WO2015144692A1 - Expression decrease of the gene coding for the ephrin receptor in aphids inhibits the transmission of the turnip yellow virus - Google Patents

Abstract

The invention relates to a method for preventing or reducing Luteoviridae family virus transmission between plants by aphids comprising silencing the ephrin receptor protein- A7 (EphA7) or one of its homo logs in said aphids.

Description

EXPRESSION DECREASE OF THE GENE CODING FOR THE EPHRIN RECEPTOR IN APHIDS INHIBITS THE TRANSMISSION OF THE TURNIP

YELLOW VIRUS

Viruses are obligate parasites that use the host cell machinery to produce their progeny. Consequently, viruses have to adapt the different steps of their infection cycle (e.g. translation of proteins, genome replication, cell-to-cell movement, and host-to-host transmission) to the characteristics of their hosts. Plant viruses have to deal with plant-specific features that restrict virus transmission to other plant hosts, such as:

the immobility of plants, leading to limited possibilities for virus transfer among hosts via direct plant-plant contact;

a solid and virus-impermeable wall made of cellulose, hemicellulose, and pectin that surrounds all cells and limits both entry and exit of viruses.

Viruses have evolved different strategies to circumvent this cell- wall barrier. According to current knowledge on viral transmission, 6% of the described plant viral species are transmitted directly through seeds, and there are some rare reports of virus transmission through pollen. However, plant viruses most often use external "tool(s)" to actively break the cell wall, thus allowing escape of virus particles. For some viruses, mechanically injured tissues from infected plants can be transferred to non-infected plant by human or animal activity, or winds. Although this strategy appears to be very efficient for some virus species (e.g. Potato virus X and Tobacco mosaic virus), it is not a common means of transmission for the vast majority of plant viruses. Indeed, most plant viruses use a partner, known as a "vector", for efficient transmission to new hosts. A vector is a mobile organism able to gain access to the plant cell cytoplasm by breaking through the cell wall and membrane using their feeding organs, and that moves frequently among plants. Root nematodes, parasitic fungi and plant-feeding arthropods, particularly insects, have been described as efficient vectors in plant-to-plant transmission of viruses.

Aphids have long been the subject of intense research because:

- they cause large economic losses;

they have adopted a complex life cycle with alternating asexual and sexual phases; - they show remarkable phenotypic plasticity.

Another important aspect is the fact that aphids are certainly by far the most frequent and efficient vectors of plant viruses. They transmit hundreds of plant pathogens, mostly viruses. Aphid's mouthparts are remarkably adapted to their feeding habits, and the thin and flexible stylet bundle is perfectly able to pass intercellularly without damaging plant tissues. Prior to feeding from the phloem of a host plant, aphids have the extraordinary capacity to sample cell content during brief intracellular probes into epidermal and/or mesophyll cells without killing them. Once the plant has been sampled and accepted by the aphid, the stylets are introduced deeply under the epidermis until they reach the sieve elements. During the steps of the feeding process, aphids produce different salivas with different compositions and functions. Remarkably, aphids can acquire viruses at all steps of this feeding process, both in superficial and in deeper tissues. Thus, aphids are organisms that can acquire and inoculate any viral taxon within plants, whatever their tissue specificity.

Depending on the aphid species and viruses studied, scientists observed the existence of different patterns of aphid- virus interactions:

- the non-persistent mode, with viruses acquired within seconds and retained for only a few minutes by their vectors (non-circulative group);

- the semi-persistent mode, with viruses acquired within minutes to hours and retained for several hours (non-circulative group);

- the persistent mode, with viruses that require minutes to hours for acquisition and that can be retained for very long periods, often until the vector dies (circulative). Circulative non-propagative transmission seems to be restricted to plant viruses. The virions cross the gut epithelium to be released into the hemo lymph, and then to the salivary gland cells to be secreted in the salivary ducts. The specificity of the non-propagative mode is that, except for gut and accessory salivary gland (ASG) cells, no other cellular types will contain virions, and no replication will ever occur in any of the virion-harboring cells. Plant viruses known or believed to exploit this mechanism of aphid transmission are members of the Nanoviridae (referred to as nanoviruses) and Luteoviridae (referred to as luteovirids) families. These viruses are phloem- limited, which makes phloem- feeding aphids suitable vectors. Nanoviruses consist of 6-8 small icosahedral particles that each contains a circular single- stranded DNA molecule. Luteovirids also have icosahedral particles but their genome is a single- stranded monopartite RNA. Although they share a similar mode of transmission, luteovirids and nanoviruses rely on different viral determinants for virus internalization in the vector: for luteovirids the capsid protein is required for efficient virus transport, whereas an additional HC (helper component) is suspected to be required for nanovirus transmission. Intestinal endocytosis and exocytosis of luteovirids rely on a sequential mechanism, starting with clathrin-mediated endocytosis, followed by transport of virions enclosed in vesicles from the apical to the basal pole of the cell, and ending with fusion of virus-containing vesicles to the basal plasmalemma. Virions are always enclosed in vesicles, this suggests that, except for membrane components, no direct contact with aphid compounds occurs.

The high vector specificity of luteovirid transmission suggests that viral determinants located on the surface of the particle determine its recognition by aphid receptors, uptake in the cells and release into the plant.

So far, luteovirid receptors have not been identified but several aphid proteins exhibiting the ability to bind purified luteovirid particles in vitro have been reported. They involve several proteins extracted from whole aphids {Myzus persicae), which have the capacity to bind Potato leafroll virus (PLRV) or Turnip yellows virus (TuYV), and other proteins extracted from the heads of Sitobion avenae, which display the ability to bind purified Barley yellow dwarf virus-MAV . Not all these proteins were identified by mass spectrometry and their involvement in the aphid transmission process was reported for only two of them: symbionin which is produced by the aphid endosymbiont, Buchnera spp., and SaM50, which is localized in the ASG cells.

Transmission by a given aphid species can be shared between several luteovirids, as is the case for M. persicae, which can transmit at least 7 of the 20 viruses listed in the Luteoviridae family. Electron microscopy observations, which showed that gut tropism can vary between luteovirids in similar aphid species, together with genetic analyses, suggest that transmission of closely related virus species can be controlled by different genes within the same aphid species or genotype.

How aphids modulate host defenses and mediate the transmission of plant viruses has to be better understood because this is crucial in agricultural area, for example in producing cereals. The green peach aphid M. persicae can feed on over 40 different plant families and is capable of efficiently transmitting over 100 types of plant viruses.

The inventors investigated the involvement of an aphid protein in the transmission of a plant virus (Turnip yellows virus, Polerovirus, Luteoviridae) by developing a technique based on plant-mediated RNAi.

By screening a M. persicae cDNA library by the yeast two hybrid system using polerovirus capsid proteins as baits, EphA7 was found as a potential partner of the structural proteins of TuYV. They have shown that it is possible to down-regulate M. persicae EphA7 gene expression by feeding the aphids on transgenic plants expressing dsRNA targeting EphA7 mRNA.

EphA7-mRNA accumulation was reduced in M. persicae fed on transgenic Arabidopsis thaliana.

Silencing of EphA7 did not affect aphid fecundity neither virus acquisition from artificial medium.

Silencing of EphA7 reduced TuYV internalization in persicae and TuYV transmission by aphids. SUMMARY

The subject-matter of the present invention is thus a method for preventing or reducing Luteoviridae family virus transmission between plants by aphids comprising silencing the ephrin receptor protein- A7 (EphA7) in said aphids, wherein EphA7 is a protein encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or one of its homologs exhibiting a sequence which is at least 60%, preferably at least 70%>, and more preferably at least 80%, the most preferably at least 90% identical to the one of EphA7. Another subject-matter of the present invention is a vector comprising SEQ ID NO:5 which expresses said insert as inverted repeats.

Another subject-matter of the present invention is a plant or part of plant genetically engineered to express an isolated nucleic acid compound comprising at least a portion that hybridizes to an EphA7 transcript under physiological conditions and decreases the expression of EphA7 in a cell.

Another subject-matter of the present invention is an isolated ephrin receptor protein-A7 (EphA7) exhibiting a protein sequence encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or one of its isolated homologs exhibiting a sequence which is at least 60%>, preferably at least 70%>, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

Another subject-matter of the present invention is an isolated dsRNA with a hairpin structure arising from the transcription of two copies of SEQ ID NO:5 in sense and antisense separated by a non-coding sequence.

Another subject-matter of the present invention is an isolated dsRNA with a hairpin structure arising from the transcription of SEQ ID NO:4. Another subject-matter of the present invention is aphids genetically engineered to have: downregulation of the expression of ephrin receptor protein- A7 (EphA7) or one of its homologs, or

expression of non- functional ephrin receptor protein-A7 (EphA7) or one of its non- functional homologs, or

expression of an antagonist of ephrin receptor protein-A7 (EphA7) or of one of its homologs wherein EphA7 exhibits a protein sequence encoded by a mRNA selected in the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3 and one of its homologs exhibits a sequence which is at least 60%, preferably at least 70%, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

Another subject-matter of the present invention is an isolated antisense polynucleotide comprising at least a portion that hybridizes under physiological conditions to an EphA7 mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or one of its isolated homologs exhibiting a sequence which is at least 60%, preferably at least 70%, and more preferably at least 80%, the most preferably at least 90% identical to the one of EphA7, and decreases the expression of EphA7 in a aphid cell.

