No. 2, and the modified miRNA or miRNA-like element comprises SEQ ID No. 3 (Table 1). In a certain aspect the deletion in the exon of the 5'-UTR comprises SEQ ID No. 6, optionally in combination with the insertion of SEQ ID No. 7 (Table 1).

Table 1.

Table 1:

The first four nucleotides of SEQ ID No. 1 (bold) are complementary to the last four nucleotides of SEQ ID No. 6, which is deleted. The PY-box is therefore partly deleted.

SEQ ID No. 2 is complementary to the last five nucleotides of SEQ ID No. 6, which is deleted. This TF binding site is therefore completely deleted.

The first 14 nucleotides of SEQ ID No. 3 (bold) are the reverse complement of the first 14 nucleotides of SEQ ID No. 6. This miRNA-like element is therefore partly deleted.

In one embodiment the invention relates to a modified RDR1 gene, wherein the modification results in enhanced expression, and which modification comprises the deletion or partial deletion of a regulatory element, which regulatory element comprises SEQ ID No. 1 and/or SEQ ID No. 2 and/or SEQ ID No. 3.

In a particular embodiment the wild-type sequence of an RDR1 gene of the invention is represented by SEQ ID No. 4 (Cucumis sativus), SEQ ID Nos. 8 and 21 (Daucus carota), SEQ ID Nos. 9 and 20 {Solatium lycopersicum), SEQ ID Nos. 10 and 19 {Citrullus lanatus), SEQ ID Nos. 11 and 22 (Cucumis melo), SEQ ID Nos. 12 and 27 (Spinacia oleracea), SEQ ID Nos. 13 and 23 (Phaseolus vulgaris), SEQ ID Nos. 14 and 29 (Spinacia oleracea), SEQ ID Nos. 15 and 28 (Beta vulgaris), SEQ ID Nos. 16 and 24 (Lactuca sativa), SEQ ID Nos. 17 and 26 (Brassica oleracea), or SEQ ID Nos. 18 and 25 (Lactuca sativa). When two SEQ ID Nos. are indicated for a certain crop species, the first one refers to the sequence 2kb upstream of the start codon, including the promoter and the 5'-UTR region, and the second one refers to the CDS, including the start codon. The invention also relates to the use of a modified RDRl gene for conferring and/or increasing virus resistance to an RNA virus in a plant which comprises the modified RDRl gene, which resistance and/or increased resistance is caused by enhanced expression of the modified RDRl gene as compared to the expression of a non-modified wild-type RDRl gene. The modified RDRl gene is capable of conferring or increasing virus resistance to a plant in which the modified RDRl gene is present, in particular to a plant selected from any of the species Phaseolus vulgaris, Beta vulgaris, Brassica oleracea, Daucus carota, Lactuca sativa, Cucumis melo, Cucumis sativus, Spinacia oleracea, Solanum lycopersicum, or Citrullus lanatus.

Enhanced expression of the modified RDRl gene in a plant is demonstrated as an increase in the mRNA level of the RDRl gene, and/or an increase in the level of RDRl protein, and/or an increase in the activity of the RDRl protein.

The invention further provides a plant comprising the modified RDRl gene of the invention, which plant shows resistance and/or increased resistance to virus due to enhanced expression of the modified RDRl gene when compared to a plant comprising the wild-type RDRl gene. The invention according to a further aspect thereof provides a plant wherein the modification of the RDRl gene comprises the deletion or partial deletion of a regulatory element upstream of the start codon of the gene, optionally in the 5'-UTR. More particularly the invention provides a plant wherein the modification of the RDRl gene comprises the deletion or partial deletion of a cis- acting element, which cis-acting element is preferably a transcription factor binding site, more preferably a transcription repressor binding site, and/or the deletion or partial deletion of a miRNA or miRNA -like element.

As used herein, showing and/or increasing resistance means that the presence of a modified RDRl gene leads to a level of virus resistance in a plant that has no resistance to a certain virus, and/or the presence of the modified RDRl gene increases the resistance in a plant that already has a level of resistance to a certain virus. The level of resistance is as compared to an isogenic plant that does not comprise the modified RDRl gene of the invention.

