ES2351917B1 - VIRAL SEQUENCE INVOLVED IN THE REGULATION OF GENE EXPRESSION, VECTOR OF EXPRESSION, CELL AND PLANT THAT UNDERSTANDS IT. - Google Patents

Abstract

Viral sequence involved in the regulation of gene expression, expression vector, cell and plant that comprises it. # The invention relates to an isolated nucleotide sequence, corresponding to an RNA sequence of the Cucumber Mosaic Virus, PepMV, involved. in the regulation of gene expression. In addition, the present invention relates to a vector comprising the above sequence, a cell or a plant comprising said sequence or vector. Also, the invention relates; to the use and method of any sequence or vector of the invention for the detection of at least one plant of the genus Solanum resistant to PepMV virus; or to the method for producing a functional or non-functional protein, in the cell or plant of the invention comprising co-transfection with a nucleotide sequence encoding a capsid protein (CP) of a potexvirus.

Description

Viral sequence involved in the regulation of gene expression, expression vector, cell and plant that comprises it.

The invention relates to an isolated nucleotide sequence, corresponding to an RNA sequence of the Cucumber Mosaic Virus, PepMV, involved in the regulation of gene expression. In several preferred embodiments, said sequence comprises; an MCS sequence recognized by at least one unique restriction enzyme; a nucleotide sequence (P) encoding a functional or non-functional protein; a nucleotide sequence encoding an RNA-dependent RNA polymerase (RpRd) and a triple gene block sequence (TGB) of a potexvirus (RpRd-TGB); or any combination of the above. In addition, the present invention relates to a vector comprising any previous sequence, a cell or a plant comprising any of said sequences or vectors. Likewise, the invention relates; to the use and method of any sequence or vector of the invention for the detection of at least one plant of the genus Solanum resistant to PepMV virus; or to the method for producing a functional or non-functional protein, in the cell or plant of the invention comprising co-transfection with a nucleotide sequence encoding a capsid protein (CP) of a potexvirus.

Prior art

The sweet cucumber mosaic virus (Pepino mosaic virus, PepMV; Potexvirus) is a common virus in tomato crops in Spain, in the rest of Europe and also in North America. It is a virus composed of a single strand of positive sense RNA with a size of 6.4 kb (Aguilar et al., 2002. Arch Virol. 147: 2009-2015). Its genome encodes 5 ORFs. The closest to the 5 ’end; supposedly encodes RNA-dependent RNA polymerase (RpRd) (163-164 kDa). The following ORFs, 2,3 and 4, which partially overlap in different reading frames, have the typical organization of potexvirus, of proteins involved in movement (triple gene block, TGB), and encode peptides of 26 kDa, 14 kDa and 9 kDa, respectively (Aguilar et al., 2002. Arch Virol, 147: 20092015). The ORF closest to the 3 ’end; the CP is coded, with a size of 25 kDa, responsible for the encapsidation of the viral genome and involved in cell-to-cell movement.

The ability of single-stranded RNA viruses to alter cellular metabolism and express proteins encoded by the viral genome led many researchers to postulate that RNA viruses could be used to produce recombinant proteins in infected plants (Pogue et al., 2002. Annu Rev Phytopathol.

40: 45-74). There are different strategies to convert the genome of a virus into a viral vector (Scholthof et al., 2002. Genetic engineering, 24: 67-85) but this type of vector often presents a series of limitations such as instability (due to homologous or non-homologous recombination phenomena) (Dawson et al., 1989. Virology, 172: 285-292), inability of the virus to homogeneously colonize all plant tissues or problems associated with biosecurity.

The possibility of manipulating the genome of a virus in vitro by means of infective clones allows to exploit the ability of some viruses to express foreign proteins, not naturally encoded by the viral genome (Scholthof et al., 1996. Annu Rev Phytopathol, 34: 299 -323). Infectious clones of RNA plant viruses are basically composed of: 1) a complementary DNA (cDNA) to the genomic RNA of the virus, 2) a promoter sequence of the transcription fused to said cDNA and 3) and a cloning vector (plasmid bacterial). There are currently several examples of viruses used as viral vectors (Lico et al., 2008. J Cell Physiol, 216: 366-377).

However, the selection of the regulatory sequences of the expression of said optimal foreign proteins is a key aspect in the effectiveness of the system. Therefore, a detailed study of the sequences of each viral type is required to delimit the sequence that is most effective. The selection of a minimal sequence of PepMV virus that allows the effective expression of foreign proteins in natural hosts of said virus, would be a powerful tool that could be part of an expression vector that allowed to produce proteins in vivo with high yield.

Explanation of the invention.

The invention relates to an isolated nucleotide sequence, corresponding to an RNA sequence of the Cucumber Mosaic Virus, PepMV, involved in the regulation of gene expression. In several preferred embodiments, said sequence comprises; an MCS sequence recognized by at least one unique restriction enzyme; a nucleotide sequence (P) encoding a functional or non-functional protein; a nucleotide sequence encoding an RNA-dependent RNA polymerase (RpRd) and a triple gene block sequence (TGB) of a potexvirus (RpRd-TGB); or any combination of the above. In addition, the present invention relates to a vector comprising any previous sequence, a cell or a plant comprising any of said sequences or vectors. Likewise, the invention relates; to the use and method of any sequence or vector of the invention for the detection of at least one plant of the genus Solanum resistant to PepMV virus; or to the method for producing a functional or non-functional protein, in the cell or plant of the invention comprising co-transfection with a nucleotide sequence encoding a capsid protein (CP) of a potexvirus.

PepMV virus has several characteristics that make it an excellent candidate to use its sequences as part of a protein expression vector of interest: 1.) Some of the proteins expressed by its genome, such as CP, accumulate at very high levels in inoculated plants; 2.) The symptoms that it induces in the inoculated plants are mild and do not lead to their death; 3.) It is present in agroecosystems naturally; 4.) It accumulates in large quantities in N. benthamiana (candidate plant for protein expression). In this work, an autonomous expression vector capable of expressing GFP in N. benthamiana plants has been obtained. By replacing the GFP gene with another of interest, the expression of the gene of interest in plants is achieved.

As described in the examples, in the present invention a region of the PepMV virus genome that acts as a promoter of the ORF RNA of the CP protein is delimited. The results obtained after testing the mutants allowed: 1) to delimit the 3 'end of this region (36 nt downstream from the start codon of the ORF of the CP) and 2) to show its implication in the ability of the mutants to Start outbreaks of infection. For the development of an expression vector, the 5 ’end was also delimited, which was located 75 nt upstream of the start codon of the CP ORF. From the viability of PepMV's designed viral vectors it could be deduced that the selected region of 111 nucleotides (SEO ID NO: 1-SEQ ID NO: 2) contained all the functional elements necessary for its activity. Therefore, constructions that were not infective with potential gene expression regulatory sequences that had 88, 106 or 124 nucleotides were discarded (Fig. 19). In addition, it is demonstrated that the replacement of the gene expression regulatory sequence, SEQ ID NO: 1-SEQ ID NO: 2, by a heterologous sequence, also produces the expression of the P sequence.

The heterologous sequence as understood in the present invention refers to a sequence from a strain other than the PepMV-Sp13 strain. For example, but not limited to, the heterologous sequence comes from the PepMV-Ch2 Virus strain. Preferably, the heterologous sequence of the present invention is a homologous sequence. Homologous sequences refer to sequences of different strains with similar phenotypic expressions that come from a common ancestral sequence.

The agrofiltration assays carried out with the sequences of the invention showed their capacity (as well as the expression vector that contains them) to express GFP (which replaces the sequence coding for the CP protein) and systemically infect plants from N Benthamiana. Complementation assays in which the CP protein was provided in trans (by co-transformation) showed that it was possible to restore cell-to-cell movement of mutants that do not express CP, thereby increasing the production capacity of proteins through this system.

Additionally, an application of high commercial interest of an expression vector that expresses a reporter gene (in this case, GFP) under the control of the gene expression regulatory sequence SEQ ID NO: 1-SEQ ID NO: 2, is its use for the search of plants resistant to the PepMV virus in collections of mutants of the genus Solanum and preferably of the species Solanum lycopersicum.

One aspect of the present invention refers to an isolated nucleotide sequence that has at least 80% identity with respect to the sequence SEQ ID NO: 1, involved in the regulation of gene expression. A preferred embodiment refers to the nucleotide sequence, wherein said sequence is SEQ ID NO: 1.

The sequence SEQ ID NO: 1 is a DNA sequence that corresponds to an RNA sequence belonging to the PepMV virus. The sequence SEQ ID NO: 1 corresponds to a fragment of the regulatory region of the expression (promoter or promoter region) of the gene encoding the capsid protein (CP) of PepMV virus (Pepino mosaic virus), Mosaic virus sweet Cucumber. PepMV belongs to the potexvirus family. As demonstrated in the examples of the present invention, this fragment has a clear function in the regulation of the expression of sequences located downstream thereof. The extension (number of nucleotides) and composition of this sequence have been selected taking into account that mutations of any of its nucleotides cause the reduction

or even the loss of the ability to promote the expression of sequences located downstream of it (see examples). However, said sequence admits some variation. For example, this sequence, in PepMV strain CH2 has seven variant nucleotides, which assumes that it is a sequence with 84% identity with respect to SEQ ID NO: 1. The sequence SEQ ID NO: 1 contains the conserved sequence GTTAAGTTT, present in both the Sp13, US2 or CH2 strains of the PepMV virus and in other viruses belonging to the same genus such as, for example, PVX, BaMV or WCIMV. The sequence with at least 80% identity with respect to SEQ ID NO: 1 is a heterologous sequence. More preferably, the heterologous sequence is a homologous sequence of SEQ ID NO:

one.

Another preferred embodiment refers to the isolated nucleotide sequence having at least 80% identity with respect to the sequence SEQ ID NO: 1 or the sequence SEQ ID NO: 1, joined at its 3 'end to the sequence SEQ ID NO : 2.

The sequence SEQ ID NO: 2 is the DNA nucleotide sequence corresponding to an RNA sequence belonging to the PepMV virus, located next to the RNA sequence encoded by SEQ ID NO: 1, that is, attached to the 3 'end of the RNA sequence, in the virus genome, encoded by SEQ ID NO: 1. The sequence SEQ ID NO: 2 contains an ATG nucleotide triplet, or translation start codon, which indicates the beginning of the coding sequence of the CP gene. In accordance with what is shown in the examples of the present invention, said sequence contributes to the expression of a downstream nucleotide sequence, that is, it is also involved in the regulation of gene expression. Specifically, the thymine located in the central position of the start codon (ATG) is important for the performance of said regulatory function.

In the present invention, the term "involved in the regulation of gene expression" refers to the effect on the functionality of a gene in terms of the control of the transcription of a DNA sequence, the control of the replication of a sequence of RNA, to the generation of messenger RNAs from polycistronic RNAs, or to the control of the translation of an RNA sequence or other sequences not described. In the present invention, the term "gene expression regulator" can be used to refer to the implication of a sequence in the regulation of gene expression. For example, among the regulatory sequences of gene expression are promoters and other less common as certain introns.

Another preferred embodiment refers to the isolated nucleotide sequence that has at least 80% identity with respect to the sequence SEQ ID NO: 1, or to the sequence SEQ ID NO: 1; or to any of the above sequences, linked to the sequence SEQ ID NO: 2 (as described above); which is linked by its 3 ’end to a nucleotide sequence (MCS), where the MCS sequence is recognized by at least one unique restriction enzyme. To refer to the MCS sequence (Multicloning Site), the term polilinker can be used. As understood in the present invention, the MCS or polilinker sequence is a short DNA sequence that contains at least one unique restriction site. That is, the restriction site is recognized by an enzyme capable of cutting only at said restriction site since said enzyme does not recognize other restriction sites in any of the sequences described in previous paragraphs or in vector sequences described below. The MCS sequence may contain other restriction sites recognized by one or more enzymes capable of recognizing other restriction sites of the nucleotide sequence of an expression vector into which any of the sequences of the invention described in preceding paragraphs is integrated. The MCS sequence does not modify the reading patterns of the coding sequences, but provides a place for the introduction of a number of nucleotides following the regulatory sequence (s) of the gene expression. This allows the cloning of one or more DNA fragments into the MCS sequence.

Hereinafter, to refer to the isolated nucleotide sequence that has at least 80% identity with respect to the sequence SEQ ID NO: 1; or to the sequence SEQ ID NO: 1; or to any previous sequence, linked to the sequence SEQ ID NO: 2 in the manner described above; or to any previous sequence linked by its 3 ’end to the MCS sequence; the term "sequence / s of the invention" or "sequence / s of the present invention" may be used.

According to another preferred embodiment, the sequence of the invention is linked at its 3 ′ end to a nucleotide sequence

(P) coding for a functional or non-functional protein. The P sequence is functionally linked to the sequence of the invention. The functional union is that which allows the beginning of the transcription of the sequence and the beginning of translation of said sequence.