Another subject-matter of the present invention is a method for preventing or reducing Luteoviridae family virus internalization by aphids comprising silencing the ephrin receptor protein- A7 (EphA7) in said aphids, wherein EphA7 is a protein encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or one of its homologs exhibiting a sequence which is at least 60%, preferably at least 70%, and more preferably at least 80%, the most preferably at least 90% identical to the one of EphA7.

DEFINITIONS RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms. The process of RNAi was described as "post-transcriptional gene silencing" (PTGS) in plant systems.

With the RNAi method, double-stranded RNA (dsRNA) can specifically lower the transcript abundance of a target gene when injected into an organism or introduced into cultured cells. RNAi involves the cleavage of dsRNA precursors into small- interfering RNA (siRNA) of approximately 21 to 26 nucleotides by the enzyme Dicer. These siRNAs are then incorporated into a RNA-induced silencing complex (RISC). Argonaute proteins, the catalytic components of RISC, use the siRNA as a template to recognize and degrade the complementary messenger RNA (mRNA).

The term "Eph receptor" refers to a tyrosine kinase receptor which belongs to the Eph family of receptors.

As used herein, the terms Ephrin and Eph are used to refer, respectively, to ligands and receptors. They can be from any of a variety of animals (e.g., mammals/non-mammals, vertebrates/non-vertebrates). The nomenclature in this area has changed rapidly and the terminology used herein is that proposed as a result of work by the Eph Nomenclature Committee, which can be accessed, along with previously-used names at web site https://eph- nomenclature.med.harvard.edu/.

The work described herein, particularly in the examples, refers to EphrinA7 and EphA7.

The ephrins (ligands) are of two structural types, which can be further subdivided on the basis of sequence relationships and, functionally, on the basis of the preferential binding they exhibit for two corresponding receptor subgroups. Structurally, there are two types of ephrins: those which are membrane-anchored by a glycerophosphatidylinositol (GPI) linkage and those anchored through a transmembrane domain. Conventionally, the ligands are divided into the Ephrin-A subclass, which are GPI-linked proteins which bind preferentially to EphA receptors, and the Ephrin-B subclass, which are transmembrane proteins which generally bind preferentially to EphB receptors.

The Eph family receptors are a family of receptor protein-tyrosine kinases which are related to Eph, a receptor named for its expression in an erythropoietin- producing human hepatocellular carcinoma cell line. They are divided into two subgroups on the basis of the relatedness of their extracellular domain sequences and their ability to bind preferentially to Ephrin-A proteins or Ephrin-B proteins. Receptors which interact preferentially with Ephrin- A proteins are EphA receptors and those which interact preferentially with Ephrin-B proteins are EphB receptors. Eph receptors have an extracellular domain composed of the ligand- binding globular domain, a cysteine rich region followed by a pair of fibronectin type III repeats. The cytoplasmic domain consists of a juxtamembrane region containing two conserved tyrosine residues; a protein tyrosine kinase domain; a sterile alpha motif (SAM) and a PDZ-domain binding motif. Eph receptors are activated by binding of clustered, membrane attached ephrins, indicating that contact between cells expressing the receptors and cells expressing the ligands is required for Eph activation.

Upon ligand binding, an Eph receptor dimerizes and autophosphorylates the juxtamembrane tyrosine residues to acquire full activation. In addition to forward signaling through the Eph receptor, reverse signaling can occur through the ephrin- ligands. Eph engagement of ephrins results in rapid phosphorylation of the conserved intracellular tyrosines and somewhat slower recruitment of PDZ binding proteins.

The Eph family comprises structurally related transmembrane receptor tyrosine kinases, each having an extracellular region, a cytoplasmic region comprising a highly conserved tyrosine kinase domain flanked by a juxtamembrane region and a carboxyl-terminal tail, which are less conserved. The various variant forms with deletions, truncations, substitutions, or insertions are included in the present definition.

Eph receptors of the EphA group, designated "EphA receptors" herein, preferentially interact with glycosylphosphatidylinositol (GPI)-linked ligands (of the Ephrin-A subclass, which presently comprises five ligands).

The term "Eph ligand" herein refers to a polypeptide which binds to and, optionally, activates (e.g. stimulates tyrosine phosphorylation of) an Eph receptor.

"Homology" is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. One such computer program is "Align 2", authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991.

The term "receptor binding domain" is used herein to designate any region of an Eph ligand retaining at least a qualitative receptor binding ability of a corresponding native sequence Eph ligand. Amino acid sequence variants and/or derivatives of a native sequence receptor binding domain are encompassed in this definition.

The term "ligand binding domain" as used herein refers to any region of an Eph receptor retaining at least a qualitative ligand binding activity of a corresponding native sequence Eph receptor. Amino acid sequence variants and/or derivatives of a native sequence ligand binding domain are encompassed in this definition. The term "antagonist" when used herein refers to a molecule which is capable of inhibiting one or more of the biological activities of a target molecule, such as an Eph receptor. Antagonists may act by interfering with the binding of a receptor to a ligand and vice versa, by incapacitating or killing cells which have been activated by a ligand, and/or by interfering with receptor or ligand activation (e.g. tyrosine kinase activation) or signal transduction after ligand binding to a cellular receptor. The antagonist may completely block receptor-ligand interactions or may substantially reduce such interactions. All such points of intervention by an antagonist shall be considered equivalent for purposes of this invention. Thus, included within the scope of the invention are antagonists (e.g. neutralizing antibodies) that bind to Eph receptor, Eph ligand or a complex of an Eph receptor and Eph ligand; amino acid sequence variants or derivatives of an Eph receptor or Eph ligand which antagonize the interaction between an Eph receptor and Eph ligand; soluble Eph receptor or soluble Eph ligand, optionally fused to a heterologous molecule such as an immunoglobulin region (e.g. an immunoadhesin); a complex comprising an Eph receptor in association with Eph ligand; synthetic or native sequence peptides which bind to Eph receptor or Eph ligand; small molecule antagonists; and nucleic acid antagonists (e.g. antisense).

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. "Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding region or variable domains thereof. Examples of antibody fragments include Fab, Fab', F (ab') 2, Fv fragments and single-domain antibody (sdAb, Nanobody); diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature 256: 495 (1975), or may be made by recombinant DNA methods. The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in

Clackson et al, Nature 352: 624-628 (1991) and Marks et al, J. Mol. Biol. 222: 581-597 (1991), for example. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies [U. S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81 : 6851-6855 (1984)].

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "Complementarity Determining Region" or "CDR" i.e. residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 26-35 (HI), 50- 65 (H2) and 95-102 (H3) in the heavy chain variable domain; [Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)] and/or those residues from a "hypervariable loop" i.e. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53- 55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987); Saerens D, Conrath K, Govaert J, Muyldermans S (2008) Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains. Journal of Molecular Biology 377: 478-488. "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (1994). The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

The term "aptamer" means a DNA or RNA molecule binding a protein specifically and with a high affinity. This definition comprises natural aptamers and chemically modified analogs.

An "isolated" molecule is one which has been identified and separated and/or recovered from a component of its natural environment.

The terms "antisense oligodeoxynucleotide" and "antisense oligo" refer to a polynucleotide which hybridizes with an area of Eph receptor or Eph ligand mRNA or DNA and thereby prevents or reduces production of Eph receptor or Eph ligand polypeptide in vitro or in vivo. This term encompasses "modified" polynucleotides, examples of which are described herein. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of a gene: at least about 600 nucleotides of sequence upstream from the 5' end of the coding region and at least about 2500 nucleotides of sequence downstream from the 3' end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

As used herein, the term "orthologs" refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode proteins having the same or similar functions. The terms "polypeptide", "peptide" or "protein" are used interchangeable throughout this specification.

To determine the percentage of homology between two amino acid sequences (e.g., one of the sequences encoded by a nucleic acid of the invention and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = numbers of identical positions/total numbers of positions x 100). An "isolated" nucleic acid molecule is one molecule, which is substantially separated from other nucleic acid molecules, which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is substantially free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism, from which the nucleic acid is derived. The term "polynucleotide" as used in accordance with the present invention relates to a polynucleotide comprising a nucleic acid molecule which encodes a polypeptide having a biologically activity as specified below.