In a certain embodiment a plant of the invention comprises a modified RDRl gene wherein the modification is a modified transcription factor binding site comprising SEQ ID No. 1 and/or SEQ ID No. 2, and/or a modified miRNA or miRNA-like element comprising SEQ ID No. 3 (Table 1).

A plant of the invention is a plant in which an orthologous RDRl gene is suitably modified, which modification results in enhanced RDRl expression, for example a plant selected from any of the species Phaseolus vulgaris, Beta vulgaris, Brassica oleracea, Daucus carota, Lactuca sativa, Cucumis melo, Cucumis sativus, Spinacia oleracea, Solanum lycopersicum, Citrullus lanatus, or Oryza sativa. A plant comprising a modified RDRl gene of the invention, which modification leads to enhanced expression of the RDRl gene, shows virus resistance and/or has increase virus resistance. The increased expression of the RDRl gene is thought to lead to enhanced formation of dsRNA, which in turn might lead to a higher rate of siRNA production. The higher level of siRNA can subsequently be effectively used in the RISC, leading to increased activation and a higher rate of degradation of viral RNA. This system enables the establishment of broad virus resistance in different crop species due to increased RDRl gene expression.

Due to this mechanism, a virus against which enhanced RDRl gene expression gives and/or increases resistance to or in a plant belongs to the group of viruses with an RNA genome, preferably single strand RNA viruses which can have a single positive-strand RNA genome ((+)RNA viruses or (+)ssRNA viruses) or a single negative-strand RNA genome ((-)RNA viruses). Common plant virus families with (+)RNA genomes comprise Potyviridae, for example the genus Ipomovirus and the genus Potyvirus, and Virgaviridae, comprising the genus

Tobamovirus. Virus families with (-)RNA genomes include Bunyaviridae, for example the Tospovirus family. Presence of the modified RDRl gene, with enhanced expression, in a plant preferably confers and/or increases resistance against one or more (+)RNA viruses, in particular of the family Potyviridae and/or the family Virgaviridae.

The invention also relates to a seed comprising a modified RDRl gene of the invention, wherein the plant grown from the seed shows resistance and/or increased resistance to one or more RNA viruses, in particular to one or more RNA viruses of the family Potyviridae and/or the family Virgaviridae. A seed of the invention is preferably a seed of any of the species Phaseolus vulgaris, Beta vulgaris, Brassica oleracea, Daucus carota, Lactuca sativa, Cucumis melo, Cucumis sativus, Spinacia oleracea, Solanum lycopersicum, or Citrullus lanatus.

Virus species belonging to the RNA viruses that cause major problems by infecting a large number of cultivated crops are for example, but not limited to, Cucumber Vein Yellowing Virus (CVYV), Cucumber Mosaic Virus (CMV), Zucchini Yellow Mosaic Virus (ZYMV), Papaya Ringspot Virus (PRSV), Watermelon Mosaic Virus (WMV), Cucumber Green Mottle Mosaic Virus (CGMMV), Tobacco Mosaic Virus (TMV), Tomato Mosaic Virus (ToMV), Pepper Mild Mottle Virus (PMMoV), Pepper Mottle Virus (PepMoV), Potato Virus Y (PVY), Potato Virus X (PVX), Soybean Mosaic Virus (SMV), Maize Dwarf Mosaic Virus (MDMV), Tomato Torrado Virus (ToTV), Pepino Mosaic Virus (PepMV), Peanut Bud Necrosis Virus (PBNV), and Tomato Spotted Wilt Virus (TSWV).