The functional protein is one that performs one or more functions in vivo or in vitro. Said function may be an expected or not function, that is, the unexpected function may be the consequence of the modification of any of its nucleotides. The non-functional protein is one that does not perform any function in vivo or in vitro or, the protein whose result of the translation does not consist immediately of functional proteins, but for example, but not limited to, a polyprotein that will give rise to one or more Functional proteins after being processed. The sequence of the present invention is capable of regulating the gene expression of the P sequence, functional or non-functional. The system presented in the present invention can be used to determine the functionality of P sequences by carrying out, for example, but not limited to, modifications in the original sequence P, as a consequence, some of the P sequences used may result not functional.

A more preferred embodiment refers to the sequence where the nucleotide sequence P codes for a functional protein reporter (or reporter). The reporter protein is a protein that can be easily detected since it is not normally present in the cell or organism in which it is expressed. The functional protein reporter is selected from the list comprising, but not limited to, beta-galactosidase, luciferase, chloramphenyl acetyltransferase (CAT), betaglucuronidase (GUS) or fluorescent green protein (GFP). A more preferred embodiment refers to the sequence where the nucleotide sequence P encodes the functional protein reporter GFP.

Another preferred embodiment refers to the sequence of the present invention linked at its 5 'end to the 3' end of a nucleotide sequence encoding an RNA-dependent RNA polymerase (RpRd or also used the term replicase) and for a triple sequence gene block (TGB) of a potexvirus. In the present invention, this sequence is called RpRd-TGB. The sequence RpRd-TGB comprises both ORF (ORF1 corresponding to RpRd; ORF2 corresponding to TGB1; ORF3 corresponding to TGB2; ORF4 corresponding to TGB3) and the gene expression regulatory sequences of said ORF. The 2,3 and 4 ORFs partially overlap in different reading frames.

In a more preferred embodiment, the nucleotide sequence encoding the RpRd-TGB sequence belongs to the PepMV virus. In this case, that is, in the case that the RpRd-TGB sequence belongs to PepMV virus, the RNA-dependent RNA polymerase (RpRd) has a size between 163 and 164 kDa and the TGB sequences encode 26 kDa peptides , 14 kDa and 9 kDa, respectively.

Another aspect of the present invention is an expression vector comprising the sequence of the invention; or said sequence linked to the sequence P; or any previous sequence linked to an RpRd-TGB sequence.

The term "vector" refers to a DNA fragment that has the ability to replicate in a given host and, as the term indicates, can serve as a vehicle to multiply another DNA fragment that has been fused to it (insert). Insert refers to a DNA fragment that is fused to the vector; In the case of the present invention, the vector may comprise any sequence described according to the above aspects and embodiments which, when fused thereto, can be replicated in the appropriate host. The vectors can be plasmids, cosmids, bacteriophages or viral vectors, without excluding other types of vectors that correspond to the definition made of vector.

According to a preferred embodiment, the expression vector is a binary vector, that is, it is a vector capable of replicating in two types of host cell. The host cell must be that cell suitable for the type of expression vector used, that is, the vector must comprise the genetic elements necessary for self-replication and / or expression in said host cell. Said vector can be replicated and / or expressed, for example but not limited, in two different species of bacteria or in one species of bacteria and another of yeast. A more preferred embodiment is the expression vector where the host cell is a bacterium. The article "a" is not intended to limit the number of strains, species or bacteria cells but indicates that the host cell belongs indeterminately to a bacterium.

Another preferred embodiment refers to an expression vector where the sequence of the invention; or the sequence of the invention linked to the sequence P; or any previous sequence linked to an RpRd-TGB sequence, is part of a tDNA. Transfer DNA (tDNA) is a segment of the Ti plasmid (tumor inducer) of the soil bacterium Agrobacterium tumefaciens, which can be transferred to plant cells, where it is integrated into the genome. The tDNA does not contain any gene important for the transfer process, except for two short sequences with imperfect repeats located at its edges (LB and RB). By means of genetic manipulation, the original sequences of the DNAt of the microorganism have been replaced, which turned the plant into a nutrient factory

A. tumefaciens, for others that usually allow the selection of transformed tissue or plant and / or for genes of scientific or biotechnological interest (NPTII, marker genes or other genes).

Hereinafter the term "vector of the invention" or "vector of the present invention" will be used to refer to any expression vector described above.

The vector of the invention may contain one or more repetitions of any of the sequences involved in the regulation of gene expression.

Another aspect of the present invention is a cell that transiently or stably comprises the sequence of the invention, or said sequence linked to the P sequence; or any previous sequence linked to an RpRd-TGB sequence; or the vector of the invention. The term "cell" as understood in the present invention refers to a prokaryotic or eukaryotic cell. Thus, the term cell comprises, at least, one parenchyma cell, meristematic cell or of any type, differentiated or undifferentiated. Likewise, a protoplast (cell of a plant that lacks a cell wall) is also included in this definition.

According to a preferred embodiment said cell belongs to a plant species. The plant cell refers to a plant eukaryotic cell and within this group, more preferably, to those cells belonging to the Plantae kingdom.

Hereinafter the term "cell of the invention / of the present invention" or "plant cell of the invention / of the present invention" will be used to refer to any cell described above.

Another aspect of the present invention relates to pollen comprising the plant cell of the invention. This aspect is of high interest since the transmission of genetic and phenotypic characters can be carried out by pollination of any plant variety compatible with the pollen referred to. In this way, a plant comprising any of the sequences described in the present invention is achieved and, after the respective crosses and selections, a plant can be obtained in which the sequence is stably integrated and in a number of copies suitable for get the same desirable characters in later generations.

Another aspect of the present invention is a plant comprising the cell of the present invention. According to a preferred embodiment, the plant belongs to the genus Solanum. According to a more preferred embodiment, the plant belongs to the species Solanum lycopersicum.

The term "plant" encompasses each part of it, which can be preserved or cultivated in isolation or in combination. The plant may contain any sequence of the invention described in previous paragraphs, in homozygosis, heterozygosis or hemicigosis.

Hereinafter, to refer to the plant described, the term "plant of the invention" or "plant of the present invention" will be used.

The plant of the invention can be achieved by biolistic-mediated genetic transformation, Agrobacterium tumefaciens or any other technique that allows the integration of any of the sequences of the invention into the plant's DNA, be it genomic, chloroplast or mitochondrial. Likewise, the plant can also be achieved by transferring any of the sequences of the invention by crossing, that is, by using pollen of the plant of the invention to pollinate any other plant that does not contain any of the sequences of the invention or pollinating the gynecees. of plants containing any of the sequences of the invention with other plant pollen that do not contain these sequences. The methods for achieving the plant of the invention are not limited exclusively to the techniques described in this paragraph. Thus, for example, the genetic transformation by Agrobacterium tumefaciens by means of agroinfiltration, can be carried out by "magnification", that is, by immersion of the whole plant in a diluted suspension of Agrobacterium tumefaciens (which comprises the sequence of the invention) and later vacuum application (0.5-1 bar) for 1-2 minutes.

Another aspect of the present invention relates to the germplasm of the plant of the invention. Germplasm is defined by that biological material that contains intraspecific genetic variability or genetic materials that can perpetuate a species or population of an organism. Thus, germplasm is the seed, tissue culture of any part of the plant or plants established in ex situ collections, without excluding any other material that is contemplated in this definition.

A further aspect of the present invention refers to the use of any sequence of the invention defined in preceding paragraphs, for the construction of an expression vector.

Another aspect of the present invention is the use of any sequence of the invention defined in preceding paragraphs, or of the vector of the invention, for the detection of at least one plant of the Solanum genus resistant to PepMV virus. According to a preferred embodiment, the plant is of the species Solanum lycopersicum.

For the detection of at least one plant of the genus Solanum or at least one plant of the species Solanum lycopersicum, resistant to PepMV virus, it is necessary to inoculate or transfect with any of the sequences of the present invention that are capable of expressing a P sequence in any of said plants and proceed to the detection of messenger RNA (mRNA) encoded by the nucleotide sequence Pola protein detection. The plant in which said product is detected, will be a plant susceptible to infection by PepMV virus. On the contrary, the plant in which said product is not detected, will show resistance against PepMV virus. It is possible to find intermediate results in terms of the detection of said products encoded by the P sequence. In this case, the quantity of product detected in the inoculated or transfected problem plant is compared with the amount of product detected in a control plant inoculated or transfected with the same sequences as the problem plant. Detection is carried out by any of the techniques known in the state of the art. The control plant is a plant sensitive or resistant to PepMV virus.

A further aspect of the present invention refers to the use of the cell of the invention or of the plant of the invention, for the production of a protein, functional or non-functional.

A preferred embodiment refers to the use of the cell of the invention or of the plant of the invention, where the production of the functional or non-functional protein comprises co-transfection, simultaneously or not, of said cell or said plant, with a sequence nucleotide encoding a capsid protein (CP) of a potexvirus. According to a more preferred embodiment, the potexvirus is PepMV virus, that is, the sequence coding for the CP protein is PepMV virus.

The production of a functional or non-functional protein can be carried out by any cell or plant comprising any sequence of the present invention where SEQ ID NO: 1 is linked at its 5 'end to the 3' end of a nucleotide sequence that encodes for an RNA-dependent RNA polymerase (RpRd) and for a triple gene block sequence (TGB) of a potexvirus (preferably PepMV virus). As explained in a previous paragraph, the ORFs of these sequences as well as the regulatory sequences of gene expression are also included (in the subsequent paragraphs that mention this sequence this definition is also understood, even if it is not mentioned).

Cotransfection is the joint transfection of a transfected cell, at the same time (simultaneous) or already previously transfected (not simultaneous), with a nucleotide sequence encoding a CP protein. Said sequence coding for CP can be co-transfected by any technique already mentioned in previous sections. Also, said sequence may be part of an expression vector.

PepMV CP protein is not necessary for multiplication at the unicellular level, but for cell-to-cell movement of PepMV virus. As demonstrated in the examples of the present invention, the contribution of the sequence coding for the CP protein contributed by cotransfection (transient or stable integration), that is, provided in trans, increases the infectivity of the transfected sequences in said plant (preferably any sequence of the present invention where SEQ ID NO: 1 is linked at its 5 'end to a 3' end of a nucleotide sequence encoding an RNA-dependent RNA polymerase (RpRd) and a triple gene block sequence ( TGB) of a potexvirus [preferably PepMV virus]). This increase in infectivity produces the expression of the P sequence of interest in a greater amount of plant cells, which means a higher yield in the production of the protein for which it encodes. In the examples of the present invention it is demonstrated that the performance of complementation tests in which CP was provided in trans showed that it was possible to restore the cell-to-cell movement of plants that do not express the sequence coding for the CP protein.

Another aspect of the present invention relates to a method for obtaining the vector of the invention, which comprises:

to.

Select any sequence described in the present invention,

b.

inserting the sequence of section (a) into an expression vector capable of replicating in at least one type of host cell, and

C.

select the vector obtained according to section (b) comprising said sequence.

The insertion of any of the sequences selected in section (a) into an expression vector can be carried out by means of cloning methods that are part of the common general knowledge, by cutting the sequences and the vector with enzymes of restriction and its subsequent ligation, so that the sequence of the vector integrates the sequence selected in section (a).

The selection of the vector obtained in section (c) can be carried out by techniques such as, but not limited to, the selection of cells containing the vectors of the invention by the addition of antibiotics to the culture medium. The resistance of these cells to substances such as antibiotics is caused by the synthesis of products encoded by a sequence contained in the sequence of the vector. For the selection of the vector obtained in the section

(c) the presence of the sequence inserted in section (b) must be determined by any technique known in the state of the art such as, but not limited to, fragment size analysis obtained by digestion with enzymes of restriction and / or sequencing of said fragments or of the vector.

Another aspect of the present invention is a method for obtaining the cell of the invention, which comprises:

to.

Obtain a cell from a biological sample,

b.

transfect the isolated host cell of section (a) with the vector of the invention, and

C.

select the transfected cell according to section (b) comprising said sequence.

In the present invention, the term "biological sample" refers to a sample of both microorganisms and plant tissue. Obtaining the cell is intrinsic to obtaining the tissue. Plant tissues are made up of differentiated cells, with exceptions, as is the case with meristematic tissues where cells are undifferentiated. Plant cells are totipotent, that is, they contain a complete copy of the genetic material of the plant to which it belongs regardless of its function or position in it, and therefore has the potential to regenerate a whole new plant.

The biological sample and, in particular, the cell, is obtained from any type of microbiological culture (for example

E. coli or Agrobacterium tumefaciens) or plant tissue.

Transfection involves the introduction of external genetic material into the nucleus of eukaryotic cells. In the present invention, the term transformation can be used as a synonym for transfection. The genetic transformation of the cells obtained in section (a) is carried out by means of techniques that are part of the common general knowledge, such as, for example, biolistic-mediated genetic transformation, Agrobacterium tumefaciens or any other technique that allows the integration of any of the sequences of the invention in the cell's DNA. By means of these techniques it is possible to introduce, in a transient or stable way, a vector that includes any of the sequences described in the present invention, so that, after successive divisions of the cell, the incorporated sequence continues to be expressed, in the case of an insertion stable, or stop expressing itself, in the case of a temporary insertion. The cell transformed with the vector of the invention can integrate any sequence described in the invention, into any of the cell's DNA; nuclear, mitochondrial and / or chloroplastic or, remain as part of a vector that has its own machinery for self-replication.