Moreover, the term "polynucleotide" as used in accordance with the present invention further encompasses variants of the aforementioned specific polynucleotides. Said variants may represent orthologs, paralogs or other homologs of the polynucleotide of the present invention. The polynucleotide variants, preferably, also comprise a nucleic acid sequence characterized in that the sequence can be derived by at least one nucleotide substitution, addition and/or deletion whereby the variant nucleic acid sequence shall still encode a polypeptide having a biological activity as specified above. Variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific nucleic acid sequences, preferably, under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization conditions are hybridization conditions in 6 x sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2 x SSC, 0.1 % SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under "standard hybridization conditions" the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA:DNA hybrids are, preferably, 0.1 x SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA: RNA hybrids are, preferably, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et al, "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants are obtainable by PCR-based techniques such as mixed oligonucleotide primer- based amplification of DNA, i.e. using degenerated primers against conserved domains of the polypeptides of the present invention. A fragment as meant herein, preferably, comprises at least 20, at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of any one of the aforementioned nucleic acid sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any one of the aforementioned amino acid sequences. Variant polynucleotides as referred to in accordance with the present invention may be obtained by various natural as well as artificial sources. For example, polynucleotides may be obtained by in vitro and in vivo mutagenesis approaches using the above mentioned specific polynucleotides as a basis. Moreover, polynucleotide being homo logs or orthologs may be obtained from various species. Binding as meant in this context refers to hybridization by Watson-Crick base pairing discussed elsewhere in the specification in detail. An oligonucleotide as used herein has a length of at most 100, at most 50, at most 40, at most 30 or at most 20 nucleotides in length which are complementary to the nucleic acid sequence of the polynucleotides of the present invention. The sequence of the oligonucleotide is, preferably, selected so that a perfect match by Watson-Crick base pairing will be obtained. The oligonucleotides of the present invention may be suitable as primers for PCR-based amplification techniques.

As used herein, the term "R A interference (R Ai)" refers to selective intracellular degradation of RNA used to silence expression of a selected target gene, i.e. the polynucleotide of the present invention. RNAi is a process of sequence-specific, post- transcriptional gene silencing in organisms initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the gene to be silenced. The RNAi technique involves small interfering RNAs (siRNAs) that are complementary to target RNAs (encoding a gene of interest) and specifically destroy the known mRNA, thereby diminishing or abolishing gene expression. RNAi is generally used to silence expression of a gene of interest by targeting mRNA, however, any type of RNA is encompassed by the RNAi methods of the invention. Briefly, the process of RNAi in the cell is initiated by long double-stranded RNAs (dsRNAs) being cleaved by a ribonuclease, thus producing siRNA duplexes. The siRNA binds to another intracellular enzyme complex which is thereby activated to target whatever mRNA molecules homologous (or complementary) to the siRNA sequence. The function of the complex is to target the homologous mRNA molecule through base pairing interactions between one of the siRNA strands and the target mRNA. The mRNA is then cleaved approximately 12 nucleotides from the 3' terminus of the siRNA and degraded. In this manner, specific mRNAs can be targeted and degraded, thereby resulting in a loss of protein expression from the targeted mRNA. A complementary nucleotide sequence as used herein refers to the region on the RNA strand that is complementary to an RNA transcript of a portion of the target gene.

The term "dsRNA" refers to RNA having a duplex structure comprising two complementary and anti-parallel nucleic acid strands. Not all nucleotides of a dsRNA necessarily exhibit complete Watson-Crick base pairs; the two RNA strands may be substantially complementary. The RNA strands forming the dsRNA may have the same or a different number of nucleotides, with the maximum number of base pairs being the number of nucleotides in the shortest strand of the dsRNA. Preferably, the dsRNA is no more than 500, more preferably less than 300, and most preferably between 100 and 300, nucleotides in length. dsRNAs of this length are particularly efficient in inhibiting the expression of the target gene using RNAi techniques. dsRNAs are subsequently degraded by a ribonuclease enzyme into short interfering RNAs (siRNAs). RNAi is mediated by small interfering RNAs (siRNAs). The term "small interfering RNA" or "siRNA" refers to a nucleic acid molecule which is a double-stranded RNA agent that is complementary to i.e., able to base-pair with, a portion of a target RNA (generally mRNA), i.e. the polynucleotide of the present invention being RNA. siRNA acts to specifically guide enzymes in the host cell to cleave the target RNA. By virtue of the specificity of the siRNA sequence and its homology to the RNA target, siRNA is able to cause cleavage of the target RNA strand, thereby inactivating the target RNA molecule. Preferably, the siRNA which is sufficient to mediate RNAi comprises a nucleic acid sequence comprising an inverted repeat fragment of the target gene and the coding region of the gene of interest (or portion thereof). Also preferably, a nucleic acid sequence encoding a siRNA or dsRNA (e.g. 250 bp) comprising a sequence sufficiently complementary to a target gene is operatively linked to an expression control sequence. Thus, the mediation of RNAi to inhibit expression of the target gene can be modulated by said expression control sequence. Preferred expression control sequences are those which can be regulated by an exogenous stimulus, such as the tet operator whose activity can be regulated by tetracycline or heat inducible promoters. Additionally, constitutive expression of the dsRNA can be achieved by using the 35S promoter of the Cauliflower mosaic virus (CaMV). Alternatively, an expression control sequence may be used which allows tissue- specific or expression of the siRNA or expression at defined time points in development, such as the phloem-specific promoters exemplified by the sucrose synthase, the sucrose-H⁺ symporter and the phloem protein-2 promoters. The complementary regions of the siRNA or dsRNA allow sufficient hybridization of the siRNA or dsRNA to the target mRNA and thus mediate RNAi. In mammalian cells, siRNAs are approximately 21-25 nucleotides in length (see Tuschl et al. 1999 and Elbashir et al. 2001). The siRNA sequence needs to be of sufficient length to bring the siRNA and target RNA together through complementary base-pairing interactions. The siRNA used with a seed specific expression system e.g. under control of the USP promoter of the invention may be of varying lengths. The length of the siRNA is preferably greater than or equal to ten nucleotides and of sufficient length to stably interact with the target RNA; specifically 15-30 nucleotides; more specifically any integer between 15 and 30 nucleotides, most preferably 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, and 30. By "sufficient length" is meant an oligonucleotide of greater than or equal to 15 nucleotides that is of a length long enough to provide the intended function under the expected condition. By "stably interact" is meant interaction of the small interfering RNA with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions). Generally, such complementarity is 100% between the siRNA and the RNA target, but can be less if desired, preferably 91 %, 92%, 93%, 94%, 95%, 96%o, 97%), 98%o, or 99%. For example, 19 bases out of 21 bases may be base-paired. In some instances, where selection between various allelic variants is desired, 100%) complementary to the target gene is required in order to effectively discern the target sequence from the other allelic sequence. When selecting between allelic targets, choice of length is also an important factor because it is the other factor involved in the percent complementary and the ability to differentiate between allelic differences. Preferably, the oligonucleotides used for RNAi approaches target the 5' or 3' untranslated region of a mRNA corresponding to a polynucleotide of the present invention.

Also in a preferred embodiment, the oligonucleotide of the present invention can be used for the generation of micro-RNAs (miRNA) or as a miRNA. These miRNAs are single-stranded RNA molecules of preferably 20 to 25, more preferably 21 to 23 nucleotides in length capable of regulating gene expression. miRNAs are physiologically encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA). Rather, they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. The mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules. Due to this complementary regions, they are capable of binding to a given target mRNA and to subsequently down-regulate expression thereof. Preferably, miRNAs to be used in accordance with the present invention comprise sequence stretches complementary to the polynucleotide sequences of the present invention referred to above. More preferably, the sequence stretches are between 20 and 30 nucleotides, more preferably, between 20 and 25 nucleotides and, most preferably, between 21 and 23 nucleotides in length. The miRNAs, preferably, are capable of binding to complementary sequences of the coding regions or of the 3' or the 5' UTR sequences of the polynucleotides of the invention. More preferably, the oligonucleotide of the present invention, thus, comprises a sequence of at least 15 nucleotides in length complementary to the nucleic acid sequence of the polynucleotide of the invention. Most preferably, the said oligonucleotide is capable of down regulating the expression of the said polynucleotide, preferably either by functioning as a double-stranded RNAi molecule or as a single- stranded miRNA molecule. Downregulation as meant herein relates to a statistically significant reduction of the mRNA detectable in a cell, tissue or organism or even to a failure to produce mRNA in detectable amounts at all. This also includes the reduction of the stability of mRNA encoding all or a part of any sequence described herein. Moreover, downregulation also encompasses an impaired, i.e. significantly reduced, production of protein from RNA sequences encoding all or a part of any sequence or even the absence of detectable protein production.

The present invention provides antisense oligonucleotides and polynucleotides complementary to polynucleotides according to the invention or one of its orthologs which hybridizes with it under high stringency conditions. Such antisense oligonucleotides should be at least about six nucleotides in length to provide minimal specificity of hybridization and may be complementary to one strand of DNA or mRNA encoding an isolated polypeptide according to the invention or a portion thereof, or to flanking sequences in genomic DNA which are involved in regulating the expression of said isolated polypeptide according to the invention. The antisense oligonucleotide may be as large as 100 nucleotides and may extend in length up to and beyond the full coding sequence for which it is antisense. The antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The action of the antisense oligonucleotide may result in alteration, primarily inhibition, of the expression of said isolated polypeptides according to the invention in cells.

A transgenic plant is defined herein as a plant which is genetically modified in some way. The altered genetic material may encode a protein comprising a regulatory or control sequence or may be or include an antisense sequence or encode an antisense RNA or siRNA which inhibits the DNA or mRNA sequence or portion thereof responsible of the expression of the polypeptide of which a modification of the expression is foreseen.

A "transgene" or "transgenic sequence" is defined as a foreign gene or partial sequence which has been incorporated into a transgenic plant.