A plant comprising a modified RDRl gene of the invention preferably shows resistance against CVYV, optionally in combination with increased resistance against CMV and/or CGMMV and/or ZYMV. Further research was performed to observe the effect of a modified RDR1 gene of the invention in cucumber ( Cucumis sativus). It was found that a cucumber plant comprising a modified CsRDRl_II gene leading to enhanced CsRDRl_II expression had virus resistance and/or increased virus resistance. Said cucumber plant particularly showed resistance and/or increased resistance to one or more RNA viruses, more specifically to one or more (+)ssRNA viruses. Said cucumber plant was observed to have CVYV resistance and optionally also showed an increase in CMV resistance and/or CGMMV resistance and/or ZYMV resistance. A plant, preferably a cucumber plant, comprising a modified CSRDR1_II gene of the invention preferably shows CVYV resistance, optionally in combination with an increase in CMV resistance (EXAMPLE 3).

The invention relates to a modified CsRDRl_II gene, which modification leads to enhanced expression of the CsRDRl_II gene, wherein the modified CsRDRl_II gene is capable of conferring and/or increasing resistance to CVYV and/or ZYMV and/or CMV and/or CGMMV when it is present in a Cucumis sativus plant. The modified CsRDRl_II gene is particularly capable to confer resistance against CVYV and to increase resistance against CMV and/or CGMMV and/or ZYMV in Cucumis sativus. A Cucumis sativus plant comprising a modified CsRDRl_II gene of the invention showing resistance against CVYV and increased resistance against CMV and/or CGMMV and/or ZYMV is part of the invention.

In a particular embodiment the modification in the CsRDRl_II gene is a deletion of SEQ ID No. 6 in the first exon of the 5'-UTR, optionally in combination with a 5 bp insertion of SEQ ID No. 7 (Table 1).

In a particular embodiment the modification in the CsRDRl_II gene is a modification that renders a regulatory element in the 5'-UTR non-functional, in particular a regulatory element comprising SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3, or a combination thereof. Non-functionality of such regulatory element can be achieved by a mutation or a deletion or a partial deletion of one or more sequences according to said SEQ ID Nos.

The invention further relates to use of a modified CsRDRl_II gene, which modification leads to enhanced expression of the CsRDRl_II gene, for conferring and/or increasing virus resistance to or in a Cucumis sativus plant, preferably conferring and/or increasing virus resistance against CVYV and/or CMV and/or CGMMV and/or ZYMV, most preferably conferring virus resistance against CVYV, optionally in combination with increased resistance against CMV and/or CGMMV and/or ZYMV.

Also part of this invention are plants, in particular Cucumis sativus plants, that comprise a modified CsRDRl_II gene as disclosed herein.

The types of modification leading to enhanced RDR1 gene expression result in virus resistance and/or increased virus resistance once the gene is present in a plant. A modified RDR1 gene of the invention can be introgressed from a plant comprising the modified RDR1 gene into a plant lacking the modified RDRl gene, using crossing when the plants are sexually compatible, optionally combined with techniques that aid the development of viable seeds or facilitate development into a plant. In a particular event a modified CsRDRl_II gene can be introgressed from a Cucumis sativus plant comprising the modified CsRDRl_II gene into a Cucumis sativus plant lacking the modified CsRDRl_II gene using standard breeding methods.

Crossing can optionally be followed by embryo rescue techniques or other techniques that result in a successful combination and introgression, which techniques are known to the person skilled in the art.

Identification of RDRl orthologues can technically be performed in many crops, methods for which are known in the art. In the present research a Blast programme was used to compare the CsRDRl_II sequence against sequences of other plant genomes. The best hits were identified as candidate RDRl genes. After this, the closest hits were further selected inter alia based on common motifs (Example 5). The closest hits are considered to be functional orthologues of the CsRDRl_II gene, which was found to convey and/or increase resistance when expression is enhanced. Based on the same mechanism, enhanced expression of a functional equivalent RDRl gene in a plant species leads to virus resistance and/or increased virus resistance in said crop.

In one embodiment a modified RDRl gene with enhanced expression is a modified RDRl gene orthologous to CsRDRl_II, examples of which orthologues are presented in Figure 4 and Figure 5 for the species Phaseolus vulgaris, Beta vulgaris, Brassica oleracea, Daucus carota, Lactuca sativa, Cucumis melo, Cucumis sativus, Spinacia oleracea, Solanum lycopersicum, Citrullus lanatus, Oryza sativa, and Arabidopsis thaliana.