The cell selection in section (c) can be carried out by means of any technique known in the state of the art such as, but not limited to, the addition of antibiotics to the culture medium that supplies nutrients to them.

The selected cells, if they are vegetal, can undergo a program of somatic organogenesis or embryogenesis whereby, after their differentiation by means of a suitable combination of plant hormones and other compounds, a stem or a complete plant is given that It contains the genetic material of the original cell from which it comes.

Another aspect of the present invention relates to a method for the detection of at least one plant of the genus Solanum, resistant to PepMV virus, comprising:

to. select at least one plant from a collection of mutant lines of the genus Solanum,

b.

inoculate at least one plant of section (a) with any sequence of the invention linked to a nucleotide sequence P, or with the vector of the invention comprising any of the sequences corresponding to this section, and

C.

select at least one PepMV virus resistant plant that does not express the functional or non-functional protein.

According to a preferred embodiment of the method, the plant of the genus Solanum belongs to the species Solanum lycopersicum.

The collection of mutant lines of the genus Solanum (or of the species Solanum lycopersicum) can be obtained, for example, but not limited, by chemical mutagenesis. Preferably the mutants belong to the F2 generation, that is, to the plants obtained from the germination of the seeds produced by the F1 plants. F1 plants are plants from seed germination obtained from parental mutant plants.

Plants of the genus Solanum (or of the species Solanum lycopersicum) have no obvious symptoms of infection after 10 days post-inoculation with the PepMV virus. The detection of viral infection therefore requires a complementary technique for the evaluation of susceptibility. The development of these constructions bearing a reporter gene would avoid the use of these complementary techniques since the detection would be carried out by means of ultraviolet light lamps for example, expressing fluorescence (due to the presence of the reporter protein) only susceptible plants, that allow virus replication.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

Description of the fi gures

Fig. 1. Shows the strategy used to generate the PepXL6agg fragment. The position and orientation of the initiators is indicated.

Fig. 2. Shows the scheme followed for the generation of PepGFPΔCP mutants.

The molds used, the ampli fi ed region, the position and orientation of the initiators in each case and the restriction sites used are indicated. 1) PepGFPΔCP1, 2) PepGFPΔCP2, 3) PepGFPΔCP3 and 4) PepGFPΔCP4.

Fig. 3. Shows the scheme followed for the generation of the pBPep501 construction.

The molds used, the ampli fi ed region, the position and orientation of the initiators are indicated.

Fig. 4. Shows the scheme followed for the generation of the construction pBPep5.128.

The molds used, the ampli fi ed region, the position and orientation of the initiators are indicated.

Fig. 5. Shows the steps followed for the development of the pBPepGFPΔCPCh2 construction.

The molds used, the ampli fi ed region, the position and orientation of the initiators in each case and the restriction sites used in the subcloning are indicated.

Fig. 6. Shows the RT-PCR product using primers CE-42 and CE-43 on total N. benthamiana RNA extract inoculated with PepMV-SP13. DNA lambda / Hind III marker (lane 0) and RT-PCR product (lane 1).

Fig. 7. Shows the result of a membrane hybridization showing the presence of PepMV in agroin fi ltered plants with clones pBPepXL6.3, pBPepXL7.7 and pBPepXL 9.1a5dpi.

In the upper part, the imprints of petioles sections of 3 agroin fi ltered plants are numbered from 1 to 3. Two imprints are included, one with an agro-fi ltered sheet (left imprint) and another with a systemically infected leaf (right imprint). C-corresponds to the imprints of agroin fi ltered plants with the negative controls, pBIN61 and pBGFP. In the lower right is a positive control of RNA transcribed from the clone pLMPepMV

0.5. Viral RNA was detected with a digoxigenin labeled probe complementary to the PepMV replicase gene.

Fig. 8. It shows the DNA constructs used to study the implication of CP in the multiplication of PepMV.

A) PepXL.6 (from the agroinfective clone), unmodified PepMV genome. B) PepXL6agg construction, the arrow indicates point mutation from ATG to AGG of the start codon of the CP. C) PepGFPΔCP construction, in which part of the ORF of the CP has been replaced by the complete ORF of the GFP.

Fig. 9. It shows the Northern-blot analysis of total RNA extracts from inoculated N. benthamiana protoplasts PepXL6 (XL6), PepXL6agg (XL6agg) and PepGFPΔCP1 (ΔCP1).

C-corresponds to total RNA extracts from uninoculated protoplasts and C + corresponds to total RNA extracts from agroinfiltrated leaf with the agroinfective clone. Viral RNA was detected by molecular hybridization with a probe labeled with digoxigenin complementary to PepMV replicase. The load control is shown in the lower panel. The size of PepMV genomic RNA (rRNA) is indicated.

Fig. 10. It shows the foci of infection initiated by PepGFPΔCP1 in cells of the epidermis and leaf mesophyll

N. benthamiana directly agroin fi ltered.

A) Cell isolated from the epidermis. B) Group of 3 mesophilic cells. C) Agrofiltered area where several foci of infection are observed. D) Area agroin fi ltrada with the construction control pBGFP. All images were taken at 6 dpi in the confocal microscope.

Fig. 11. Shows the viral constructs used in the agrofiltration tests in N. benthamian.

A) Clone pBPepGFPΔCP1, B) Clone pBPepGFPΔCP2 C) Clone pBPepGFPΔCP3 and D) pBPepGFPΔCP4. The expression of the cloned inserts between the left (LB) and right (RB) ends of the Ti plasmid of A. tumefaciens is controlled by the promoter and the 35S terminator. All constructs were transformed into A. tumefaciens (strain C58C1 and virulence plasmid pCH32).

Fig. 12. Shows leaves of agri-infiltrated N. benthamiana with the different combinations of plasmids used.

The agroin fi ltered regions are marked with a broken line. Within each agroin fi ltered region, the combination used is indicated. ΔCP1: pBPepGFPΔCP1, ΔCP2: pBPepGFPΔCP2, ΔCP3: pBPepGFPΔCP3, ΔCP4: pBPepGFPΔCP4, p19: pB19 and CP: pBINCPep. In the image on the left, agroin fi ltrations are shown in the absence of CP and in the image on the right, agroin fi ltrations in the presence of CP.

Fig. 13. It shows the number of foci of infection initiated after agroinfiltration by PepMV mutants that do not express CP.

Clones used: pBPepGFPΔCP1 (ΔCP1), pBPepGFPΔCP2 (ΔCP2), pBPepGFPΔCP3 (ΔCP3) and pBPep GFPΔCP4 (ΔCP4). Infiltrations were performed in the presence of p19 (clone pB19). Isolated cells that expressed GFP were counted on 0.5 cm2 a6 and 10 dpi leaf discs. The experiment was repeated three times and each measurement results from the average of the values obtained in 9 leaf / construction discs. A, B, B, C, C, a, b and c represent homogeneous groups according to the average value of the number of cells expressing GFP / construction; constructions with the same letter do not show significant differences at the same 95% probability level, according to a Tukey test.

Fig. 14. It shows the restoration of the movement of PepMV mutants that do not express the CP by the contribution of the CP in trans by agrofiltration.

Clones used: pBPepGFPΔCP1 (ΔCP1), pBPepGFPΔCP2 (ΔCP2), pBPepGFPΔCP3 (ΔCP3) and pBPep GFPΔCP4 (ΔCP4). The agroin fi ltration of the constructions was performed in the presence of CP (clone pBINCPep) and p19 (clone pB19). Images taken at 6 (Panel A) and 10 dpi (Panel B) under UV light illumination.

Fig. 15. It shows the insertion of the GFP ORF into the PepMV genome and representation of the subgenomic RNAs that supposedly should be generated.

The GFP ORF should be expressed from a duplication of the CP subgenomic RNA promoter. The original ORFs, the region proposed as promoter of the subgenomic RNA (SGP and SGPd) of the CP, and the ORF of the GFP are shown in the figure. The approximate size of subgenomic RNAs is indicated on the right.

Fig. 16. Shows the scheme of PepMV constructions marked with GFP generated. Up pBPep501 and down pBPep5.128.

Fig. 17. It shows the comparison between the number of infection foci initiated by PepGFPΔCh2 and other PepMV mutants that do not express CP after agroinfiltration.

Clones used: pBPepGFPΔCP1 (ΔCP1), pBPepGFPΔCP2 (ΔCP2), pBPepGFPΔCP3 (ΔCP3), pBPepGFPΔ CP4 (ΔCP4) and pBPepGFPΔCPCh2 (ΔCPCh2). Infiltrations were performed in the presence of p19 (clone pB19). Isolated cells that expressed GFP were counted on 0.5 cm2 a6 and 10 dpi leaf discs. The experiment was repeated three times and each measurement results from the average of the values obtained in 9 leaf / construction discs. A, A, B, B, C, C, a, b, b, c and c represent homogeneous groups according to the average value of the number of cells expressing GFP / construction; constructions with the same letter do not show significant differences at the same 95% probability level, according to the Tukey Test.

Fig. 18. It shows the restoration of the movement of the PepGFPΔCPCh2 chimeric mutant (B) and comparison with the PepGFPΔCP1 mutant (A).

The agroin fi ltration of the constructions was performed in the presence of CP (clone pBINCPep) and p19 (clone pB19). Images taken at 6 and 10 dpi under UV light illumination.

Fig. 19. Shows the designs of the DNA constructs containing different regulatory sequences of the gene expression whose infectivity was tested.

Constructions 1,2 and 3 (pBINPepGFP1, 2 and 3) were not infective, that is, they did not express the GFP gene.The strategy for GFP expression is the same in all cases, that is, duplication of the regulatory sequence of the gene expression. Construction 4 (pBPep501) was infective.

The size of the gene expression regulatory sequences (SGP; Subgenomic Promoter) of each of the constructs is detailed below:

1.-124 nt; 72 nt (3 ’ORF4) + 38 nt UTR region + 14 nt (5’ CP).

2.-106 nt; 54 nt (3 ’ORF4) + 38 nt UTR region + 14 nt (5’ CP).

3.-88 nt; 36 nt (3 ’ORF4) + 38 nt UTR region + 14 nt (5’ CP).

4.-111 nt; 37 nt (3 ’ORF4) + 38 nt UTR region + 36 nt (5’ CP).

Examples

Materials and methods

The materials and methods necessary to carry out the present invention are first described since it is necessary to know this section in order to understand the examples described below.

1. Isolated viral and plant material

The viral isolate used for the development of the agroinfective clone (PepMV-Sp13) was revived from infected and lyophilized material from N. benthamiana (Aguilar et al., 2002. Arch Virol, 147: 2009-2015). For this, N. benthamiana plants were inoculated for approximately 25 days (5-6 leaves). After inoculation the plants were kept in a greenhouse with day and night conditions from 16 h at 25-26ºC and 8 h at 19ºC, respectively.

2. Development of a clone agroinfective of PepMV

For the generation of an agroinfective clone of PepMV, the steps detailed below were followed.

2.1. Synthesis of a complementary DNA (cDNA) to the total genome length

Generation of the cDNA corresponding to the total length of PepMV mRNA was performed by RT-PCR using as a template the total RNA obtained with TRI-reagent and primers with specific nucleotide sequences of the 5 'and 3' ends of the PepMV-Sp13 viral genome .

For the preparation of total leaf RNA, 0.1 g of fresh plant tissue was split. The tissue was triturated with liquid nitrogen in 1.5 ml eppendorf tubes and homogenized with 1 ml of TRI-reagent (Sigma). In the case of preparations from protoplasts, the resulting pellet was homogenized with 0.25 ml of TRI-reagent. In both cases the total RNA preparations were carried out following the manufacturer's instructions.

The primers were: CE-42 complementary to the 5 'end of the virus sequence, which contains a Bam HI target, and CE-43, complementary to the 3' end containing a 20 dTTP tail followed by the restriction targets: Xma I, Sma I, Apa I, Sac II and Kpn I (Table 1).

For the synthesis of the first cDNA chain, 3 μl (approx. 2 μg) of total RNA was used. The reverse transcription reaction was carried out in a final volume of 20 μl containing 4 μl of the reverse transcriptase buffer concentrated 5 times, 7.5 μl of the 10 μM EC 43 initiator, 1.5 μl of a mixture of 10 mM dNTPs, 1.5 μl of 100 mM DTT, 20 U of RNAse inhibitor (Roche) and 100 U of reverse transcriptase (Roche). This mixture was incubated at 43 ° C for 70 min. After the retrotranscription reaction, the product was purified using GENECLEAN turbo columns (Q-Biogene) to remove the salts from the reaction.

The first cDNA chain was amplified by PCR in a final volume of 50 μl. In the PCR, 6 μl of the reverse transcription product, 0.8 μM of each initiator (EC 42 and EC 43), 200 μM of the mixture of dNTPs and 3.75 U of Pfu Pyrobest (Takara) were used in the buffer supplied by the manufacturer, in a final volume of 50 μl. The DNA was denatured for 2 min at 94 ° C and 20 cycles of 15 s of denaturation at 94 ° C, 30 s of hybridization at 55 ° C and 12 min of polymerization at 72 ° C were performed. At the end of the 20 cycles an elongation time of 10 min was given at 72 ° C.