Transgenic plants made in accordance with the present invention may be prepared by DNA transformation using any method of plant transformation known in the art. Plant transformation methods include direct co-cultivation of plants, tissues or cells with Agrobacterium tumefaciens or direct infection; direct gene transfer into protoplasts or protoplast uptake; electroporation; particle bombardment; injection into meristematic tissues of seedlings and plants; injection into protoplasts of cultured cells and tissues. Generally a complete plant is obtained from the transformation process. Plants are regenerated from protoplasts, callus, tissue parts or explants, etc.

Legends of figures Figure 1: Ephrin receptor A7 organization, expression and detection in M. persicae. A)

Predicted mR A splicing variants (VI to V3) of EphA 7 gene are depicted by exon (boxes) and intron ( V ); proportions are only kept for coding sequence (CDS, grey boxes), not for intron, neither untranslated region (UTR, open-boxes). Hairpin cloning and transgene detection primers are represented by open-arrows; the filled-arrows correspond to qRT-PCR primers. Dual-line corresponds to the cDNA fragment picked-up from the M. persicae cDNA library.* indicates a shorter exon (5 amino acids) in VI compare to V2 and V3. Eph LBD: Ephrin receptor Ligand-Binding Domain; FN3: Fibronectin type-Ill domain; TMD: Transmembrane domain; SAM: Sterile alpha motif domain. B) EphA 7 mRNA detection in M. persicae. RT-PCR products obtained from total RNA extracted from whole aphids (one sample) or from aphid guts (two samples) were loaded on an agarose gel and stained with ethidium bromide. C-: PCR negative control obtained by adding H₂0 instead of cDNA in the PCR mixture; C) Evaluation of EphA 7 mRNA accumulation in M. persicae instars by qRT- PCR. Data represent the mean ± standard deviation of triplicate results from two independent experiments. EphA 7 mRNA expression was arbitrarily fixed to 1.00 in M. persicae adults. Figure 2: siRNA detection in A. thaliana transgenic plants expressing an RNA hairpin targeting EphA7 in the aphid M. persicae. Total RNA was extracted from 4-week old seedlings of F3 heterozygous A. thaliana transgenic plants which survived the BASTA^®F1 treatment. Each lane was loaded with 20 μg of RNA and the blot was hybridized with a DIG-labeled probe complementary to the EphA 7- fragment introduced into the transgenic plants. The blot was further stripped and hybridized with a DIG-labeled U6 probe to control equal loading. Col-0: total RNA extract from a non-transformed A. thaliana Col-0 plant.

Figure 3 Reduction of EphA7-mRNA accumulation in aphids fed on transgenic A. thaliana. M. persicae fed on Hp-EphA7, Hp-LacZ or Hp-GFP for 10 days were harvested and analyzed for down-regulation of EphA 7 mRNA by qRT-PCR. Data represent the mean ± standard deviation of triplicate results. Data from one representative experiment (Exp. l in Table 1) out of 4 are shown. The asterisk above the bar indicates a significant difference in EphA 7 mR A accumulation compared to the controls (aphids fed on Hp-LacZ or Hp-GFP) (Student's t-test, n=3, p<0.05).

Figure 4: Effect of EphA7 silencing on M. persicae fecundity. M. persicae fed on

Hp-EphA7 or on Hp-GFP plants for 10 days were transferred to non-transformed Col-0 and nymphs production was recorded daily during 5 days. Data shown represent the mean value of nymphs produced daily per aphid ± standard deviation. This experiment was performed using 10 aphids per condition.

Figure 5: TuYV uptake by aphids fed on dsRNA-expressing A. thaliana. A) Aphids fed for 10 days on Hp-EphA7, Hp-LacZ or Hp-GFP plants were transferred onto a TuYV purified suspension (50 μg/ml) for 24 h. Aphids were then harvested and the amount of viral genomes in the aphids was evaluated by qRT-PCR. Data correspond to one experiment and represent the mean ± standard deviation of triplicates. B) Virus acquisition was evaluated in M. persicae fed on wild-type Col-0 or on Hp-LacZ plants. Otherwise, this experiment has been performed as in (A). Figure 6: TuYV particles internalized into EphA 7-silenced aphids. Aphids fed for 10 days on Hp-EphA7, Hp-LacZ or Hp-GFP were transferred onto a TuYV purified suspension (50 μg/ml) for 24 h before being deposited onto test plants (Col-0) for 7 days. Aphids were then harvested and the amount of viral genomes in the aphids was evaluated by qRT-PCR. Data correspond to one experiment and represent means of 3 technical replicates ± standard deviation of a pool of 10 aphids per sample.

Figure 7: siRNA detection in A. thaliana transgenic plants expressing an RNA hairpin targeting LacZ. Total RNA was extracted from 4-week old seedlings of F3 heterozygous A. thaliana transgenic plants which survived the BASTA^®F1 treatment. Each lane was loaded with 20 μg of RNA and the blot was hybridized simultaneously with a DIG-labeled probe complementary to the LacZ-fragment introduced into the transgenic plants and a DIG-labeled U6 probe.

Figure 8: TuYV uptake by EphA 7-silenced aphids. EphA 7-silenced M. persicae were transferred onto a TuYV purified suspension (50 μg/ml) for 24 h. Aphids were then harvested and the amount of viral genomes in the aphids was evaluated by qRT-PCR. Data correspond to mean ± standard deviation of triplicates. Figure 9: TuYV particles internalized into EphA 7-silenced aphids. Aphids fed for 10 days on Hp-EphA7 or Hp-LacZ were transferred onto a TuYV purified suspension (50 μ /ηι1) for 24 h before being deposited onto test plants (Col-0) for 7 days. Aphids were then harvested and the amount of viral genomes in the aphids was evaluated by qRT-PCR. Data correspond to one experiment and represent means of 3 technical replicates ± standard deviation of a pool of 10 aphids per sample.

DETAILED DESCRIPTION The first subject-matter of the invention is a method for preventing or reducing Luteoviridae family virus transmission between plants by aphids comprising silencing the ephrin receptor protein- A7 (EphA7) in said aphids, wherein EphA7 is a protein encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3.

Another subject-matter of the invention is a method for preventing or reducing Luteoviridae family virus transmission between plants by aphids comprising silencing one of the homo logs of the ephrin receptor protein- A7 (EphA7) in said aphids, wherein EphA7 is a protein encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 and wherein said homolog exhibits a sequence which is at least 60%, preferably at least 70%, and more preferably at least 80%, the most preferably at least 90% identical to the one of EphA7.

Preferably, the method according to the invention is a method for preventing or reducing Luteoviridae family virus transmission between plants from a commercially important crop including Asteraceae (including the food crops lettuce, chicory, globe artichoke, Jerusalem artichoke, sunflower, yacon, safflower, tragopogon), Cucurbitaceae (commonly known as gourds or cucurbits and includes crops like cucumbers, squashes (including pumpkins), luffas, melons and watermelons), Brassica (including swedes, turnips, kohlrabi, cabbages, brussels sprouts, cauliflower, broccoli, mustard seed and oilseed rape), Leguminous Crops (including dry beans, dry broad beans, dry peas, chickpea, garbanzo, bengal gram dry cowpea, black- eyed pea, blackeye bean, pigeon pea, toor, cajan pea, congo bean, lentil, bambara groundnut, earth pea vetch, common vetch Lupins, soybean, peanut), Chenopodiaceae (beet, spinach), Amaryllidaceae (onions, leek), Apiaceae (carrots), Grain crops (rice, barley, wheat, oats, corn), Solanaceae (including potato, tomato, courgette/zucchini, aubergine/eggplant, hot pepper, sweet pepper, tobacco), fruit trees (apple tree, orange tree, Clementine tree), cotton plants, pine tree or vineyard.

More preferably, the method according to the invention is a method for preventing or reducing Luteoviridae family virus transmission between cereal plants, for example rice plants, corn plants, oats plants and wheat plants.

Aphid species that can transmit definitive or tentative members in the Luteoviridae family are described in Herrbach E. 1999. Vector- Virus Interactions. In: H.G. Smith and H. Barker (Eds), The Luteoviridae, CABI, Wallingford, UK, pp. 85-88. They comprise: Sitobion avenae, Rhopalosiphum padi, Myzus persicae, Aphis gossypii, Macrosiphum euphorbiae, Myzus nicotianae, Acyrtho siphon pisum, Schizaphis graminum, Rhopalosiphum maidis, Cavariella aegopodii, Aphis craccivora, Aphis glycines, Aulacorthum solani, Hyadaphis foeniculi, Toxoptera aurantii, Acyrthosiphon pelargonii, Aphis craccivora, Aphis rubicola, Chaetosiphon fragaefolii, Melenaphis sacchari, Megoura viciae, Myzus (Sciamyzus) ascolonicus, Myzus (Nectarosiphon) certus,, Myzus (Phorodon) humuli, Brachycaudus helichrysi, Myzus ornatus, Brevicoryne brassicae, Aulocorthum circumflexum, Ceruraphis eriophori; Acyrthosiphon (Metopolophium) dirhodum, Rhopalosiphum rufiabdominalis, Sipha agropyrella, Rhopalosiphum insertum, Macrosiphum (Sitobion) fragariae, Neomyzus circumflexus. Luteoviridae family viruses are described in the Ninth Report of the International Committee on Taxonomy of Viruses (Editors Andrew M.Q. King, Michael J. Adams, Eric B. Carstens, and Elliot J. Lefkowitz, International Union of Microbiological Societies, Virology Division Elsevier, Amsterdam, 2012).