In one embodiment the invention relates to a method for producing a plant that shows resistance and/or has increased resistance against one or more RNA viruses, in particular against one or more RNA viruses of the family Potyviridae and/or the family Virgaviridae, comprising modifying an RDRl gene whereby the modification leads to enhanced expression, in particular modifying an RDRl gene represented by SEQ ID No. 4 (Cucumis sativus), SEQ ID Nos. 8 and 21 (Daucus carota), SEQ ID Nos. 9 and 20 (Solanum lycopersicum), SEQ ID Nos. 10 and 19 (Citrullus lanatus), SEQ ID Nos. 11 and 22 (Cucumis melo), SEQ ID Nos. 12 and 27 (Spinacia oleracea), SEQ ID Nos. 13 and 23 (Phaseolus vulgaris), SEQ ID Nos. 14 and 29

(Spinacia oleracea), SEQ ID Nos. 15 and 28 (Beta vulgaris), SEQ ID Nos. 16 and 24 (Lactuca sativa), SEQ ID Nos. 17 and 26 (Brassica oleracea), or SEQ ID Nos. 18 and 25 (Lactuca sativa).

As mentioned earlier, orthologues of RDRl genes have been identified and grouped already in various crops. The present research has specifically identified RDRl genes that most closely resemble the CsRDRl_II sequence by comparing common protein motifs. Each orthologous RDRl gene harbours regulatory elements, in particular cis-regulatory elements, upstream of its start codon, in the promoter and/or 5'-UTR sequence, in the CDS, and/or in the 3'- UTR. Sequence motifs of regulatory elements are known to or can be recognized by the skilled person and can be identified using techniques known to the skilled person. Suitable modification of a regulatory element leads to enhanced expression of the gene, and such orthologous modified RDR1 genes are also genes of the invention.

Suitable modification comprises modification of a transcription factor (TF) binding site for a transcriptional repressor, which modification leads to reduction or absence of transcriptional repression; modification of a transcription factor binding site for a transcriptional activator, which modification leads to induction or enhancement of transcription; modification of a microRNA binding site, which modification leads to reduction or absence of gene repression; and or modification of a small RNA sequence, in particular a microRNA sequence, which modification leads to reduction or absence of gene repression.

Modification comprising the at least partial deletion or alteration of a TF binding site, a microRNA binding site, or a microRNA sequence will commonly result in non-functionality of said regulatory element. When such element is involved in gene repression, non-functionality will reduce repression and lead to enhanced expression of the gene as compared to a gene having a functional regulatory element.

In one embodiment the modification is introduced into an endogenous RDR1 gene that is orthologous to CsRDRl_II by means of mutagenesis. Mutagenesis comprises the random introduction of at least one modification by means of one or more chemical compounds, such as ethyl methanesulphonate, nitrosomethylurea, hydroxylamine, proflavine, N-methyl-N- nitrosoguanidine, N-ethyl-N-nitrosourea, N-methyl-N-nitro-nitrosoguanidine, diethyl sulphate, ethylene imine, sodium azide, formaline, urethane, phenol and ethylene oxide, and/or by physical means, such as UV -irradiation, fast-neutron exposure, X-rays, gamma irradiation, and/or by insertion of genetic elements, such as transposons, T-DNA, retroviral elements.

Mutagenesis also comprises the more specific, targeted introduction of at least one modification by means of homologous recombination, oligonucleotide -based mutation induction, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems.

A modified RDR1 gene of the invention can alternatively be introduced into a plant using genetic modification. Genetic modification comprises transgenic modification or transgenesis, using a gene from a non-crossable species or a synthetic gene, and cisgenic modification or cisgenesis, using a natural gene, coding for an (agricultural) trait, from the crop plant itself or from a sexually compatible donor plant.