TABLE 1

Initiators used in the amplification of the full length cDNA of PepMV

2.2. Cloning in pTOPO-XL

The PCR ampli fi ed fragments were purified from a 0.8% agarose gel with violet crystal recommended for the purification of 3-10 kb fragments offered by the TOPO-XL PCR Cloning Kit (Invitrogen).

Once the bands were purified, they underwent an A-tailing reaction in order to add a dATP at the 5 ’ends of the insert and prepare it for ligation in the pTOPO-XL. The addition of the dATPs at the ends was performed in a reaction volume of 20 μl. For this, 16 μl (approx. 150 ng) of the purified PCR product, 0.8 μl of dATP at a final concentration of 0.2 mM, 2 μl of 10x reaction buffer of the Taq polymerase and 10 units of Ecotaq were used (Ecogen). The mixture was incubated at 72 ° C for 30 min. After the incubation, the reaction was purified with the ULTRAKIT ™ 15 (MO BIO) kit for salt removal.

The purified band was subjected to a standard ligation reaction following the manual of the pTOPO-XL Kit. The final product was transformed into Escherichia coli TOP10 by electroporation (Gene pulser II and capacitance extender, Bio-Rad laboratories, CA, USA) following the kit instructions.

The colonies obtained were analyzed after multiplication in LB medium (Luria-Bertoni). Plasmid DNA preparation was carried out by cultures from isolated colonies in 3 ml of LB medium at 37 ° C overnight. The cultures were centrifuged for 3 min at 7,500 rpm at 4 ° C. The resulting pellet was resuspended in 250 μl of resuspension buffer 1 (50 mM Tris-CI pH8.0; 10 mM EDTA; 100 μg / ml RNAse A, Qiagen). Subsequently 250 µl of lysis buffer 2 (200 mM NaOH, 1% SDS) was added, stirred 6-7 times and 250 µl of neutralization buffer 3 (AcNa 3M pH5.5) was added, stirred 6-7 times and allowed to incubate on ice 5 min. After incubation the tubes were centrifuged at 13,000 rpm for 15 min at 4 ° C. Supernatants were collected and 1 ml of 100% Et-OH was added to precipitate the DNA. It was then centrifuged at 14,000 rpm, 15 min at 4 ° C. The final pellet was resuspended in 50 ml sterile H2O.

An aliquot of the DNA obtained was digested with the restriction enzymes Bam HI and Eco RI (New England Biolabs) to perform a restriction analysis and check the direction of the insert. The clones carrying the insert were subsequently digested with the restriction enzymes Bam HI and Xma I, single-cut targets located at the ends of the virus genome and releasing the insert from the vector thus preparing it for cloning into the binary vector pBIN61. The selected clones were named pTPepXL6, pTPepXL7 and pTPepXL9.

2.3. Cloning in pBIN61

The binary vector pBIN61 and the clones carrying the inserts were digested with the restriction enzymes Bam HI and Xma I. For this, 1.5 μg of DNA was used in each case, to which 10 units of each enzyme were added and the buffer recommended by the manufacturer for double digestions in a final volume of 50 μl. The reaction was carried out for 3 hours at 37 ° C. The digested DNAs were subjected to 0.7% agarose gel electrophoresis, the gel bands of interest were recovered and purified using the GENCLEAN Turbo kit (Q-biogene). Once the bands were purified, the DNA concentration was estimated by electrophoresis.

100 ng of dephosphorylated vector and 100 ng of insert (1: 2 ratio) were added to the ligaments to which 40 ligase units (T4 DNA ligase; New England Biolabs) were added plus the appropriate buffer in a final volume of 20 μl. The mixture was incubated at 16 ° C overnight to obtain the maximum number of transformants. The ligation product was concentrated at a final volume of 3 μl by means of ethanol precipitation without glycogen salts (Roche) and transformed into E. coli TOP10 by electroporation.

For electroporation, the standard conditions recommended for E. coli (25 μF capacitance, 200 Ω resistance, 2500 V) in 0.2 cm cuvettes were followed. 1.5 µl of the concentrated ligation reaction was added to 40 µl of competent cells. After electroporation, the cells were transferred to 1 ml of LB and incubated at 37 ° C for 1 h with gentle shaking (100 rpm), to allow the cells to express the antibiotic resistance gene. They were then seeded in Petri dishes with LB-Agar medium containing kanamycin (50 ng / ml) and incubated overnight at 37 ° C.

Plasmid DNA preparation was performed in the same manner as for pTOPO-XL clones, that is, from colonies isolated in 3 ml of LB. The pellet in this case was resuspended in 30 μl. 4 μl aliquots of the DNA obtained were digested with the restriction enzymes Bam HI and Eco RI (New England, Biolabs) to perform the restriction analysis. Three clones were selected again from each ligation reaction. The constructions were named: pBPepXL6.1, pBPepXL6.2, pBPepXL6.3, pBPepXL7.7, pBPepXL7.8, pBIPepXL7.9, pBPepXL9.1, pBPepXL9.2 and pBPepXL9.3.

3. DNA constructs derived from the infective clone of PepMV

To carry out studies on the involvement of PC and possible regulatory regions of gene expression in viral multiplication, a series of constructions were developed that gave rise to PepMV mutants that did not express CP, called pT7PepXL6agg and pBPepGFPΔCP1, 2 , 3, and 4 (to which different modifications were introduced) and pT7PepGFPΔCP1, in addition to the construction pBINCPep.5, in which the PepMV CP is cloned. For the development of the viral vector, two carrier constructs of the GFP gene were generated that would give rise to two versions of the same vector, called: pBPep501 and pBPep5.128. For the assay of a promoter (in the present invention, gene expression regulatory sequence) heterologous the construction pBPepGFPΔCPCh2 was generated. Below are the steps taken to obtain each of the mentioned constructions in detail.

3.1. Construction pT7PepXL6agg

The PepXL6agg fragment was obtained by directed mutagenesis using the overlapping PCR method (Fig. 1). The mutation consisted in the change of the second nucleotide of the codon of translation initiation of the CP, from ATG to AGG. Three PCRs were carried out. In PCR1, 5,640 nt were amplified, which contain the replicase gene (in the present invention, RNA-dependent RNA polymerase [RpRd]), TGB and the first 11 nt of the capsid protein (CP protein) ; The initiators used were CE-42 (Table 1) and CE-199 (Table 2) carrying the desired mutation. In parallel, in PCR2 the complete CP and the 3’UTR end were amplified using the CE-198 initiators (with the same nucleotide change as the CE199) and the CE-43 (Table 1).

PCRs were carried out with 5 ng of template (plasmid pTPepXL6), 2.5 U of Pfu Pyrobest (Takara), 0.8 μM of the initiators, 200 μM of the dNTPs in the buffer supplied by the manufacturer and in a final volume of 50 μl. The mixture was subjected to the following PCR cycles: one cycle for 2 min at 94 ° C, 15 cycles of 15 s of denaturation at 94 ° C followed by 30 s of hybridization at 51 ° C and 10 min (PCR1) or 4 min (PCR2) of polymerization at 72 ° C. At the end of the 15 cycles an elongation time of 12 min (PCR1) or 6 min (PCR2) was given at 72 ° C.

For PCR3 the template used was the mixed product of previously purified PCRs 1 and 2. The PCR3 mixture was denatured for 2 min at 94 ° C and first 3 cycles of 30 s of denaturation at 94 ° C, 1 min of hybridization at 40 ° C and 12 min of polymerization at 72 ° C were performed, then subjected to 15 cycles of 15 s of denaturation at 94 ° C followed by 30 s of hybridization at 52 ° C and 12 min of polymerization at 72 ° C. At the end of the 15 cycles an elongation time of 12 min was given at 72 ° C.

The resulting fragment was cloned into the pTOPO-XL vector, giving rise to clone pTPepXL6agg. Then to place the construction under the control of the T7 transcription promoter, said promoter was cloned. The resulting fragment was cloned into the pTOPO-blunt vector giving rise to clone pT7PepXL6agg.

TABLE 2

Initiators used for generation of the PepXL6agg mutant. Underlined nucleotides introduce the corresponding mutation

3.2. PBPepGFPΔCP constructions

Four different constructs were generated in which PepMV CP was replaced by the GFP: pBPepGFPΔCP1, pBPepGFPΔCP2, pBPepGFPΔCP3 and pBPepGFPΔCP4 (Fig. 2). That is, the constructions achieved comprise the sequences SEQ ID NO: 1-SEQ ID NO: 2 linked by the 3 'end (corresponding to the 3' end of SEQ ID NO: 2) to a nucleotide sequence (P) encoding a Functional or non-functional protein and, in turn, this sequence block SEQ ID NO: 1-SEQ ID NO: 2-P, is connected by its 5 'end (corresponding to the 5' end of SEQ ID NO: 1) to the RpRd block -TGB belonging to the PepMV virus. That is, these constructions have a structure RpRd-TGB-SEQ ID NO: 1-SEQ ID NO: 2-P. In this case, the P sequence is the sequence that codes for the GFP protein. The main steps for the generation of these constructions were the following:

Step 1

Overlapping PCR that gave rise to a GFP-3'UTR fragment (in the case of constructions 1 and 2) or GFP-120.3'UTR fragment (in the case of constructions 3 and 4), carrier of the fused GFP gene to the 3 'end of PepMV

To obtain the GFP-3'UTR fragment, the GFP gene (PCR1) was first amplified with the primers CE-236 (Table 3) and CE-456 (Table 3), which includes an additional sequence of 18 nt complementary to the 3'UTR end of PepMV. In parallel, in PCR 2 the 3'UTR end of PepMV was amplified using the CE-457 primers (Table 3), which includes an additional 18 nt sequence complementary to the 3 'end of the GFP gene and CE-43 (Table one). The initiators CE-456 / CE-457 complementary to each other, allowed the overlapping of the previous fragments using the combination of the initiators CE-236 and CE-43.

An additional 120 nt of the C-terminal end of the CP was included in the GFP-120.3’UTR fragment. This fragment was generated in the same way as GFP-3’UTR using as complementary initiators CE-460 and CE-461.

As plasmid of PCR1 a plasmid pTOPO-GFP was used and as plasmid of PCR2 plasmid pTPepXL6 was taken. PCRs were carried out with 5 ng of mold, 2.5 U of Pfu Pyrobest (Takara), 0.4 μM of the initiators, 200 μM of the dNTPs in the buffer supplied by the manufacturer and in a final volume of 50 μl. The conditions of the PCRs were the same as those used in the generation of the pT7PepXL6agg construct by adjusting the hybridization temperature conditions and polymerization times to the temperature of the initiators (Tm) used and the size of the amplified fragment in each case.

Step 2

Overlapping PCRs for the generation of PepGFPΔCP fragments

PepGFPΔCP fragments were generated through overlapping PCRs. The plasmids, ampli fi ed regions and specific primers used in each case are specified in Fig. 2.

In PCR1, 1,100 nt of the PepMV genome were amplified, which contain part of the TGB and the start of the ORF of the CP with the Pep303 and CE-458 / CE-351 initiators (Table 3) as appropriate, which include an additional sequence of 18 nt complementary to the 5 'end of the GFP gene. To obtain the PepGFPΔCP1 and 3 fragments, plasmid pTPepXL6 was taken as the template for PCR1 with the combination of Pep303 and CE-458 initiators, which places the GFP ORF (start codon modified to AGG) at 36 nt of the codon of the beginning of the CP. To obtain the PepGFPΔCP 2 and 4 fragments as a template for PCR1, plasmid pTPepXL6agg was used with the combination of Pep303 and CE-351 initiators, which places the GFP ORF (with its own ATG) at 38 nt of the codon start of the CP (start codon modified to AGG), and also includes a Sac II target just upstream of the start codon of the GFP ORF.

In PCR2, the GFP-3'UTR or GFP-120.3'UTR fragment was amplified using the CE-459 primers (in the case of PepGFPΔCP1y3) or CE-353 (in the case of PepGFPΔCP2y4) (Table 3) that include an additional sequence of 18 nt complementary to the 5 'end of the CP and the CE-43. The initiators CE-458 / CE-459 and CE-351 / CE-353 complementary to each other, allowed the overlapping of the above fragments using the combination of the Pep303 and CE-43 initiators.

PCRs were carried out with 5 ng of template, 2.5 U of Pfu Pyrobest (Takara), 0.4 μM of the initiators, 200 μM of the dNTPs in the buffer supplied by the manufacturer and in a final volume of 50 μl. The conditions of the PCRs were the same as those used in the generation of the pT7PepXL6agg construct by adjusting the hybridization temperature conditions and polymerization times to the Tm of the initiators used and the amplified fragment in each case.