The Luteoviridae family comprises the genus Luteovirus, the genus Polerovirus and the genus Enamovirus.

The genus Luteovirus comprises the following species: Barley yellow dwarf virus-MA V, Barley yellow dwarf virus-PAS, Barley yellow dwarf virus-PA V, Bean leafroll virus (Legume yellows virus) (Michigan alfalfa virus) (Pea leafroll virus), Rose spring dwarf-associated virus, Soybean dwarf virus, (Subterranean clover red leaf virus), Soybean dwarf virus-Tas-1 (SbDV-Tas-1), Barley yellow dwarf virus-GAV (BYDV-GAV). The genus Polerovirus comprises the following species: Beet chlorosis virus (BChV-2a), Beet mild yellowing virus (BMYV-2ITB), Beet western yellows virus (Malva yellows virus) (Turnip mild yellows virus) (BWYV-US), Carrot red leaf virus (CtLRV-UKl), Cereal yellow dwarf virus-RPS (CYDV-RPS-Mexl), Cereal yellow dwarf virus-RPV (CYDV-RPV-NY), Chickpea chlorotic stunt virus (CpCSV-Et-f -aml), Cucurbit aphid-borne yellows virus (CABYV-N), Melon aphid-borne yellows virus (MABYV-BJ), Potato leafroll virus (Solanum yellows virus) (Tomato yellow top virus) (PLRV-U ), Sugarcane yellow leaf virus (ScYLV- FL), Tobacco vein distorting virus (TVDV-CN), Turnip yellows virus (TuYV-FLl), Cotton leafroll dwarf virus (CLRDV), Suakwa aphid-borne yellows virus (SABYV). The genus Enamovirus comprises the following species: Pea enation mosaic virus-1 (PEMV- 1-WSG).

The family Luteoviridae also comprises the following unassigned species: Barley yellow dwarf virus-GPV (BYDV-GPV-04FX6) (WYDV-RPV), Barley yellow dwarf virus-RMV (BYDV-RMV-IL), Barley yellow dwarf virus-bv (BYDV-SGV-NY), Chickpea stunt disease associated virus (CpSDaV-IC), Groundnut rosette assistor virus (GRAV-M16GCP), Indonesian soybean dwarf virus (ISDV-IND), Sweet potato leaf speckling virus (SPLSV- Peru), Tobacco necrotic dwarf virus (TNDV-JA).

The family Luteoviridae also comprises other related viruses which have not been approved as species: Chickpea yellows virus (CpYV), Lentil stunt virus (LSV). The term "silencing" means down-regulating or antagonizing, at least partly.

In one embodiment, said silencing of the method according to the invention is temporary.

"Temporary" means reversible in several weeks, preferably several days.

Preferably, said silencing of EphA7 consists in administering said aphids an effective amount of: - an antagonist, preferably antibody or an aptamer, which binds to said EphA7 protein and inhibits its receptor activity in a aphid cell; or

- an oligonucleotide capable of down regulating the expression of EphA7 protein in a aphid cell or

- an antisense oligonucleotide or polynucleotide comprising at least a portion that hybridizes to an EphA7 transcript under physiological conditions and decreases the expression of EphA7 in a aphid cell. Preferably, said silencing of one of the homologs of EphA7 consists in administering said aphids an effective amount of:

- an antagonist, preferably antibody or an aptamer, which binds to said EphA7 homolog protein and inhibits its receptor activity in a aphid cell; or - an oligonucleotide capable of down regulating the expression of said EphA7 homolog protein in a aphid cell or

- an antisense oligonucleotide or polynucleotide comprising at least a portion that hybridizes to an EphA7 homolog transcript under physiological conditions and decreases the expression of said EphA7 homolog in a aphid cell.

Preferably, silencing is due to feeding of aphids on transgenic plants according to the invention.

Most preferably, silencing is due to reduction of EphA7 expression in aphids fed on transgenic plants expressing a nucleic acid compound down-regulating the expression of EphA7 in an aphid cell, for example an R A hairpin comprising two copies of SEQ ID NO:5 in sense and antisense separated by a non-coding sequence.

Another subject-matter of the present invention is a vector comprising SEQ ID NO:5 which expresses said insert as inverted repeats.

Preferably, appropriate template vectors are described in: - Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, et al. (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant Journal 27: 581-590, - Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM (2002) High-throughput vectors for efficient gene silencing in plants. Functional Plant Biology 29: 1217-1225, Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30: 289-295,

- Eamens AL, Waterhouse PM (2011) Vectors and methods for hairpin R A and artificial microRNA-mediated gene silencing in plants. Methods in molecular biology 701 : 179-197,

Himmelbach A, Zierold U, Hensel G, Riechen J, Douchkov D, et al. (2007) A set of modular binary vectors for transformation of cereals. Plant Physiology 145: 1192- 1200,

Zhu Wenhua, Xie Meng, Lin Yuheng, Yang Mingyu, Ma Qiumin, et al. (2011) Construction of Tobacco Bax Inhibitor- 1 ihpRNA Gene Silencing Vector and Transformation into Agrobacterium tumefaciens EHA105. Journal of Northeast Agricultural University (English edition) 18(2): 58-64,

- Yan P, Shen W, Gao X, Li X, Zhou P, et al. (2012) High-Throughput Construction of

Intron-Containing Hairpin RNA Vectors for RNAi in Plants. PLoS ONE 7(5): e38186. doi: 10.1371/journal.pone.0038186,

http://www.arabidopsis.org/abrc/catalog/vector_l .html.

Preferably, appropriate template vectors are selected in the group consisting of: pHANNIBAL and pKANNIBAL (first used to generate ihpRNA constructs), GATEWAY cloning system- based RNAi vectors such as the pHELLSGATE series and the plPK series (widely used for generating ihpRNA constructs), pGSA2285, pRNAi-GG, pFGC1008, pFGC5941, pGSA1131, pGSA1165, pGSA1204, pGSA1252, pGSA1276, pGSA1285, pGSA1403, pGSA1427, pGSA1561, pGSA1783, pMCG161. Another subject-matter of the present invention is a plant or part of plant genetically engineered to express: an oligonucleotide capable of down-regulating the expression of ephrin receptor protein-A7 (EphA7) or one of its homo logs in a aphid cell, or

an antagonist of ephrin receptor protein- A7 (EphA7) or of one of its homo logs, or - an antisense oligonucleotide or polynucleotide comprising at least a portion that hybridizes to an EphA7 transcript or a EphA7 homo log transcript under physiological conditions and decreases the expression of EphA7 or one of its homo logs in a aphid cell wherein EphA7 exhibits a protein sequence encoded by a mRNA selected in the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3 and one of its homologs exhibits a sequence which is at least 60%, preferably at least 70%>, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

In one embodiment, said plant or part of plant is genetically engineered to express an isolated nucleic acid compound comprising at least a portion that hybridizes to an EphA7 transcript under physiological conditions and decreases the expression of EphA7 in a aphid cell, preferably expressing an RNA hairpin structure decreasing the expression of EphA7 in a aphid cell, for example an RNA hairpin structure comprising two copies of SEQ ID NO: 5 in sense and antisense separated by a non-coding sequence.

Another subject-matter of the present invention is an isolated ephrin receptor protein-A7 (EphA7) exhibiting a protein sequence encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or one of its isolated homologs exhibiting a sequence which is at least 60%>, preferably at least 70%>, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

Another subject-matter of the invention is an isolated polynucleotide comprising SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3, analogs, paralogs or orthologs thereof Another subject-matter of the present invention is an isolated dsRNA with a hairpin structure arising from the transcription of two copies of SEQ ID NO:5 in sense and antisense separated by a non-coding sequence.

Another subject-matter of the present invention is an isolated antisense polynucleotide comprising at least a portion that hybridizes under physiological conditions to an EphA7 mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 or one of its isolated homologs exhibiting a sequence which is at least 60%>, preferably at least 70%, and more preferably at least 80%, the most preferably at least 90% identical to the one of EphA7, and decreases the expression of EphA7 in a aphid cell. This antisense polynucleotide can be, for example, the complementary sequence of SEQ ID NO:5 . Preferably, said non-coding sequence is an intron selected in the group consisting of: - FAD2-1A intron 1, and soybean FATB intron (patent application WO2004001000 A2);

Pdk, pyruvate orthophosphate dikinase (Rosche E, Westhoff P (1995) Genomic structure and expression of the pyruvate, orthophosphate dikinase gene of the dicotyledonous C-4 plant Flaveria trinervia (Asteraceae). Plant Molecular Biology 29: 663-678);

Solanum esculentum RNA-directed RNA polymerase 1 (SeRDRl) intron 3 (Dalakouras A, Moser M, Zwiebel M, Krczal G, Hell R, et al. (2009) A hairpin RNA construct residing in an intron efficiently triggered RNA-directed DNA methylation in tobacco. Plant Journal 60: 840-851);

Second intron of ran gene from Nicotiana sylvestris (G. Tonkovska, A. Atanassov, I. Atanassov. (2004) A hybrid cloning procedure for construction of plant transformation vectors for ihpRNA-mediated gene silencing. Biotechnology & Biotechnological Equipment 18(3): 174-178);

Chalcone synthase intron

Castor bean catalase intron (Nizampatnam NR, Dinesh Kumar V (2011) Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522. Plant Mol Biol 76: 557- 573; Shekhawat UKS, Ganapathi TR, Hadapad AB (2012) Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection. Journal of General Virology 93: 1804-1813);

First intron of the rice a-amylase 3D gene (Kim NS, Kim TG, Kim OH, Ko EM, Jang YS, et al. (2008) Improvement of recombinant hGM-CSF production by suppression of cysteine proteinase gene expression using RNA interference in a transgenic rice culture. Plant Mol Biol 68: 263-275).