In one embodiment the modified RDR1 gene is an exogenous RDR1 gene which can be introduced into a plant by a transgenic method or a cisgenic method. The invention also relates to a modified recombinant RDRl gene, wherein the expression of said modified recombinant RDRl gene is driven by a strong promoter, which promoter is operably linked to an RDRl gene sequence, which gene sequence includes the 5'-UTR, the CDS, and/or the 3'-UTR. Many examples of strong constitutive promoters are known in the art; some of the most commonly used ones are e.g. the cauliflower mosaic virus 35S-promoter

(pCaMV 35S) and modified versions thereof, ubiquitin promoters from various plant species, actin promoters from various plant species, and the promoter of Elongation Factor 1 alpha (EIF1 ).

In one embodiment the invention relates to a gene construct, which gene construct comprises a selectable marker, a promoter sequence, an RDRl gene sequence, and a terminator sequence.

The present invention establishes that enhanced activity of an RDRl gene leads to and/or increases virus resistance of a plant, preferably to one or more RNA viruses, more preferably to one or more (+)ssRNA viruses, even more preferably to one or more viruses belonging to the family Potyviridae and/or Virgaviridae.

In one embodiment modified, in particular enhanced, RDRl gene expression leads to and/or increases resistance against CVYV and/or CMV and/or CGMMV and/or ZYMV in a plant belonging to the genus Cucumis, in particular to Cucumis sativus and/or Cucumis melo. In a preferred embodiment modified, in particular enhanced, RDRl gene expression leads to resistance against CVYV and increases resistance against CMV and/or CGMMV and/or ZYMV in a plant belonging to the genus Cucumis, in particular to Cucumis sativus and/or Cucumis melo. In a most preferred embodiment modified, in particular enhanced, RDRl gene expression leads to resistance against CVYV and increases resistance against CMV in a plant belonging to the genus Cucumis, in particular to Cucumis sativus.

Enhanced activity or upregulation of the gene can be obtained in many ways. A promoter or 5'-UTR region of an RDRl gene, which promoter or 5'-UTR is modified in such a way that transcription is increased, can be combined with a non-modified, optionally endogenous, RDRl gene sequence.

RDRl gene expression can also be upregulated by means of inducing overexpression, for example using a construct comprising a strong promoter enhancing gene expression in combination with the cDNA sequence of the RDR gene (EXAMPLE 4).

In a further embodiment, any of the transgenic approaches aimed at overexpressing an RDRl gene may involve the use of inducible promoter sequences. In this approach external factors or treatments can be used to induce and/or modulate the temporal and/or spatial expression pattern of a transgene, by virtue of the effect these external factors or treatments have on the activity of the promoter sequence to which the transgenic nucleic acid sequence is operably linked. The inducible promoter may be selected from the group comprising heat -inducible promoters, chemical-inducible promoters, steroid-inducible promoters, alcohol-inducible promoters or others. Treatment of transgenic plants with the agent(s) and/or condition(s) that induce(s) the activity of the inducible promoter (such as a heat shock or heat treatment, specific chemicals, specific steroids such as dexamethasone, or alcohol) activates the inducible promoter, which results in the transient (over)expression of the transgene.

Transgenic approaches for incorporating the modified RDRl gene including the promoter and the 5'-UTR in other plant species, or using the CDS of an RDRl gene in combination with another strong promoter and/or leader sequence which enhance gene expression, can lead to enhanced RDRl expression or overexpression in a recipient plant. An enhanced or overexpressed RDRl gene incorporated in a plant can lead to and/or increase virus resistance of that plant.

Apart from enhancing the transcriptional activity of an RDRl gene, the same effect may be achieved by duplication of the RDRl locus in the genome. In such a case highly similar transcripts are being transcribed from two or more separate loci, which may lead to a higher steady-state level of the mRNA and of the encoded protein(s).