TABLE 3

Initiators used for the generation of PepGFPΔCP mutants

Step 3

The fragments resulting from PCR 3 were cloned into the pTOPO-blunt vector and subsequently sequenced. Once the sequences were confirmed, the clones were digested with Xmn I and Xho I (fl anch the modified region within the pTPepXL6) and subcloned into pTPepXL6. In this way the clones were obtained: pTPepGFPΔCP1, 2, 3 and

4. Finally, the fragments were placed under the control of the 35S promoter in pBIN61, between the BamH I and Xma I targets giving rise to the clones pBPepGFPΔCP1, 2,3 and 4.

3.3. Construction pT7PepGFPΔCP1

For the generation of the pT7PepGFPΔCP1 construct, the T7 promoter sequence was inserted at the 5 ’end of the pTPepGFPΔCP1 clone, obtained in the previous section.

3.4. PBINCPep construction

The pBINCPep construct will be used to co-transform (co-transfect) plants along with other constructs that lack the sequence coding for PepMV CP protein. It was obtained from the amplification of the ORF of the CP of plasmid pTPepXL6 with the primers CE-356, complementary to the 5 'end of the CP, and an oligo d (T) (Table 4). The fragment obtained was cloned in pBIN61 previously digested with Sma I which allows blunt end ligation.

The amplification of the CP was carried out by PCR. 5 ng of 2.5 U mold of Pfu Pyrobest (Takara), 0.5 μM of the initiators, 200 μM of the dNTPs were used in the buffer supplied by the manufacturer and in a final volume of 50 μl. The mixture was subjected to the following PCR cycles: one cycle for 2 min at 94 ° C, 20 cycles of 15 s of denaturation at 94 ° C followed by 30 s of hybridization at 40 ° C and 2 min of polymerization at 72 ° C. At the end of the 20 cycles an elongation time of 5 min was given at 72 ° C.

TABLE 4

Initiators used to generate the pBINCPep clone

3.5. PepMV constructions: GFP: pBPep501 and pBPep5.128

Two PepMV constructs carrying the GFP gene were generated: pBPep501 and pBPep5.128. The strategy used in the two cases was basically the same as for the generation of the pBPepGFPΔCP constructs, that is, through overlapping PCRs and subsequent subcloning in pTPepXL6. For the generation of construction pB-Pep501 (Fig. 3) the plasmid pTPepGFPΔCP1 with the combination of Pep303 and CE-502 initiators (Table 5) and in PCR2 the plasmid pTPepXL6 with the combination of primers were taken as templates in PCR1 CE-501 (Table 5) and CE-43 (Table 1).

For the generation of the construction pBPep5.128 (Fig. 4), plasmids pTPepXL.6 with the combination of initiators Pep303 and CE-503 (Table 5) and in PCR2 the plasmid pTPepGFPΔCP1 were taken as templates in PCR1 combination of initiators CE-504 (Table 5) and CE-43.

TABLE 5

Initiators used for generation of Pep501 and Pep5.128 mutants

PCRs were carried out with 5 ng of mold, 2.5 U of Pfu Pyrobest (Takara), 0.4 μM of the initiators, 200 μM of the dNTPs in the buffer supplied by the manufacturer and in a final volume of 50 μl. The conditions of the PCRs were the same as those used in the generation of the pT7PepXL6agg construct by adjusting the hybridization temperature conditions and polymerization times to the Tm of the initiators used and the amplified fragment in each case.

3.6. PBPepGFPΔCPCh2 Construction

The PepGFPΔCPCh2 fragment was the result of the substitution of the sequence selected as SGP (Subgenomic Promoter) of the PepMV-Sp13 CP (ie, SEQ ID NO: 1-SEQ ID NO: 2 of the present invention) by the homologous sequence PepMV-Ch2 (PeMU08 / 44), which has more than 80% identity with respect to SEQ ID NO: 1-SEQ ID NO: 2. For this purpose, the steps detailed below are followed and shown in Fig . 5:

1) Amplification of the homologue of SEQ ID NO: 1-SEQ ID NO: 2 belonging to PepMV-Ch2 with the pair of CE-497 initiators (includes an additional sequence of 17 nt complementary to the 3 'end of PepMV-Sp13 ORF 4 ) and CE-498 (includes an additional 18 nt sequence complementary to the 5 'end of the GFP ORF). Plasmid PeMU08 / 44 was used as the reaction template.

2nd) Amplification of the GFP ORF with the CE-499 primers (includes additional 18 nt sequence complementary to the 3 'end of the homologous sequence of SEQ ID NO: 1-SEQ ID NO: 2 belonging to PepMV-Ch2) and CE -43. The plasmid pTPepGFPΔCP1 was used as the reaction template.

3rd) Overlapping PCR of fragments 1 and 2 obtained in the previous steps using the pair of primers CE497 and CE-43.

4th) Amplification of part of the PepMV-Sp13 TGB with the Pep303 and CE-500 initiators (includes an additional sequence of 16 nt complementary to the 5 'end of the homologous sequence of SEQ ID NO: 1-SEQ ID NO: 2 belonging to PepMV-Ch2).

5th) Overlapping PCR of the fragments obtained in steps 3 and 4 using the pair of Pep303 and CE initiators

43. 6th) PCR cloning result of step 5 in pTOPOblunt. 7th) Subcloning to pTPepXL6 between targets Xmn I and Xho I, giving rise to the construction pTPepGFPΔCP1. 8th) Subcloning to pBIN61 between the BamH I and Xma I targets.

TABLE 6

Initiators used for generation of PepGFPΔCPCh2 construction

4. Isolation and inoculation of N. benthamiana protoplasts

The N. benthamiana protoplast isolation protocol that was used is an adaptation of the protocol described in Plant Virology Protocols, (Weston and Turner, 1998. Methods in Molecular Biology. Edited by GD Foster and SC Taylor. Totowa, NJ: Humana Press , 81: 301-305). For the isolation of N. benthamiana protoplasts, we started from 5-week-old plants grown in phytonron with a photoperiod of 16 h of light at 25 ° C and 8h of darkness at 18 ° C. The fully expanded sheets were surface sterilized by immersion, for 30 min, in a 10% sodium hypochlorite solution to which 2-3 drops of Tween-20 were added. The disinfectant solution was removed by three successive washes (5, 10 and 15 min respectively) with sterile distilled water. The disinfection and washing processes were carried out under gentle and discontinuous manual agitation. Once disinfected, cross sections were made from the central nerve to the edge of the leaf, leaving an approximate distance between 1-2 mm cuts. The cut leaves were placed (beam up) in large Petri dishes (15 cm in diameter), containing 25 ml of MMC buffer (13% Mannitol, 5 mM MONTH and 10 mM CaCb, pH 5.8) and thus maintained 30 minutes at room temperature. After this time the MMC buffer was removed and 25 ml of enzyme solution was added per plate. The enzymatic solution consisted of 0.5 g Onozuka Cellulase R-10 (Yakult Honsha), 0.25 g Macerozime R-10 (Duchefa Biochemies), and 100 ml MMC buffer, at pH 5.8. Sterilization of the enzyme medium was performed by filtration through filters of 45 μm pore size (Millipore Corporation, Bedford, Madison, USA). The plates were sealed with para fi lm and incubated in the dark at 25 ° C overnight. After the digestion, the crude suspension of protoplasts was removed from the plates, taking care not to pick up plant debris, and distributed in 50 ml falcon tubes. They were centrifuged at 1,000 rpm for 4 min at 20 ° C. The supernatant was removed and the precipitate was gently resuspended in 15 ml MMC buffer. The protoplast suspension was centrifuged again at 1,000 rpm for 4 min at 20 ° C. The resulting precipitate was resuspended in 6 ml of MMC buffer and placed on a sucrose mattress (15 ml falcon tube which contained 7 ml of 20.5% sucrose). After centrifugation at 1,500 rpm for 15 min, the protoplast ring formed at the interface was collected with a Pasteur pipette, resuspended in 50 ml of cold electroporation buffer (150 mM NaCl, 4 mM CaCl2, 0.4 M Mannitol and 10 mM Hepes, pH 5.8) and the counting was carried out. They were centrifuged at 1,000 rpm for 4 min and the precipitate was resuspended so that it contained 1x106 protoplates / 500 µl of electroporation buffer. They were distributed in eppendorf tubes and kept on ice.

The protoplasts were inoculated with RNA transcribed by electroporation. For this, 5 and / or 10 μg of transcribed RNA were mixed with 1x106 protoplasts. Electroporation was carried out in the Gene pulser II and capacitance extender (Bio-Rad laboratories, CA, USA) at 1,000 μF, 180 V and 100 Ω. After electroporation, the protoplasts were transferred to a 1.5 ml eppendorf tube, left on ice for 20 min and centrifuged at 70 g, 5 min at 4 ° C.

The resulting pellet was resuspended in 1.5 ml of Aoki and Takebe culture medium (13% Mannitol, 5 mM MES and 10x Sales Aoki and Takebe: 2 mM KH2PO4,10mMKNO3,10mMMgSO4,10 μM KI, 1 μM CuSO4, 100 mM CaCl2) and were seeded in Petri dishes with a diameter of 2.5 cm that had previously been coated with a solution containing 1% agarose, 13% mannitol and 1% sucrose. The plates were incubated in the dark at 25 ° C. After the incubation, the protoplasts were collected and centrifuged at 70 g, 5 min at 4 ° C. The resulting pellet was immediately frozen with liquid nitrogen and RNA was extracted.

5. Agroinfiltration tests

5.1. Agrobacterium transformation

Agrobacterium tumefaciens cells (strain C58C1 and virulence plasmid pCH32) were transformed by electroporation with the corresponding vector in each case (bearing any of the sequences described in the present invention). Electroporations were carried out on the Gene pulser II and capacitance extender electroporator, in ice-cold pails and in accordance with the manufacturer's instructions (25 μF capacitance, 400 Ω resistance, 2500 V). 0.5 μl of plasmid was added to 20 μl of Agrobacterium electrocompetent cells. After electroporation, the cells were transferred medium LB and cultured for 3 h at 28 ° C with gentle agitation (80 rpm) to allow the cells to express the antibiotic resistance genes. They were seeded in Petri dishes with LB-Agar medium containing the corresponding antibiotics and grown at 28 ° C for 48

h.

5.2. Agroinfiltration

Agrofiltration consists of a transformation (or transfection) of N. benthamiana leaves by infiltration of Agrobacterium tumefaciens cells transformed according to the previous section. The isolated colonies of transformed Agrobacterium were inoculated in 3 ml of LB medium with the antibiotics tetracycline (5 ng / ml) and kanamycin (50 ng / ml) and were incubated at 28 ° C overnight with vigorous agitation (150 rpm). The cells were precipitated by centrifugation at 5,000 rpm for 10 min at 4 ° C and resuspended to an OD600 of 0.6 in the agroinfiltration solution (10 mM MgCl2; 10 mM MES pH 5.6 and 150 μM acetosyringone [Sigma]) . The cultures were incubated for a minimum of 2 room temperature before agrofiltration. In co-filtrations of PepMV mutant constructs in the presence of CP, equal volumes of both cultures were mixed before agrofiltration.

Agrofiltration was performed on the underside of N. benthamiana leaves (approximately 25 days) with the help of a 1 ml syringe.

6. Extraction of total RNA from leaves and protoplasts from N. benthamiana

For the preparation of total leaf RNA, 0.1 g of fresh plant tissue was split. The tissue was triturated with liquid nitrogen in 1.5 ml eppendorf tubes, homogenized with 1 ml of TRI-reagent (Sigma). In the case of preparations from protoplasts, the resulting pellet was homogenized with 0.25 ml of TRI-reagent. In both cases the total RNA preparations were carried out following the manufacturer's instructions.

7. PepMV viral RNA detection

7.1. Synthesis of probes

The probes used to detect PepMV RNA were obtained by in vitro transcription of the DNA inserted into plasmids pGPepORF1 + and pGPepCP0.3. Plasmid pGPepORF1 + carries a 3,500 bp fragment corresponding to a region of the virus genome viral replicase gene. Plasmid pGPepCP0.3 carries a complementary 850 bp insert to part of the TGB and part of the CP.

The probe used for the detection of GFP was obtained from plasmid pG208GFP (Baulcombe et al., 1995. Plant J, 7: 1045-1053). This plasmid carries a complementary fragment to the GFP10yalaCPde PVX gene.

To prepare the probes, techniques known in the state of the art using DIG-11-UTP are used to label the isolated sequences.

7.2. Dot blot and tissue-print analysis

The tissue-print sample analysis was performed on leaf petiole imprints and dotblot analysis on total RNA extracts, both in positively charged nylon membranes (Roche). The process of hybridization and chemiluminescence detection was carried out essentially following the protocol proposed by Roche (RNA labeling and detection kit): after the fixation of nucleic acids with ultraviolet light (CL-1000 “Ultraviolet Crosslinker” UPV) the membrane for two hours in standard buffer at 68 ° C and hybridized overnight at 68 ° C with the probe in this same buffer, to which the probe was added. After hybridization the membranes were washed once with 2XSSC, 0.1% SDS for 5 min at room temperature and twice with 1XSSC, 0.1% SDS, for 15 min at 68 ° C. The detection process was then carried out at room temperature and with gentle agitation: the membrane was rinsed in a washing solution for 5 min (0.1 M maleic acid, 0.15 M NaCl, pH 7.5 and 0.3% tween 20 v / v) 15 min in solution I (0.1 M maleic acid, 0.15 M NaCl, pH 7.5), 30 min in solution II (solution I plus 1% blocking agent), 30 min in solution II plus the DIG antibody conjugated to alkaline phosphatase (PA) at the recommended dilution, and 2x15 min in wash solution. Finally the membrane was equilibrated for 5 min in solution III (0.1 M Tris-HCl, 0.1 NaCl, pH 9.5), the luminescent PA substrate (CSPD, Roche, 1: 100 dilution in solution was added III) and incubated in darkness 5 min. The emitted light impressed X-ray films (Roche) that were developed according to conventional methods.