Most preferably, said non-coding sequence is chalcone synthase intron (sequence included into SEQ ID NO:4).

Another subject-matter of the present invention is an isolated dsRNA with a hairpin structure arising from the transcription of SEQ ID NO:4.

Another subject-matter of the present invention is aphids genetically engineered to have: downregulation of the expression of ephrin receptor protein- A7 (EphA7) or one of its homologs, or

expression of non- functional ephrin receptor protein-A7 (EphA7) or one of its nonfunctional homologs, or

- expression of an antagonist of ephrin receptor protein-A7 (EphA7) or of one of its homologs wherein EphA7 exhibits a protein sequence encoded by a mR A selected in the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3 and one of its homologs exhibits a sequence which is at least 60%, preferably at least 70%>, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

"Non- functional" ephrin receptor means an ephrin receptor which does not bind with its ligands or does not activate cells upon binding by its ligands.

In one embodiment, said aphids according to the invention are fed on transgenic plants according to the invention. Another subject-matter of the present invention is a method for preventing or reducing Luteoviridae family virus internalization by aphids comprising silencing the ephrin receptor protein- A7 (EphA7) in said aphids, wherein EphA7 is a protein encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3.

Another subject-matter of the present invention is a method for preventing or reducing Luteoviridae family virus internalization by aphids comprising silencing one of the homologs of the ephrin receptor protein- A7 (EphA7) in said aphids, wherein EphA7 is a protein encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, and wherein said homolog exhibits a sequence which is at least 60%>, preferably at least 70%, and more preferably at least 80%, the most preferably at least 90% identical to the one of EphA7.

EXAMPLES

Material and methods

Generation of A. thaliana transgenic plants A sequence of 249 bp (nt 1198 to 1446 on the EphA 7 coding sequence) was amplified by reverse transcription (RT) polymerase chain reaction (PCR) on total M. persicae RNA extracted using a commercial purification kit (RNeasy Plant Mini Kit, animal tissue protocol, Qiagen). Two primers containing 5' restriction site extensions were used, the reverse primer 5 -TAGGATCCATTTAAATAGATAGCGTGATCTCGGACG-3 ' = SEQ ID NO:6 (nt 1427-1446 on EphA 7 coding sequence in bold) with the two restriction sites BamHI and Swal and the forward primer 5 -TTTCTAGAGGCGCGCCGGTCCATCCGTTGTGTTCAT-3 ' = SEQ ID NO:7 (nt 1198-1217 on EphA 7 coding sequence in bold) with the restriction sites Xbal and Ascl underlined. The amplified DNA fragment was introduced in the sense and antisense orientation into the vector pFGC5941 (GenBank accession AY310901.1) in a sequential process: the PCR-amp lifted DNA fragment was digested with the Ascl and Swal restriction enzymes and introduced into the pFGC5941 digested with the same enzymes. After ligated the digested fragments, a recombinant plasmid was selected after its introduction into E. coli (DH5a). The recombinant plasmid bearing the sense sequence was further digested with Xbal and BamHI. A similar enzymatic digestion was performed onto the PCR-amplified cDNA. After ligation and transformation of E. coli (DH5a), presence of the sense and antisense sequence into the final recombinant plasmid named pFGC:EphA7 was controlled by sequencing. A similar procedure was followed to introduce a 276 bp fragment of the lacZ gene in sense and antisense orientation into the pFGC5941 vector. The lacZ cDNA fragment was amplified from the pUC18 plasmid (GenBank accession L09136.1) using the following primers (forward 5'-GTTCTAGAGGCGCGCCCGGCATCAGAGCAGATTGTA-3 '= SEQ ID NO:8 and reverse 5 -TAGGATCCATTTAAATTAGAGTCGACCTGCAGGCAT-3 = SEQ ID NO:9). As above, the bold sequence is complementary to the lacZ sequence and the underlined characters represent the restriction sites BamHI, Swal, Xbal and Ascl. The resulting plasmid was referred to as pFGC:LacZ.

The pFGC:EphA7 and pFGC:LacZ recombinant plasmids were further introduced into A. tumefaciens C58C1 which was further used to transform thaliana (Col-0) by floral dip as described by Martinez-Trujillo et al. (2006). Seeds were sown and seedlings were sprayed with phosphinothricin (BASTA^® Fl) to select for transformants. F2 seeds were sown and treated with BASTA^® F 1. The surviving plants were used in our experiments. Insertion of the transgene in the plants resistant to BASTA^® Fl treatment was verified by PCR on DNA prepared from leaves using a protocol adapted from Murray and Thompson (1980). Briefly, one or two leaves of transgenic A. thaliana were ground in 300 μΐ of CTAB buffer (2% CTAB, 1% PVP, 100 mM Tris pH 8.0, 20 mM EDTA, 1.4M NaCl). The mixture was incubated 10 min at 65°C and an equal volume of chloroform:isoamyl alcohol (24: 1) was added. After a centrifugation of 5 min at 18400 , the DNA present in the supernatant was precipitated by an incubation of 10 min at -20°C in the presence of one volume of isopropanol. DNA was recovered after a centrifugation of 10 min at 18400 g at 4°C. The pellet was washed in 70% ethanol and resuspended in 30 μΐ of 10 mM Tris pH 8.0. The primers used in the PCR reaction were those previously mentioned for the construction of the recombinant plasmids.

Aphid transmission experiments from purified virus using silenced aphids Virus-free colonies of M. persicae were reared on Capsicum annuum. M. persicae adults were transferred onto four weeks old A. thaliana transgenic plants expressing an RNA hairpin structure targeting EphA 7 gene of M. persicae, the bacterial lacZ gene or the green fluorescence protein gene (GFP) (Pitino et al, 2011) for two days for the production of offspring. The adults were then removed and the larva remained on the transgenic plants for 10 days. These aphids were then membrane-fed on purified virus as described previously (Bruyere et al, 1997) using a virus concentration of 50 μg/ml. Turnip yellows virus (TuYV, formerly referred to as Beet western yellows virus) was purified from agroinfected Montia perfoliata (Leiser et al, 1992) according to the method of van den Heuvel and associates (1991). After a 24 h acquisition period, aphids were transferred onto healthy A. thaliana Col-0 for a 7 day inoculation period. These test plants were assayed for TuYV infection 3 weeks later by double antibody sandwich (DAS) enzyme-linked immunosorbent assays (ELISA) (Clark and Adams, 1977) using TuYV-specific antiserum (Loewe) at a dilution of 1/400 (v/v).

Detection of siRNA

To evaluate siRNA accumulation in the transgenic A. thaliana plants, total RNA was extracted from leaves using TRIzol^® Reagent (Sigma). 10 μg of total RNA were mixed with an equal volume of deionized formamide and denatured during 5 min at 95°C. The RNA were resolved on a 17.5% polyacrylamide gel (acrylamide-bisacrylamide solution 19: 1/ 7 M urea in Tris-Borate-EDTA buffer 0.5X) and blotted onto an Hybond NX membrane (Amersham) by liquid transfer (Bio Rad, Criterion Blotter) for 75 min at 80V/300mA. RNA were fixed on the membrane by UV-cross linking (120000 μ.Ι/αη2, Spectrolinker XL-1000 UV Crosslinker). DNA probes were prepared by a PCR amplification of a 249 bp fragment (nt 1198-1446 on EphA 7 coding sequence) using the following primers: forward 5'-TTTCTAGAGGCGCGCCGGTCCATCCGTTGTGTTCAT-3' and reverse

5 '-TAGGATCCATTTAAATAGATAGCGTGATCTCGGACG-3 ' . For the LacZ probe, the forward 5 '-CGGCATCAGAGCAGATTGTA-3 ' and reverse

5 '-TAGAGTCGACCTGCAGGCAT-3 'primers were used in the PCR reaction. In a second step, the PCR products were further labeled using Klenow fragment (Roche) with [DIG]dUTP (Roche). To control equal loading of RNA, blots were hybridized with a U6 probe which was synthesized with the U6 forward 5 '-ATTGTCCCTTCGGGGAC-3 ' and U6 reverse 5 '-AAATTTGGACCATTTCTC-3 'primers. The signals were detected using the chemiluminescence kit (CDP-Star, Roche). Evaluation of mRNA or virus accumulation by RT- or qRT-PCR