FIGURES

Figure 1 - Cucumis sativus CsRDRl _II DNA (SEQ ID No. 4, including 2kb upstream) and protein sequence (SEQ ID No. 5)

Figure 2 - deleted sequence in the 5'-UTR of the modified CsRDRl _II gene. The

46 bp sequence ctttccatggccataacaccaaatattctctattgaatgtaatttc (SEQ ID No. 6) is present in the 5'- UTR of the susceptible ("vatbaar") plant, but is deleted in the resistant plant. In addition, an insertion is present of a much shorter 5 bp sequence: acctg (SEQ ID No. 7).

Figure 3 - Sequence of the microRNA-like element of CsRDRl _II (Table 1, SEQ ID No. 3) and alignment with potential target sequences at the 3' ends of the CDS's of CsRDRl _II and two other RDRl genes of Cucumis sativus. (SEQ ID Nos. 52-55).

Figure 4 - DNA sequences of CsRDRl _II orthologues in Phaseolus vulgaris, Beta vulgaris, Brassica oleracea, Daucus carota, Lactuca sativa, Cucumis melo, Cucumis sativus, Spinacia oleracea, Solanum lycopersicum, Citrullus lanatus, Oryza sativa, Arabidopsis thaliana. Figure 4a gives the sequences 2kb upstream of the start codon (SEQ ID Nos 8-18), Figure 4b gives these sequences aligned. Figure 4c gives the gene sequences starting from ATG (SEQ ID Nos 19-29), Figure 4d gives these sequences aligned. The overview below indicates which SEQ ID Nos are linked to which species in the aligned sequences in Figures 4b, 4d and 5b. Species SEQ ID No. SEQ ID No. SEQ ID No.

in Fig 4b in Fig. 4d in Fig. 5b

Bean_pv_218_vl_evml9451 30

Bean_pv_218_vl_evml 9448 * 13 23 31

Carrot_dc_DHl_v2_evm53328* 8 21 32

Watermelon_cl97102vl_evml0088 33

Watermelon_cl97102vl_evml0073 34

Cucumber_cs9930v2_emv_l 5008 35

Cucumber_cs9930v2_emv_14972 36

Melon cm MR1 v3 evm gene21282 37

Melon cm MR1 v3 evm gene38010 38

Cucumber_cs9930v2_emv_l 4138 *

Watermelon_cl97102vl_evm32343_evm32342* 10 19 39

Tomato_Solyc05g007510.2.1 * 9 20 40

Lettuce_Lsa022576.1* 16 24 41

Lettuce_Lsa032017.1* 18 25 42

Spinach_so_virovlay_vl_EVM2_26836_26835* 14 29 43

Beet_bv_KWS2320_vl .2_EVM3286* 15 28 44

Spinach_so_virovlay_vl_EVM2_25439* 12 27 45

Watermelon_cl97102vl_evm33604 46

Cucumber_cs9930v2_emv_l 4137 47

Melon_EVM_2019* 11 22 48

Rice_RDR 1 _ORYS J 49

Broccoli_bo_blat_vl_EVM18712* 17 26 50

Arabidopsis_RDR 1 _AR ATH 51

Figure 5 - Protein sequences (Figure 5a) and aligned protein sequences (Figure 5b) of CsRDRl _II orthologues in Phaseolus vulgaris, Beta vulgaris, Brassica oleracea, Daucus carota, Lactuca sativa, Cucumis melo, Cucumis sativus, Spinacia oleracea, Solanum lycopersicum, Citrullus lanatus, Oryza sativa, Arabidopsis thaliana. (SEQ ID Nos 30-51).

Figure 6 - Phylogenetic tree of CsRDRl _II and its identified orthologous genes. CsRDRl _II is presented as Cucumber _cs9930v2_emv_14138. Per species, the gene most similar to CsRDRl _II is indicated with *. The other genes are slightly more distant, but still contain the same motifs. Figure 7 - Relative expression report of the upregulation of the CsRDRl _II gene presented as an increase in the mRNA level. In Figure 7a the results are given, whereby P(H1) is the Probability of the alternate hypothesis that difference between sample and control groups is due only to chance. TRG is the Target, REF is the Reference.