7.3. Northern blot analysis

For the Northern blot analysis, 5 μg of total RNA was denatured for 4 min at 65 ° C in the corresponding volume of the 5X loading buffer (bromophenol, EDTA, formaldehyde, glycerol, FA and sterile water), then kept on ice so far of being loaded on the gel. Denatured samples were subjected to 1.5% agarose gel electrophoresis in buffer containing 20 mM HEPES, 1 mM EDTA pH 8.0 and 6% formaldehyde. Electrophoresis was performed at 30 V for approximately 4 h. After electrophoresis, the RNAs were transferred to a nylon + membrane, fixed by ultraviolet light and hybridized with DIG-labeled RNA probes as described in the previous section.

8. Sequence analysis

Sequence analysis and alignment was carried out using the ClustalXW programs available at www.ebi.ac.uk/clustlw (Thompson et al., 1997. Nucl Acids Res, 25: 4876-4882) and Bioedit v.7.0 available at http://www.mbio.ncsu.edu/BioEdit. From the alignments, the percentages of nucleotide similarities with ClustalXW were calculated.

9. GFP display

The fluorescence emitted by the GFP on N. benthamiana sheets was visualized with a 100 W Handheld long-wave ultraviolet lamp (UV products, Upland, CA 91786, Black Ray model B 100AP) and with a Leica TCS inverted confocal laser miscroscope SP2 (Leica Microsystems, Germany).

Example 1

Obtaining an “agroinfective” clone of PepMV

For the development of an infective clone of PepMV, a strategy based on the following steps was proposed: 1) obtaining cDNA covering the full length of the genome of the virus, 2) cloning the cDNAs into an intermediate vector (pTOPO-XL) and 3) subcloning into a binary vector (pBIN61) so that the cDNA is under the control of the 35S promoter (it should be noted that within the cited cDNA are the regulatory sequences of the gene expression SEQ ID NO: 1-SEQ ID NO: 2). This binary vector allows the synthesis of viral RNA in vivo.

Based on the sequence of the PepMV-Sp13 isolate, primers CE-42 and CE-43 were designed (Table 1). These primers included Bam HI and Xma I targets for the 5 'and 3' ends, respectively, so that the cDNAs obtained could be directionally cloned into pBIN61. Using these primers in RT-PCR experiments on a total RNA extract of an infected N. benthamiana plant, cDNAs corresponding to the total length of the genomic RNA (mRNA) of PepMV-Sp13 (6.4 Kb) were obtained. In addition to the DNA fragment of the expected size, other smaller fragments were generated in the RT-PCR, possibly the product of non-specific initiation of the initiators (Fig. 6).

The 6.4 Kb DNA fragment was purified, ligated and cloned in E. coli in plasmid pTOPO-XL as described in the corresponding materials and methods section. It was decided to proceed with three clones resulting from the ligation, since, despite using high-fidelity polymerases in the amplification and a low number of cycles, deleterious mutations could be introduced that would be individualized in each independent clone. The clones generated were pTPepXL6, pTPepXL7 and pTPepXL9. Next, the 6.4 Kb DNA fragment was subcloned into pBIN61 between the Bam HI and Xma I targets. Again three clones were selected for each of the starting clones giving rise to nine clones: pBPepXL6.1, pBPepXL6 .2, pBPepXL6.3, pBPepXL7.7, pBPepXL7.8, pBIPepXL7.9, pBPepXL9.1, pBPepXL9.2 and pBPepXL9.3.

A. tumefaciens was transformed with each of the constructs in pBIN61, and the liquid cultures of the resulting strains were infiltrated in N. benthamiana plants. After 5 days post-agroinoculation (dpi) symptoms were observed in 100% of the agroin fi ltered plants with the clones pBPepXL6 (1 to 3) and pBPepXL9 (1 to 3). Agro-infiltrated plants with pBPepXL7 clones (1 to 3) showed symptoms at 7 dpi. The symptoms were similar to those typically induced by PepMV-Sp13.

To verify the presence of viruses in the agroinoculated plants, a hybridization test was performed (Fig. 7). The analysis was carried out on imprints of petioles sections of agroinoculated leaves and non-inoculated leaves at 5 dpi. The result of hybridization was, as expected, positive for all plants, so it could be deduced that the clones were effectively infective.

Of the clones obtained, pBPepXL6.3 (from now on pBPepXL6) was chosen to work in subsequent tests. Partial sequencing of this clone revealed the existence of mutations that caused amino acid changes and silent mutations with respect to the published PepMV-Sp13 sequence. A total of 4 mutations were detected, of which only one, located in the TGB3 gene, results in an amino acid change. However, these appeared not to affect the infectivity of the clone.

Example 2

Study of the involvement of CP in the multiplication of PepMV

Since the construction of a viral vector based on PepMV could affect the expression of the CP gene (see below), it was previously decided to conduct a study on the involvement of CP in the multiplication of PepMV.

To carry out this study, constructions that alter the expression of the CP were designed from the agroinfective clone of PepMV. Two types of modifications were introduced. On the one hand, a point mutation was introduced in the supposed start codon of the CP ORF, changing it from ATG to AGG (Fig. 8, B). Quite possibly this mutation cancels the initiation of the CP translation since the following ATG codons of the PepMV CP ORF are not in a favorable context for the start of its translation. On the other hand, part of the ORF of the CP was replaced by the complete ORF of the GFP, placing it at 36 nt of the start codon of the CP and in the same reading pattern (Fig. 8, C). The effect of the modifications made was evaluated in N. benthamiana protoplasts and by agrofiltration tests on N. benthamiana leaves. With the first type of assays we aimed to determine effects on directly inoculated single cells, and with the second type of assays we also wanted to analyze possible effects of mutations in cell-to-cell and long-distance movement of the virus.

Example 3

Effect of CP on the multiplication of viral RNA in prototypes of N. benthamiana

To study the effect of mutations in isolated cells, clones pT7PepXL6agg (PepXL6agg), pT7PepGFPΔCP1 (PepGFPΔCP1) and pT7PepXL6 (PepXL6, infective clone) were generated, so that each of the DNA constructs was placed under the control of the promoter of transcription T7.

First, the behavior of PepXL6agg was studied. To that end, N. benthamiana protoplasts were inoculated by electroporation with transcribed RNAs synthesized in vitro from pT7PepXL6agg and pT7PepXL6. After 24 h post-inoculation, total RNA extractions were performed.

Northern blot analysis of the samples obtained showed a notable difference in viral RNA accumulation between total RNA extracts from protoplasts inoculated with PepXL6 RNA and those inoculated with PepXL6agg RNA (Fig. 9). These results, which were consistently repeated in three independent experiments, indicated that the modi fi cation introduced in PepXL6agg causes a reduction in the levels of viral RNA accumulation in inoculated cells. This reduction could be due to a direct or indirect involvement of the CP in the unicellular multiplication of PepMV, but they could also be the result of the instability of the viral RNA due to the non-translatability of the CP ORF. To investigate this aspect, PepGFPΔCP1 was prepared and tested (Fig. 9).

In samples from total RNA extracts of protoplasts inoculated with PepXL6 and PepGFPΔCP1 RNA (Fig. 9, lanes 1 and 3, respectively) similar levels of genomic viral RNA (gRNA) were detected, significantly higher than those obtained with PepXL6agg ( Fig. 9, street 2). The results obtained indicate that CP is not necessary for the multiplication of PepMV at the unicellular level and suggests that the reduced accumulation of PepXL6agg viral RNA is due to the instability of the RNA associated with the non-translability of the CP ORF.

This fact allows the cloning of a nucleotide sequence P that codes for a functional or non-functional protein, such as a reporter protein since, as mentioned, said CP viral protein is not necessary for virus multiplication. As described in the present invention, the sequences SEQ ID NO: 1-SEQ ID NO: 2, located in the region upstream of the start codon of the CP gene and downstream thereof, are selected to allow protein production encoded by a nucleotide sequence whose expression is regulated by SEQ ID NO: 1-SEQ ID NO: 2.

Example 4

Effect of PC on the multiplication of viral RNA in agro-infiltrated tissue

Next we study the behavior of PepGFPΔCP1 in agro-infiltrated tissue. For this, the same construction used in the previous section was placed under the control of the 35S promoter in pBIN61, giving rise to pBPepGFPΔCP1.

This construction was tested by agroin fi ltration in fully expanded N. benthamiana leaves. As agrofiltration controls, clones pBGFP (expresses the ORF of the GFP) and pBIN61 (negative control) were used. pBPepGFPΔCP1 and pBGFP were co-agro-infiltrated with pB19 (p19), which expresses the ORF of the P19 of Tomato bushy stunt virus (TBSV), suppressor of RNA silencing, to prevent decay of GFP expression after several days post- inoculation. These plants were exposed to UV light using a hand lamp and observed by confocal laser microscopy at 4.6 and 10 dpi.

Under the UV lamp, none of the agroin fi ltered plants with pBPepGFPΔCP1 emitted green fluorescence, while the positive control pBGFP generated fluorescence at 4 dpi. However, observation of the agroin fi ltered regions with pBPepGFPΔCP1 by confocal laser microscopy showed the expression of GFP in cells isolated from 4 dpi (Fig. 10).

When looking at the images in the fi gure, it can be seen how the foci of infection initiated by PepGFPΔCP1 remained restricted to a single cell or a few after several days post-infiltration. Green fluorescence was observed in cells isolated from the epidermis and mesophilic cells (Fig. 10, panels A and B, respectively). Less frequently, groups of up to three cells appeared (Fig. 10, panel B); These groups did not increase in size at any time. On the other hand, it is worth noting the small number of cells that expressed GFP in a certain area of agroin fi ltered tissue with pBPepGFPΔCP1 (Fig. 10, panel C) compared to the high number of cells (almost all) that expressed fluorescence in agroin fi ltered tissue with the clone pBGFP (Fig. 10, panel D). These results confirm that CP is not necessary for the multiplication of PepMV in inoculated cells, but for cell-to-cell movement of the virus. In addition, these results indicate that the expression of a foreign gene (GFP) is possible from the sequences SEQ ID NO: 1-SEQ ID NO: 2 of the PepMV genome.

Example 5

Supply of the CP in trans through agroin fi ltration

The previous results showed the ability of PepGFPΔCP 1 to multiply and initiate foci of infection. This section describes the restoration of the movement of PepGFPΔCP1 through a new agroin fi ltration test in which the CP was provided in trans_ (by cotransformation).

To carry out this test, the PepMV CP gene was cloned under the control of the 35S promoter in pBIN61 resulting in the pBINCPep construct. Co-infiltrations of pBPepGFPΔCP1 plus p19 were performed in the presence and absence of the CP. The agro-fi ltered sheets were observed by confocal laser microscopy and exposed to UV light by a hand lamp at 4.6 and 10 dpi.

The contribution of CP in trans through agroinfiltration restored the movement of the PepGFPΔCP1 mutant in a very efficient way. After 4 dpi in the agroin fi ltered regions in the presence of CP, foci of infection formed by groups of about 4-9 cells were detected. Also at 4 dpi other foci were observed in which the infection remained restricted to one or three cells. At 6 dpi, the foci of infection increased in size and groups of cells of different sizes could be distinguished. Finally at 10 dpi, almost all of the foci were formed by groups of more than 20 cells, visible under UV light. In the agroin fi ltered regions in the absence of CP, the foci of infection remained restricted to a single cell, after 10 dpi.

These results indicated that it is possible to restore the movement of PepMV mutants by providing CP in trans. In addition, these results confirmed that the number of initial foci of infection caused by the agrofiltration of clones derived from PepMV is small.

Northern blot analysis of total RNA extracts from agroin fi ltered regions in the absence and presence of CP confirmed the restoration of mutant movement by the contribution of CP in trans.

Example 6

Effect of modi fi cations in the 5 ’and 3’ terminal regions of the PepGFPΔCP1 GFP ORF on the number of infection foci visible after agroinfiltration

In this section we study whether modifications in the 5 ’and 3’ terminal regions of the CP gene could affect the efficiency of a PepMV-derived viral vector after agrofiltration.

To carry out the experiment, three constructs based on pBPepGFPΔCP1 were specifically designed, which included different types of mutations (Fig. 11). The number of cells expressing GFP after agrofiltration of the clones was established as an efficiency measurement parameter, assuming that this is equivalent to the number of foci of infection that can be initiated after agroinoculation.