For real-time qPCR, total RNA was extracted from 10 to 20 aphids using the RNeasy plant mini kit (Qiagen, Courtaboeuf, France). To measure EphA7 expression in the M. persicae instar larvae, RNA was extracted from 40 LI, 30 L2 or L3 and 20 adults or L4. RNA was eluted in 30 μΐ of RNase free water and quantified with the Nanodrop 2000 (Thermo Scientific). Complementary DNA (cDNA) was synthesized using specific primers (see below) with the Superscript III reverse transcriptase kit (Invitrogen) starting from 1 μg of total RNA for absolute virus quantification or 2 μg to measure cellular messenger RNAs accumulation. qRT-PCR reactions were performed in 96-well optical plate in a total volume of 10 μΐ SYBR Green Master mix (Roche) on a CI 000 Touch^® Thermal Cycler (BIO-RAD) according to the manufacturer's instructions. To evaluate mRNA accumulation of EphA 7, the forward primer 5 '-ATGTTGGTAAAGGCGTCCGAGA-3 ' (nt 1414-1435 on EphA 7 coding sequence, exon 8) and the reverse primer 5 '-ACTCGTCACCTCGGGGATAGAAC-3 ' (nt 1518-1540 on EphA 7 coding sequence, exon 9) were used. The mean value was normalized using the RpL7 (ribosomal protein L7) and L27 (60S ribosomal protein L27) genes as internal controls with the following primers: forward RpL7 5 '-GCGCGCCGAGGCTTAT-3', reverse RpL7 5 '-CCGGATTTCTTTGCATTTCTTG-3 ' and forward L27

5 '-CCGAAAAGCTGTCATAATGAAGAC-3 ' , reverse L27

5 '-GGTGAAACCTTGTCTACTGTTACATCTTG-3 ' genes as internal controls (Pitino et al, 2011). To analyze the presence of EphA 7-mKNA in M. persicae intestinal cells, guts were extracted from aphids by pulling with tweezers the head (foregut, anterior midgut and part of the posterior midgut sampled) or the cauda (hindgut and part of posterior midgut sampled). Guts collected by both procedures were mixed and total RNA was extracted using the Pico Pure RNA isolation kit (Arcturus^®, Applied Biosystems). The RT-PCR reaction was performed using the same primers as the one used for the qRT-PCR with 25 cycles of amplification. Absolute virus quantification was realized by using a dilution series of 10⁸ to 10³ viral RNA copies obtained from RNA extracted from purified TuYV virions. The forward (BPqtFO) 5 '-AAGACAATCTCGCGGGAAG-3 ' and the reverse (BPqtRl) 5 '-GGAGACGAACTCCAAAATGAC-3 ' primers amplified a sequence on the TuYV genome (GenBank accession NC 003743) from nt 3694-3830.

Results

EphA 7: a putative partner of polerovirus structural proteins

By screening a M. persicae cDNA library by the yeast two hybrid system using polerovirus capsid proteins as baits, a peptide of 243 amino acids was found as a potential partner of the structural proteins of TuYV. This sequence shared 99.2% of amino acid identity with an orthologous gene on the A. pisum genome sequence annotated as the ephrin receptor protein- A7 (GenelD: 100166161). On M. persicae genome (clone O of M. persicae blast server, Aphidbase.com) three scaffolds encompassing the complete EphA 7 sequence (Mpl087439_TGAC_Vl .l_scaffold_n°824, 880, 888) were identified, although the sequence has not yet been annotated as an ephrin receptor for M. persicae. The extracellular domain of EPHA7 contains a globular ligand-binding domain and two fibronectin type III repeats (Figure 1 A). The intracellular cytoplasmic part consists of a short transmembrane domain, the protein kinase domain and a sterile alpha motif (SAM) domain (Figure 1A). If we refer to EphA 7 A. pisum sequence, three mRNA of different lengths, synthesized by alternate splicing, are potentially expressed from the M. persicae EphA 7 sequence (Figure 1 A). Dealing with the functional domains, the only difference between the three variants is the absence of the SAM domain in variant 3. The three EphA 7 mRNA variant sequences in A. pisum and in M. persicae share high amino acid sequence homology (more than 98% of identity). The nucleotide sequence of the EphA 7 fragment found in the M. persicae cDNA library, which product binds to TuYV structural proteins, covers the two fibronectin type III repeats (Figure 1A).

EphA 7 mRNA was detected by RT-PCR in total RNA extract prepared from whole M. persicae or from dissected guts as shown in Figure IB. Moreover, expression of EphA 7 was observed by qRT-PCR in all the M. persicae instars (LI, L2, L3, L4 instars and adults) (Figure 1C).

Reduction of EphA 7 -mRNA accumulation in Myzus persicae fed on transgenic A. thaliana Two 250 bp fragments from the EphA 7 coding sequence were introduced into the pFGC5941 vector which expresses the inserts as inverted repeats under the control of the 35 S promoter of Cauliflower mosaic virus (CaMV). This recombinant plasmid was used to transform A. thaliana by floral dip and the transgenic plants were referred to as Hp-EphA7. We also obtained A. thaliana control plants that were transformed with a sense and antisense 276 bp sequence targeting the E. coli lacZ gene. The transgenic plants were named HP-LacZ. Transcription of the sense and antisense sequences of EphA 7 or lacZ in the transgenic plants results in the synthesis of dsRNA with a hairpin structure. Processing of the dsRNA produced in the transgenic plants into siRNA was analyzed by northern blot. Four plants from four independent heterozygous lines, two lines of Hp-EphA7 and two lines of Hp-LacZ, were selected after BAST A^® Fl treatment. Presence of the transgene was first controlled by PCR (not shown) and northern blot analysis of the small RNAs population synthesized in the transgenic lines showed the presence of siRNA derived from EphA 7 and lacZ dsRNA (Figure 2 and Figure 7). Accumulation of siRNA in the four plants of the line 1 of Hp-EphA7 was slightly higher compared to the amount of siRNA present in the four plants of the second transgenic line (Hp-EphA7 line 2, Figure 2). No obvious difference in the accumulation of siRNA in the eight plants of the two lines Hp-LacZ was observed even if the accumulation of siRNA varied between plants (Figure 7).

To analyze down-regulation of M. persicae EphA 7, nymphs born on the transgenic plants expressing a hairpin RNA structure targeting EphA 7 or lacZ genes were kept on these plants for 10 days before collecting some aphids for RNA extraction and qRT-PCR. Nymphs fed on plants expressing an RNA hairpin targeting the GFP were also included as a control in some experiments (Pitino et al, 201 1, Saskia Hogenhout, see acknowledgments). Down-regulation of EphA 7 in aphids kept on the two heterozygous lines Hp-EphA7 was observed in four independent experiments (Figure 3 showing the results of one representative experiment, and Table 1 the results of all the experiments). The reduction of EphA 7 mRNA accumulation varied from 48 to 86% in aphids fed on the line 1 of Hp-EphA7 (4 independent experiments, Table 1) whereas 29 to 64% of EphA 7 mRNA reduction was monitored in aphids kept on the line 2 of Hp-EphA7 (3 independent experiments, Table 1). This difference in EphA 7 down- regulation could be linked to the amount of siRNA accumulating in the transgenic plants which is higher in line 1 compared to line 2 (Figure 2).

Table 1: Relative expression of EphA 7 mRNA in aphids fed on transgenic A. thaliana

^†mean ± standard deviation of triplicate results. In brackets the percentage of down-regulation

expression.

^#the accumulation of EphA 7 mRNA has been arbitrarily set up to 1 in the transgenic plants Hp-GFP or Hp-LacZ. When both controls were used, expression in Hp-LacZ was fixed to 1.

Silencing of EphA 7 does not affect aphid fecundity neither virus acquisition from artificial medium

In order to investigate if down-regulation of EphA 7 mRNA in the silenced aphids could impact significantly aphid physiology, we first evaluated nymph production by EphA 7- silenced aphids. Nymphs exposed to transgenic A. thaliana plants became adults after 10 days and started to produce their own progeny. Production of nymphs was thereafter recorded daily during 5 days after transferring individual adults to non-transformed Col-0 plants. Reduction of EphA 7 mRNA expression was controlled before transferring silenced aphids on Col-0 plants and also at the end of the experiment (not shown). Figure 4 and table 2 show that nymphs production did not vary significantly between EphA 7-silenced aphids and control aphids previously fed on Hp-GFP plants.

To evaluate if the feeding behavior could be affected by the reduction of EphA 7 expression, virus uptake by EphA 7-silenced aphids from an artificial medium containing purified TuYV particles was evaluated. 24 h after TuYV acquisition, aphids were sampled and the virus content in the aphids was directly investigated by qRT-PCR without a gut clearing step. The amount of TuYV measured by qRT-PCR represents therefore the viral particles present in the gut lumen and those already internalized into the aphid's body. As shown in Figure 5 A and Figure 8, the amount of purified TuYV present in the aphids 24 h after virus acquisition varies significantly between the different samples. Even if we observed a relative constant virus acquisition by EphA 7-silenced aphids in the experiment presented in Figure 5 A, important variations were observed between control-aphids previously fed on Hp-GFP or on Hp-LacZ (up to a 10 fold difference in virus accumulation between aphids fed on Hp-LacZ). In another experiment, variation of virus uptake was observed between aphids fed on Hp-EphA7 (Figure 8). These variations in TuYV uptake were also monitored using nymphs fed during 10 days on wild-type Col-0 (Figure 5B) suggesting that virus uptake fluctuations are not due to a prior acquisition of dsRNA. Whether these differences originate from the experimental set-up, from an uneven distribution of the purified virus particles in the artificial medium or from intrinsic biological properties of each aphid need to be further addressed. In conclusion, we did not observe a major difference in TuYV acquisition from EphA 7-silenced aphids when compared to control-aphids.