The graph of Figure 7b is a Boxplot, wherein the box represents the interquartile range, or the middle 50% of observations. The dotted line represents the median gene expression, whiskers represent minimum and maximum observations.

Figure 7c gives the non-normalised results, which do not have expression values normalised to the selected housekeepers.

EXAMPLES EXAMPLE 1

RDR1 modification in Cucumis sativus

In the genome of Cucumis sativus two adjacent RDR1 genes were identified on chromosome 5, which RDR1 genes were inversely oriented. A population of Cucumis sativus plants were genetically analysed and the RDR1 genes on chromosome 5 were resequenced.

Resequencing showed that one of the RDR1 genes, indicated with CsRDRl _I, was monomorphic in the whole population. The other RDR1 gene however, indicated with CsRDRl -II, was monomorphic in the coding region, but was polymorphic in the region upstream of the ATG start codon, containing the promoter and the 5'-UTR. The Cucumis sativus CsRDRl _II gene and protein sequence are presented in Fig. 1.

The detected polymorphism was a deletion of 46 bp at about 1000 bp upstream of the ATG codon, in the region which defines the 5'-UTR. Instead of this stretch, a short 5bp insertion was present at this location (Fig. 2). To determine if the deletion had an effect on gene expression, the steady state level of the CsRDRl _II mRNA was determined. The analysis showed an approximate 100-fold higher level of steady state CsRDRl _II mRNA in Cucumis sativus plants that had the deletion as compared to the mRNA level in plants that had the wild-type CsRDRl _II gene (Fig. 7).

It was assumed that the deletion in the region upstream of the CDS has disrupted the functionality of a repressor of transcription initiation, which disruption leads to higher accumulation levels of the mRNA.

EXAMPLE 2

Detection of regulatory elements

Further study of the region in which the deletion has occurred indeed showed the presence of several cis -regulatory elements. As a consequence of the 46 bp deletion, one of these elements, identified as a pyrimidine box or PY-box, was partly deleted. The partial deletion renders the PY-box non-functional. The PY-box is a TF binding site that can be involved in repression of mRNA transcription. The non-functionality of the PY-box then results in a higher level of steady state mRNA by reducing gene repression (Fig. 2).

Additional PY-boxes are present in the region upstream of the gene's start codon. It is highly feasible that an additional modification in one of the other PY-boxes could result in even higher mRNA levels. Alternatively, a modification in other cis-regulatory elements of a wild- type RDR1 gene, such as the other PY-boxes, might result in the same effect as observed in the present research.

In addition, detailed study showed the possible presence of another regulatory element in the modified region upstream of the CDS of the CsRDRl_II gene. A 23 nt stretch was identified which could possible act as a microRNA. This element, herein called "microRNA-like", appeared to have a near-complementary reverse target sequence at the 3 'end of the CDS of the CsRDRlJI gene (Table 1, SEQ ID No. 3; Fig. 3).

The Cucumis sativus genome further contains at least two other RDR1 genes with a DLDGD motif. Both these genes surprisingly showed a highly complementary 23 nt stretch in the 3 'end of their CDS regions, indicating the element of the CsRDRl_II gene to be a very likely candidate for a microRNA (Fig. 3).

The 46 bp deletion in the modified CsRDRl_II gene comprised part of this microRNA-like element. Partial deletions usually render an element non-functional. The present element has the capability to act in a cis-regulatory and/or a trans -regulatory manner. Non- functionality of such a cis- and/or trans-acting microRNA has an effect on the mRNA level of the CsRDRl_II gene and/or other targeted genes.

A third regulatory element that was positioned into the modified region is a transcription factor (TF) binding site with a conserved WAAAG core motif, whereby W can be either A or T. Transcription factors binding to this site belong to the C2C2-Dof protein family, which are known to participate in the regulation of gene expression in several processes, including plant defense mechanisms. The identified 46 bp deletion includes part of this binding site, which leaves this TF binding site inactive, likely influencing the transcription and subsequent expression of the gene.