The constructs were cloned under the control of the 35S promoter in pBIN61 resulting in the clones: pBPepGFPΔCP1, pBPepGFPΔCP2, pBPepGFPΔCP3 and pBPepGFPΔCP4 (Fig. 11). In pBPepGFPΔCP1, the GFP ORF (with the modified start codon from ATG to AGG) was introduced in phase with the CP ORF, at 36 nt start codon of the CP, so that the upstream region of the GFP remained unmodified and the GFP was expressed as a fusion protein that would contain the 12 amino-terminal amino acids of the CP. In pBPepGFPΔCP2, the start codon of the CP from ATG to AGG was modified and the GFP ORF (with its own ATG) was introduced at 38 nt of the start codon of the CP (it also contains just upstream of the start codon from the GFP a Sac target

II: CCGCGG) pBPepGFPΔCP3 and pBPepGFPΔCP4 are identical to pBPepGFPΔCP1 and pBPepGFPΔCP2, respectively, but include 120 nt of the 3 ′ end of the CP ORF following the GFP ORF. These 120 nt should not be expressed since the termination codon of the GFP was maintained. The result of the sequencing of the modified regions confirmed the existence of the desired mutations.

Agrofiltration was performed on N. benthamiana leaves with each of the constructions in the presence and absence of the CP. In order to compare the effect of the mutations within the same context and avoid the possible in fl uence of external factors, the different combinations of clones were compared within the same leaf (Fig. 12). All treatments were co-infiltrated in the presence of p19.

To estimate the number of foci of infection initiated with each PepMV mutant, 0.5 cm2 leaf discs were taken from the agroin fi ltered regions at 6 and 10 dpi. The observation of the discs under a confocal microscope allowed to count the number of isolated cells expressing GFP within each disc.

The values obtained relative to the number of foci of infection initiated with each of the mutants are represented graphically in Fig. 13. As can be seen in this fi gure, significant differences were found between them, PepGFPΔCP1 prevailing for initiating a greater number of foci. of infection Significant differences such as those found between PepGFPΔCP1 and PepGFPΔCP2 should be noted, since they differ in very few nucleotides (see Fig. 11). In addition, it also highlights, at 6 dpi, the delay in the initiation of foci of infection observed with PepGFPΔCP3 and PepGFPΔCP4 compared to the other two mutants. These results indicate that the modifications introduced in PepGFPΔCP1 may give rise to significant differences in the number of infection foci initiated and the time required to establish them after agroinfiltration.

An agroinfiltration experiment was then carried out with the different constructions generated to which the pBINCPep construction was added (expresses the PepMV CP). The results obtained from the trans complementation of cell-to-cell movement of mutants that do not express CP are shown in Fig. 14.

All mutants generated visible foci of infection under UV light at 6 dpi (Fig. 14, panel A), the size of the foci being similar in all cases. At 6 dpi, it can also be seen how PepGFPΔCP1 and PepGFPΔCP2 initiated more foci of infection than PepGFPΔCP3 and PepGFPΔCP4, as occurred in the previous trial. The agroin fi ltered region with PepGFPΔCP1 (Fig. 14, panel B) stands out for presenting a greater number of foci of infection at both 6 and 10 dpi (where almost all of the agrofiltered tissue emits green fluorescence) compared to the rest of the constructions. The results obtained with this complementation test are consistent with those obtained in the previous trial and indicate that the alteration of the 5 ’and 3’ terminal regions of the PepGFPΔCP 1 GFP ORF affects the number of infection foci visible after agrofiltration. Therefore, modifying these regions could affect the efficiency of a viral vector based on PepMV.

Example 7

Design of a viral vector (vector of the invention) derived from PepMV

With the information obtained in previous sections the PepMV agroinfective clone was manipulated to insert the GFP gene. As a strategy to express the new ORF, it was decided to duplicate the gene gene regulatory sequence of the PepMV CP gene, that is, the sequences SEQ ID NO: 1-SEQ ID NO: 2 (SGP and SGPd) (Fig. 15 ).

7.1. Prediction of the gene expression regulatory sequence (subgenomic promoter; SGP) of PepMV CP gene

It has been shown for some potexviruses that gene expression regulatory sequences contain a highly conserved 8 nt sequence (5’-GTTAAGTTT-3 ’), which has been shown to be indispensable for their activity. In the case of PepMV, the regions that act as promoters of sgRNAs (encoded by TGBs and CP) are unknown. However, the results obtained in previous sections show the possibility of expressing the ORF of the GFP by placing it 36 nt downstream of the start codon of the ORF of the CP. Thus, the 3 ’end of the gene expression regulatory sequence was delimited at the distance of 36 nt, with the 5’ end being delimited.

The alignment of the gene expression sequences of the CP of different potexviruses together with the sequence upstream of the start codon of the PepMV CP showed that for the latter the reason for 8 nt mentioned above is also preserved.

On the other hand, stem-loop structures (Stem Loop; SL) that facilitate interaction with transcriptase are often found within the gene expression regulatory sequences. Generally the combination of the primary sequence and the secondary structure is required for transcription in vivo. Thus, a secondary structure prediction of a sequence covering a total of 150 nt, 114 nt upstream of the first ATG of the CP ORF and 36 nt downstream was made. An analysis of this possible secondary structure showed the possible existence of three SLs within the analyzed region. The SL1 is located upstream of the conserved sequence and the SL2 and 3 downstream, the latter being 8 nt upstream of the ATG of the CP.

Based on the previous analyzes, it was finally selected as a candidate region to contain an effective gene expression regulatory sequence, a region of 111 nt (SEQ ID NO: 1-SEQ ID NO: 2): 37 nt from the 3 'end of the ORF4 plus 38 nt of the UTR region plus 36 nt of the start of the CP. The selected region contains the 8 nt conserved motif and the SL 2 and 3 present up to the start codon of the CP plus 36 nt of the nucleotide sequence encoding said protein.

7.2. Generation of PepMV clones: GFP. GFP expression through duplication of the gene expression regulatory sequence of the CP gene

As described in a previous example, the strategy selected for the expression of the ORF of the GFP was through the duplication of the supposed regulatory sequence of the gene expression of the CP. Two different versions of a possible viral vector were designed (Fig. 16).

The strategy used for the development of the constructions was through overlapping PCRs (see corresponding section of materials and methods). DNAs from the agroinfective clone (pBPepXL6) and from the construction pBPepGFPΔCP1 were used as reaction templates. Thus, two new constructions were obtained (Fig. 16): (1) pBPep501 (Pep501), in which the GFP ORF is located upstream of the CP ORF and

(2) pBPep5.128 (Pep5.128) in which the GFP ORF is downstream of the CP ORF (at the 3 ’end of the genome). In both cases, the GFP and CP ORFs are fused to the regulatory sequence of the gene expression SEQ ID NO: 1-SEQ ID NO: 2 (in Fig. 16 it is called SGP). These constructs were cloned under the control of the 35S promoter in pBIN61.

The constructions were agrofiltered in N. benthamiana leaves in the presence of p19. From 3 dpi, the agro-infiltrated plants were exposed to UV light to monitor the infection.

At 3 dpi, the observation of some of the leaves agrofiltered by confocal microscopy revealed the existence of foci of infection formed in most cases by groups of about 4-9 cells, not visible under a UV lamp. None of the plants showed typical infection symptoms of PepMV after 10 dpi while the agroinoculated plants with the agroinfective clone showed symptoms after 5 dpi. However, the UV light illumination of the entire plants showed expression of GFP both in agroin fi ltered and in non-agroin fi ltered leaves. In addition, no differences were observed between agroin fi ltered plants with pBPep501 and pBPep5.128.

7.3. Selection and testing of a heterologous gene expression regulatory sequence

It was decided to use a gene expression regulatory sequence from another PepMV isolate, PepMV-Ch2 (Ling, 2007. Virus Genes, 34: 1-8). PepMV-Ch2 has characteristics that make it a good candidate to contain a valid gene expression regulatory sequence, including: (1) It has a sequence homology of 78% with respect to Sp13 and (2) being an isolated PepMV, presumably it contains elements that act in conserved cis, necessary for the expression of sgRNA of the CP.

The complete sequence of PepMV-Ch2 was studied in order to locate the region corresponding to the regulatory sequence of the CP gene expression. The analyzes revealed the existence of a conserved region of 20 nt at the 3 ’end of ORF 4 (TGB3) between the two isolates. This region coincides with the beginning of the region selected as the regulatory sequence of the gene expression of the CP of Sp13 and also contains the 8 nt motif conserved between potexvirus.

Next, the 5 ’and 3’ ends of the heterologous gene expression regulatory sequence were delimited (giving the same size as the PepMV-Sp13 gene expression regulatory sequence) and sequence alignment was performed. The result of the alignment between both regions showed percentage identity of the sequence SEQ ID NO: 1-SEQ ID NO: 2 of 58.4% between PepMV-Sp13 and -Ch2. When the percentage identity of the SEQ ID NO: 1 and SEQ ID NO: 2 sequences was checked separately, it was observed that the sequence SEQ ID NO: 1 corresponding to PepMV-Sp13 had an identity percentage of 84% with respect to the heterologous sequence corresponding to PepMV-Ch2. However, the sequence SEQ ID NO: 2 corresponding to PepMV-Sp13 had an identity percentage of 48.78% with respect to the heterologous sequence corresponding to PepMV-Ch2.

Therefore, the possibility of using the gene expression regulatory sequence from the PepMV-Ch2 isolate and its efficiency to express the GFP gene from the PepMV-Sp13 genome was proved. For this, a chimeric PepMV construct (PepGFPΔCPCh2) was designed, based essentially on the design of PepGFPΔCP1, in which the GFP ORF is under the control of the regulatory sequence of the Ch2 gene expression.

Said construction was cloned under the control of the 35S promoter in pBIN61 resulting in the clone pBPepGFPΔCPCh2.

Agrofiltration tests were performed with pBPepGFPΔCPCh2 in N. benthamiana plants. Agrofiltration was performed in the presence of p19. The agro-fi ltered regions were observed by confocal microscopy at 6 and 10 dpi. The emission of green fluorescence by cells of the epidermis at 4 dpi in the agro-infiltrated tissues demonstrated PepGFPΔCPCh2's ability to replicate and initiate foci of infection. These results indicate that the expression of a foreign gene from the PepMV-Sp13 genome from the Ch2 SGP is possible.

Once the infectivity of PepGFPΔCPCh2 was established, it was decided to estimate the number of infection foci initiated and to make a comparison with the data obtained for the regulatory sequence of PepMV-Sp13 gene expression. For this, 0.5 cm2 leaf disks were taken from the agroin fi ltered regions at 6 dpi and 10 dpi and the number of isolated cells expressing GFP within each disk was counted. The comparison of the results obtained with PepGFPΔCPCh2 and those obtained in previous tests with PepGFPΔCP1, 2,3 and 4 are shown in Fig. 17.

The result of the statistical analysis showed that at 6 dpi the number of infection foci initiated by PepGFPΔCPCh2 was significantly less than the number of foci initiated by PepGFPΔCP1. However, at 10 dpi it was observed how the difference between PepGFPΔCPCh2 and PepGFPΔCP1 was reduced, placing PepGFPΔCPCh2 as the second mutant that initiated the greatest number of foci of infection.

The behavior of PepGFPΔCPCh2 was also studied by providing CP in trans through agroinfiltration compared with the PepGFPΔCP1 construct. Under UV light, the results obtained with PepGFPΔCPCh2 were similar to those obtained with PepGFPΔCP1. The comparison of the results obtained with the two constructions at 10 dpi is shown in Fig. 18.

In Fig. 18 it can be seen how both the size and density of the foci of infection generated at 10 dpi was very similar with the two constructions. The results obtained indicated that it was possible to restore the movement of the PepGFPΔCPCh2 chimeric mutant in an efficient way. These results also indicated that the replacement of the regulatory sequence of PepMV-Sp13 gene expression with that of PepMV-Ch2 in PepGFPΔCP1 does not: it causes significant differences in the number of foci of infection initiated or in the efficiency of movement restoration.

The results obtained in this section suggest that a gene expression regulatory sequence (SEQ IDNO: 1, or SEQ ID NO: 1-SEQ ID NO: 2) heterologous could be used in a viral vector derived from PepMV.

Claims (30)

one.

Isolated nucleotide sequence that has at least 80% identity with respect to the sequence SEQ ID NO: 1, involved in the regulation of gene expression.

2. Nucleotide sequence according to claim 1, wherein said sequence is SEQ ID NO: 1.

3.

Sequence according to any one of claims 1 or 2, joined by its 3 'end to the end of the sequence SEQ ID NO: 2.

Four.

Sequence according to any one of claims 1 to 3, linked at its 3 'end to a nucleotide sequence (MCS), wherein MCS is recognized by at least one unique restriction enzyme.

5. Sequence according to any of claims 1 to 4, joined by its. 3 ’end to a nucleotide sequence (P) coding for a functional or non-functional protein.

6.

Sequence according to claim 5, wherein the nucleotide sequence P codes for a functional protein reporter.