Table 2: Nymphs production from M. persicae EphA 7-silenced aphids

#number of days after adult aphids were deposited onto Col-0 plants. dpi=days after infestation

†mean ± standard deviation of nymphs daily produced on Col-0 from M. persicae previously fed 10 days on transgenic Hp-EphA7 or Hp-GFP.

Silencing of EphA 7 reduces TuYV internalization in M. persicae and TuYV transmission by aphid

The ability of the EphA 7-silenced aphids to transmit TuYV was then analyzed. As previously described, aphids were first allowed to feed on dsRNA-expressing plants for 10 days to reduce expression of EphA 7 before being transferred on purified virus for 24 h. The viruliferous aphids were then deposited on test plants (Col-0) for 7 days before being removed to measure virus accumulation by qRT-PCR. As the transfer of viruliferous aphids on test plants induces a gut clearing of virus particles, this analysis evaluates the amount of virus particles that have crossed the intestinal apical plasmalemma. We observed a two times reduction in the accumulation of viral genomes in aphids maintained on Hp-GFP plants (mean value 5.45 10⁴) compared to the amount of virus found in aphids fed on Hp-LacZ (mean value 1.23 10⁵) (Figure 6). Although both lines were considered as controls in our experiments, ingestion of dsRNA targeted the GFP by aphids could have an unexpected effect on virus internalization. Additional experiments are however required to assess this hypothesis. Interestingly, in the experiment presented in Figure 6, the amount of viral genomes internalized in EphA 7-silenced aphids was significantly reduced when compared to the virus level accumulating in control-aphids fed on Hp-GFP (more than 4 times less viral genomes in EphA 7-silenced aphids) or aphid fed on Hp-LacZ (about 10 times less viral genomes in EphA 7-silenced aphids). In an additional experiment presented in Figure 9, the amount of viral particles in EphA 7-silenced aphids was reduced by 6 folds compared to the non-silenced aphids fed on Hp-LacZ. Taken together, these experiments suggest that TuYV uptake into the intestinal cells has been significantly affected in the EphA 7-silenced aphids.

To further investigate the ability of the viruliferous aphids previously fed on the transgenic plants Hp-EphA7, Hp-GFP and Hp-LacZ to transmit the virus to test plants, an ELISA assay using TuYV-specific antibodies was performed 3 weeks after virus inoculation on the aphid- inoculated plants. In two independent experiments, 84 to 95% of the test plants were infected when control aphids, initially fed on Hp-GFP or on Hp-LacZ, were used to inoculate the virus (Table 3). In contrast, the percentage of infected plants dropped significantly (from 19 to 54% of infected plants) when viruliferous EphA 7-silenced aphids were used to transmit the virus to the test plants (Table 3).

Table 3: TuYV transmissibility by EphA 7-silenced aphids

^†Number of infected Col-0 test plants/total plants inoculated with viruliferous

aphids (5 aphids per test plant). In brackets, percentage of infected plants.

Claims

1. Method for preventing or reducing Luteoviridae family virus transmission between plants by aphids comprising silencing the ephrin receptor protein- A7 (EphA7) or one of its homologs in said aphids, wherein EphA7 is a protein encoded by a mR A selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 and said homo log exhibits a sequence which is at least 60%, preferably at least 70%, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

2. The method of claim 1, wherein said silencing is temporary.

3. The method of claim 1 or 2, wherein said silencing consists in administering said aphids an effective amount of:

- an antagonist, preferably antibody or an aptamer, which binds to said EphA7 protein and inhibits its receptor activity in a aphid cell; or

- an oligonucleotide capable of down regulating the expression of EphA7 protein in a aphid cell or

- an antisense oligonucleotide or polynucleotide comprising at least a portion that hybridizes to an EphA7 transcript under physiological conditions and decreases the expression of EphA7 in a aphid cell.

4. The method of any of claims 1 to 3, wherein silencing is due to reduction of EphA7 expression in aphids fed on transgenic plants expressing a nucleic acid compound down- regulating the expression of EphA7 in a aphid cell, for example an R A hairpin comprising two copies of SEQ ID NO:5 in sense and antisense separated by a non-coding sequence.

5. The method of any of claims 1 to 4, wherein the virus is selected in the group consisting of the genus Luteovirus, the genus Polerovirus and the genus Enamovirus.

6. The method of any of claims 1 to 5, wherein the virus is selected in the group consisting of Barley yellow dwarf virus-MAV (BYDV-MAV), Barley yellow dwarf virus-PAS (BYDV- PAS), Barley yellow dwarf virus-PAV (BYDV-PAV), Bean leafroll virus (BLRV), Rose spring dwarf-associated virus, Soybean dwarf virus (SbDV), Barley yellow dwarf virus-GA V (BYDV-GAV), Beet chlorosis virus, Beet mild yellowing virus (BMYV), Beet western yellows virus (BWYV), Carrot red leaf virus (CaRLV, CRLV, CtRLV), Cereal yellow dwarf virus-RPS (CYDV-RPS), Cereal yellow dwarf virus-RPV (CYDV-RPV), Chickpea chlorotic stunt virus, Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus, Potato leafroll virus (PLRV), Sugarcane yellow leaf virus, Tobacco vein distorting virus (TVDV), Turnip yellows virus (TuYV), Cotton leafroll dwarf virus, Suakwa aphid-borne yellows virus, Pea enation mosaic virus-1 (PEMV-1), Barley yellow dwarf virus-GPV (BYDV-GPV), Wheat yellow dwarf virus-RPV, Barley yellow dwarf virus-RMV (BYDV- RMV), Barley yellow dwarf virus-bv, Chickpea stunt disease associated virus, Groundnut rosette assistor virus (GRAV), Indonesian soybean dwarf virus (ISDV), Sweet potato leaf speckling virus (SPLSV), Tobacco necrotic dwarf virus (TNDV), Chickpea yellows virus, Lentil stunt virus.

7. The method of claim 6, wherein the virus is Turnip yellows virus.

8. The method of any of claims 1 to 7, wherein aphids are selected in the group consisting of Sitobion avenae, Rhopalosiphum padi, Myzus persicae, Aphis gossypii, Macrosiphum euphorbiae, Myzus nicotianae, Acyrthosiphon pisum, Schizaphis graminum, Rhopalosiphum maidis, Cavariella aegopodii, Aphis craccivora, Aphis glycines, Aulacorthum solani, Hyadaphis foeniculi, Toxoptera aurantii, Acyrthosiphon pelargonii, Aphis craccivora, Aphis rubicola, Chaetosiphon fragaefolii, Melenaphis sacchari, Megoura viciae, Myzus (Sciamyzus) ascolonicus, Myzus (Nectarosiphon) certus,, Myzus (Phorodon) humuli, Brachycaudus helichrysi, Myzus ornatus, Brevicoryne brassicae, Aulocorthum circumflexum, Cerur aphis eriophori; Acyrthosiphon (Metopolophium) dirhodum, Rhopalosiphum rufiabdominalis, Sipha agropyrella, Rhopalosiphum insertum, Macrosiphum (Sitobion) fragariae, Neomyzus circumflexus.

9. Vector comprising SEQ ID NO: 5 which expresses said insert as inverted repeats.

10. Plant or part of plant genetically engineered to express: an oligonucleotide capable of down-regulating the expression of ephrin receptor protein-A7 (EphA7) or one of its homo logs in a aphid cell, or

an antagonist of ephrin receptor protein- A7 (EphA7) or of one of its homo logs, or an antisense oligonucleotide or polynucleotide comprising at least a portion that hybridizes to an EphA7 transcript or a EphA7 homo log transcript under physiological conditions and decreases the expression of EphA7 or one of its homo logs in a aphid cell wherein EphA7 exhibits a protein sequence encoded by a mR A selected in the group consisting of SEQ ID NO: l , SEQ ID NO:2, SEQ ID NO:3 and one of its homologs exhibits a sequence which is at least 60%, preferably at least 70%>, and more preferably at least 80%>, the most preferably at least 90% identical to the one of EphA7.

11. The plant or part of plant of claim 10, wherein said oligonucleotide capable of down- regulating the expression of ephrin receptor protein- A7 (EphA7) is an R A hairpin structure comprising two copies of SEQ ID NO: 5 in sense and antisense separated by a non-coding sequence.

12. The plant or part of plant of claim 10 or 11, selected in the group consisting in cereals, potatoes, beets and cucurbits.

13. Isolated ephrin receptor protein- A7 (EphA7) exhibiting a protein sequence encoded by a mRNA selected in the group consisting of SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3.

14. Isolated polynucleotide comprising SEQ ID NO: l, SEQ ID NO:2 or SEQ ID NO:3.

15. Isolated dsRNA with a hairpin structure arising from the transcription of two copies of SEQ ID NO:5 in sense and antisense separated by a non-coding sequence.

16. Isolated dsRNA with a hairpin structure arising from the transcription of SEQ ID NO:4.