EXAMPLE 3

Virus resistance in relation to modified CsRDRl_II

Cucumis sativus plants that contained two wild-type RDR1 genes as described in Example 1, designated CsRDRl_I and CsRDRl_II, were compared for virus resistance to plants containing at least a modified CsRDRl_II gene. The modification is the deletion as described in the above Examples.

A PCR assay was designed to be able to discriminate between the two CsRDRl_II alleles in a population of plants. Plants containing the wild-type CsRDRl_II gene and the modified CsRDRlJI allele were screened for resistance against CVYV, CMV, CGMMV, and ZYMV.

There was a perfect correlation between the presence of the modified CsRDRl_II allele, and consequently the enhanced expression of the gene, and resistance against CVYV in cucumber. In addition, the presence of the modified CsRDRl_II allele influenced the resistance against CMV and/or CGMMV and/or ZYMV. For these viruses, the modified CsRDRlJI allele, and therefore enhanced expression of the gene, contributes to the level of resistance. In the actual presence and level of resistance against CMV, ZYMV, and/or CGMMV other resistance genes are involved, but once those other genes are present, the combination with enhanced CsRDRl_II gene expression leads to an increased or enhanced level of resistance. EXAMPLE 4

Transgenic overexpression of an RDR1 gene results in CVYV resistance in melon

An overexpression construct for an orthologous RDR1 gene of melon (Fig. 4) is designed, comprising the full cDNA of the RDR1 gene, operably linked to the cauliflower mosaic virus (CaMV) 35S promoter, and to the nos terminator. The cDNA sequence (including the 5'UTR) had been obtained by RT-PCR from a cDNA library of melon leaves. The construct further comprises the ΝΡΤΠ gene (conferring kanamycin resistance) as a plant selection marker (under control of the NOS promoter).

This construct is subsequently introduced into the genome of a melon line that is susceptible to CVYV, using the improved transformation protocol for Cantaloupe melons developed by Guis et al., 2000 (Sci. Hort. 8: 91-99). Ten independent TO melon plants harbouring at least one copy of the transgenic construct are selected, and these plants are grown in a growth cabinet alongside a set of untransformed plants of the same genetic background. All plants are inoculated with the CVYV virus, and subsequently monitored for symptoms.

In the untransformed control plants a distinct vein yellowing is observed, whereas the transformed lines show no symptoms or only minor symptoms. The expression level of the

RDR1 orthologue is investigated by means of semi -quantitative RT-PCR, and this reveals that the symptom-free transgenic lines have the highest expression level of the RDR1 gene, whereas the transgenic lines with minor symptoms have expression levels that are intermediate between the wildtype level and the level in the symptom-free transgenic lines. This confirms that a good correlation exists between the elevated expression level of the RDR1 gene in melon on the one hand, and the level of resistance to the CVYV virus on the other hand. EXAMPLE 5

Identification of CsRDRl _II orthologues

RDR1 genes are known to be represented in a wide variety of crop species.

Orthologues of the CsRDRl _II gene were identified by using a nucleotide Blasting programme (BLASTN) to compare the CsRDRl _II sequence with the sequences of other crop species. The protein annotation programme UniProt was subsequently used to functionally predict the best hits, which resulted in 5-6 candidate genes for each crop. These genes were selected for further analysis. The presence of several motifs that are commonly conserved in RDR1 proteins, as well as a specific focus on similarity at the start and end regions of the gene sequences, was used to further specify the most similar genes. DNA and protein sequences of the orthologues that were identified through this method are represented in Figure 4 and Figure 5.

The protein motifs that were identified in CsRDRl _II and in its orthologues as mentioned in Figure 5 are the following: CSGS, DLDGD, AVDF(PA)KTG, ASAWY,

A(FY)QIRY. The most similar orthologous gene was found in watermelon (Citrullus lanatus). The relationship and therefore the similarity between the various orthologues is represented as a phylogenetic tree, made with the programme Clustal, in Figure 6.