7. Sequence according to claim 6, wherein the functional protein reporter is GFP.

8.

Sequence according to any one of claims 1 to 7, joined at its 5 'end to the 3' end of a nucleotide sequence encoding an RNA-dependent RNA polymerase (RpRd) and a triple gene block sequence (TGB) of a potexvirus (RpRd-TGB).

9.

Sequence according to claim 8, wherein the nucleotide sequence encoding the RpRd-TGB sequence belongs to the PepMV virus.

10.

Expression vector comprising the sequence according to any of claims 1-9.

eleven.

Expression vector according to claim 10, wherein said vector is binary.

12.

Expression vector according to claim 11, wherein said binary vector is expressed in a bacterium.

13.

Expression vector according to any one of claims 10 to 12, wherein the sequence according to any of claims 1 to 10 forms part of a cDNA.

14. Cell that includes transiently or stably:

to.

the sequence according to any of claims 1-9, or

b.

the vector according to any of claims 10 to 13.

fifteen.

Cell according to claim 14, which belongs to a plant species.

16.

Pollen comprising the plant cell according to claim 15. 17. A plant comprising the cell according to any of claims 14 or 15.

18.

Plant according to claim 17 belonging to the genus Solanum.

19.

Plant according to claim 18 belonging to the species Solanum lycopersicum.

twenty.

Germplasm of the plant according to any of claims 17 to 19.

twenty-one.

Use of the sequence according to any one of claims 1 to 9 for the construction of an expression vector.

22

Use of the sequence according to any of claims 1-9, or of the expression vector according to any of claims 10 to 13, for the detection of at least one plant of the Solanum genus resistant to PepMV virus.

2. 3.

Use of the sequence or vector according to claim 22, wherein the plant is of the species Solanum lycopersicum.

24.

Use of the cell according to any of claims 14 or 15, or of the plant according to any of claims 17 to 19, for the production of a functional or non-functional protein.

25. Method for the production of a functional or non-functional protein comprising:

to.

Select the cell according to any of claims 14 or 15, or the plant according to any of claims 17 to 19, and

b.

co-transfect, simultaneously or not, said cell or said plant, with a nucleotide sequence encoding a capsid protein (CP) of a potexvirus.

26. Method according to claim 25, wherein the potexvirus is PepMV virus. 27. Method for obtaining the expression vector according to any of claims 10 to 13 comprising:

to.

Select the sequence according to any of claims 1-9,

b.

inserting the sequence of section (a) into an expression vector capable of replicating in at least one type of host cell, and

C.

select the vector obtained according to section (b) comprising said sequence.

28. Method for obtaining the cell according to any of claims 14 or 15, comprising:

to.

Obtain a cell from a biological sample,

b.

transfecting the host cell isolated from section (a) with a vector comprising the sequence according to any of claims 1-9, and

C.

select the transfected cell according to section (b) comprising said sequence.

29. Method for the detection of at least one plant of the genus Solanum, resistant to PepMV virus, comprising:

to.

select at least one plant from a collection of mutant lines of the genus Solanum,

b.

inoculate at least one plant of section (a) with the sequence according to any one of claims 5 to 9, or with the corresponding expression vector according to any of claims 10 to 13, wherein said sequence or vector comprises the nucleotide sequence P and

C.

select at least one PepMV virus resistant plant that does not express the functional or non-functional protein.

30. Method according to claim 29, wherein the plant of the genus Solanum belongs to the species Solanum lycopersicum. SEQUENCE LIST <110> Higher Council for Scientific Research <120> Viral sequence involved in the regulation of gene expression, expression vector, cell and plant that comprises it <130> 1641.521 <160> 26 <170> PatentIn version 3.5 <210> 1 <211> 31 <212> DNA <213> PepMV cucumber mosaic virus -Sp13 <400> 1 <210> 2 <211> 80 <212> DNA <213> PepMV cucumber mosaic virus -Sp13 <400> 2 <210> 3 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-42 <400> 3 <210> 4 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-43 <400> 4 <210> 5 <211> 23 <212> DNA <213> Initiator CE-198 cucumber mosaic virus PepMV -Sp13 <400> 5 <210> 6 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-199 <400> 6 <210> 7 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-236 <400> 7 <210> 8 <211> 19 <212> DNA <213> Pep303 cucumber mosaic virus PepMV initiator -sp13 <400> 8 <210> 9 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-351 <400> 9 <210> 10 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-353 <400> 10 <210> 11 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-456 <400> 11 <210> 12 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-457 <400> 12 <210> 13 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-458 <400> 13 <210> 14 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-459 <400> 14 <210> 15 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-460 <400> 15 <210> 16 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-461 <400> 16 <210> 17 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-356 <400> 17 <210> 18 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Oligo dT <400> 18 <210> 19 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-501 <400> 19 <210> 20 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-502 <400> 20 <210> 21 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-503 <400> 21 <210> 22 <211> 38 <212> DNA <213> Initiator CE-504 PepMV Cucumber Mosaic Virus -Sp13 <400> 22 <210> 23 <211> 33 <212> DNA <213> Initiator CE-497 cucumber mosaic virus PepMV -Ch2 <400> 23 <210> 24 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-498 <400> 24 <210> 25 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Initiator CE-499 <400> 25 <210> 26 <211> 33 <212> DNA <213> Artificial sequence <220> <223> CE-500 Initiator <400> 26 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200930391 SPAIN Date of submission of the application: 06.30.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12N15 / 40 (01.01.2006) C12N15 / 83 (01.01.2006) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

TO

AGUILAR, J. M., HERNÁNDEZ-GALLARDO, M. D., CENIS, J. L. et al. Complete sequence of the 1-3,9,10,14-21,

Cucumber mosaic virus RNA genome. Archives of Virology. October 2002, Vol. 147, No. 10, pages 2009-2015. ISSN 0304-8608. <Doi. 10.1007 / s00705-002-0848-9> &amp; EMBL database. Accession number AF484251, Version 2. [online] 29.05.2009 [retrieved on 04.11.2010] Recovered from the Internet: <URL: http: //www.ebi.ac.uk/cgi-bin/sva/sva.pl ? session =% 2Ftmp% 2FSESSION106261289996205-1 &amp; index = 4 & view = 888598337>

24-28

TO

COTILLON, A.-C., GIRARD, M., DUCOURET, S. Complete nucleotide sequence of the genomic 1-3,9,10,14-21,

RNA of a French isolate of Cucumber mosaic virus (PepMV). Archives of Virology. October 2002, Vol. 147, No. 11, pages 2231-2238. ISSN 0304-8608. <Doi. 10.1007 / s00705-002-0873-8>

24-28

TO

SKRYABIN, K. G., MOROZOV, S. Y., KRAEV, A. S. et al. Conserved and variable elements in 1-3,10,14-17,20,

RNA genomes of potexviruses. FEBS Letters November 1988, Vol. 240, No. 1-2, pages 33-40. ISSN 0014-5793.

21,24,25,27,28

TO

EP 1897953 A1 (ICON GENETICS GMBH) 12.03.2008, the whole document. 4-8,10-17,20,21,

24,25,27,28

TO

CHAPMAN, S., KAVANAGH, T., BAULCOMBE, D. Potato virus X as a vector for gene expression 4-8,10-17,20,21,

in plants. The Plant Journal July 1992, Vol. 2, No. 4, pages 549-557. ISSN 0960-7412.

24,25,27,28

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 28.01.2011

Examiner E. Relaño Reyes Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200930391 Minimum documentation sought (classification system followed by classification symbols) C12N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, MEDILINE, BIOSIS, EMBASE, NPL, COMPENDX, INSPEC, XPESP, XPOAC, REGISTRY, HCAPLUS, EMBL, naGeneSeq State of the Art Report Page 2/5  WRITTEN OPINION Application number: 200930391 Date of Written Opinion: 28.01.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-30 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-30 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/5  WRITTEN OPINION Application number: 200930391 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

AGUILAR, J. M. et al. Archives of Virology. October 2002, Vol. 147, No. 10, pages 2009-2015. 10.2002

D02

COTILLON, A.-C. et al. Archives of Virology. October 2002, Vol. 147, No. 11, pages 2231-2238. 10.2002

D03

SKRYABIN, K. G. et al. FEBS Letters November 1988, Vol. 240, No. 1-2, pages 33-40. 11.1998

D04

EP 1897953 A1 12.03.2008

D05

CHAPMAN, S. et al. The Plant Journal July 1992, Vol. 2, No. 4, pages 549-557. 07.1992

In D01 the complete sequence of a strain of PepMV is disclosed. D02 analyzes the sequence of a strain of PepMV, indicating a possible promoter sequence for CP. In D03 an alignment of the sequences of several potexviruses (PVX, WCIMV and PoAMV) is shown reaching a consensus sequence of the possible CP promoter. D04 develops a series of gene constructs derived from potexvirus, taking as an example the potato X virus (PVX). These are included in binary vectors, such as Ti plasmids. One of the most advantageous constructions, includes in sense 5’-3 ’: RpRd-TGB-GFP. Not being able to move from one cell to another, the RpRd-TGB-CP-GFP construction was designed, among others. The GFP protein is expressed under the control of the CP subgenomic promoter. As this promoter includes the protein initiation codon, it was mutated. In D05 the feasibility of using PVX as a vector is investigated. For this, two constructions are developed, containing both RpRd-TGB at the 5 ’end. Next, one includes the GUS gene in substitution of CP, preserving the first nucleotides encoding the CP protein, since they are thought to be part of the promoter. The other presents the GUS gene linked to the putative CP promoter and then that same promoter sequence linked to CP. The vector with the deletion in the CP protein, although expressing GUS, cannot be moved from one cell to another. 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application aims at a nucleotide sequence of SEQ. ID. NO. 1, involved in the regulation of gene expression (claims 1 and 2). This sequence belongs to the sweet cucumber mosaic virus (PepMV) and is located between nucleotides 75 and 26 upstream of the initiation codon of the capsid protein (CP). It is also the object of the invention, a gene construct in which the SEQ. ID. NO. 1 is joined by its 3 ’end to the SEQ. ID. NO. 2, as found in PepMV, presenting the resulting promoter activity sequence (claim 3). In addition, gene constructions are included in which or the SEQ. ID. NO. 1 or the SEQ. ID. NO. 1-SEQ. ID. NO. 2, are attached at their 3 'end to an MCS sequence together with a protein of interest, and / or at their 5' end to an RNA-dependent RNA polymerase (RpRd) and PepMV TGB sequences (claims 4 at 9). Other additional objects of the invention are: the expression vector, the transformed cell, or the pollen, the plant or the germplasm comprising said cell (claims 10 to 20). Finally, the use of the sequence is also included: in the construction of an expression vector; in the selection of a plant of the genus Solanum resistant to PepMV; or in the production of a protein (claims 21 to 24); as well as a method of producing a protein, obtaining an expression vector or a transformed cell, or selecting a PepMV resistant plant, which comprises the use of SEQ. ID. NO. 1 (claims 25 to 30). State of the Art Report Page 4/5  WRITTEN OPINION Application number: 200930391 NEW AND INVENTIVE ACTIVITY (Art. 6.1 AND 8.1 LP11 / 1986) The object of the present application, according to claim 1, is a sequence with at least 80% identity with the SEQ. ID. NO. 1, and involved in the regulation of gene expression. In D01 the complete PepMV sequence is disclosed, with nucleotides from 5557 to 5588 having a 100% identity with the SEQ. ID. NO. 1. However, the possibility that this region has a promoter function is not noted. D02 analyzes the sequence of a PepMV strain. In this document, it is indicated that at 44 nt upstream of the CP initiation codon, the sequence 5’-AC / GGGGUUAAGUUUUCC (7nt) GAA-3 ’is found, which has a 100% identity with the SEQ. ID. NO. 1. This sequence has also been found at the 5 ’end of the ORF2 of this virus, and very similar ones have been found in other potexviruses, which seems to indicate that it could be a promoter sequence. However, no experimental data supporting this hypothesis is shown. In D03 the sequences of several potexviruses (PVX, WCIMV and PoAMV) are aligned. By comparing the conserved regions and the variables, they obtain a consensus sequence with respect to the possible CP promoter. In this consensus sequence, the central region 5’-GGTTAAGTT-3 ’shows 100% identity with nucleotides from 9 to 19 of the SEQ. ID. NO. 1. As in D02, no experimental data is presented in which the functionality of said sequence is checked. Therefore, from documents D01, D02 or D03, it is not deduced with some certainty that the SEQ. ID. NO. 1 have promoter activity. Accordingly, claim 1 meets the requirements of novelty and inventive activity. Claim 2 is dependent on claim 1, and like this, it is also new and has inventive activity. As claim 1 is new and inventive, the gene construct or expression vector comprising it (claims 3 to 13), as well as the cell transformed with the sequence or plant comprising said cell (claims 14 to 20) also have novelty and inventive activity. Finally, for the same reasons mentioned above, the different uses of the SEQ. ID. NO. 1 (claims from 21 to 24) or the methods comprising the use of the sequence (claims from 25 to 30) also meet the requirements of novelty and inventive activity. State of the Art Report Page 5/5