WO2001053475A2 - Isolation and characterization of a n. crassa silencing gene and uses thereof - Google Patents

Isolation and characterization of a n. crassa silencing gene and uses thereof Download PDF

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WO2001053475A2
WO2001053475A2 PCT/IT2001/000008 IT0100008W WO0153475A2 WO 2001053475 A2 WO2001053475 A2 WO 2001053475A2 IT 0100008 W IT0100008 W IT 0100008W WO 0153475 A2 WO0153475 A2 WO 0153475A2
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nucleic acid
isolated nucleic
acid molecule
domain
gene
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WO2001053475A3 (en
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Carlo Cogoni
Giuseppe Macino
Caterina Catalanotto
Gianluca Azzalin
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Universita' Degli Studi Di Roma 'la Sapienza'
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to the isolation and characterization of a Neurospora crassa gene encoding for an essential activity in the co-suppression process and to uses and applications thereof in vegetal, animal and fungine fields.
  • the production of transgenic organisms is of large utility both in basic and applied biological research.
  • the transgenic DNA is usually integrated in the genome and transferred as a Mendelian character.
  • the transgene introduction induces gene silencing phenomena (Flavell, R.B. 1994), i.e. the repression of the expression of the transgene itself and/or of one or more endogenous homologous genes.
  • the gene silencing (suppression of gene expression) can act at two levels: transcriptional (trans- inactivation) where transgenes contain sequences homologous to the silenced gene promoter (Vaucheret, 1993); and post-transcriptional (co-suppression) which requires ho ologies between coding regions (Flavell, 1994; Stam et al . , 1997; Baulcombe, 1996).
  • transcriptional trans- inactivation
  • co-suppression which requires ho ologies between coding regions
  • the silencing induced by a transgene requires an almost complete sequence homology (from 70% to 100%) between transgene and silenced gene sequences (Elkind, 1990) .
  • the al -1 gene "quelling" in Neurospora is- characterized in that: 1) the- gene silencing is reversible further to the loss of transgene copies; 2) the reduction of mRNA basal level results from a post-transcriptional effect; 3) transgenes containing at least a region of 132 base pairs which is identical to the region encoding for the target gene are sufficient to induce the "quelling”; 4) the duplication of promoter sequences is ineffective to induce the silencing; 5) the "quelling” exhibits a dominant behavior in eterocarions containing both transgenic and untransformed nuclei, indicating the involvement of a trans-acting diffusible molecule among the nuclei; 6) the expression of an aberrant RNA transcribed by the transgenic locus is strictly correlated to silencing, suggesting that the "quelling" can be induced and/or mediated by a transgenic RNA molecule.
  • nuclei wherein the albino-1 gene is silenced are able to induce the al -1 gene silencing of the other not transformed nuclei, all sharing the same cytoplasmic environment (Cogoni et al., 1996).
  • TMV Tobacco Etch Virus
  • TMV Tobacco Etch Virus
  • Garrick et al. (1998) produced mouse transgenic lines wherein 100 transgenic copies are present in a unique locus and are repeats-arrayed in direct tandem.
  • the transgene expression has been disclosed to be inversely proportional to the number of occurring copies, indicating that silencing phenomena dependent on repeat copies are present also in mammalians.
  • dsRNAi double stranded RNA interference
  • dsRNAi double stranded RNA interference
  • RNA- induced silencing and DNA-induced silencing can be transmitted from cell to cell. Therefore the identification of Neurospora genes which are involved in the silencing is the first step to modulate the same process in plants, animals and fungi. The silencing modulation is of great relevance when transgenic organisms able to express the desired phenotype are produced.
  • the authors of the present invention have already isolated Neurospora crassa strains mutated at essential functions for gene silencing (Cogoni and Macino, 1997); 15 independent isolated mutants define three complementation groups, thus identifying the qde-1 , qde-2 and qde-3 genes ⁇ qde stands for "quelling"-deficient) , whose products are essential to the silencing machinery.
  • qde genes are essential to the Neurospora silencing, as suggested by the fact that silencing of three independent genes ⁇ al -1, al-2 and qa -2) is impaired by qde mutations (Cogoni and Macino, 1997).
  • the present invention can be applied with reference to two general scopes: 1) silencing potentiation as a tool for inactivating more effectively and durably a desired gene, .and 2) silencing suppression to obtain a better expression of the introduced transgenes.
  • the isolated qde-2 gene can be introduced alone or with qde-1 and/or qde-3 genes in plants, animals or fungi, in order to inactivate the expression of selected genes.
  • the aim is to activate a sequence-specific silencing mechanism both in deficient organisms and in organisms wherein the same is not very efficient.
  • the gene silencing can be induced also by introducing specific double stranded DNA or RNA sequences, homologous to the gene to be inactivated.
  • the over- expression of one or more genes controlling the phenomenon can lead to higher efficiency and/or stability thereof. Therefore the introduction of qde-2 gene or of homologous genes thereof in organisms can constitute a tool to repress more effectively gene functions. Particularly this approach is specially useful in plants wherein the co-suppression is usually used for the "knock-out" of gene functions.
  • the gene silencing potentiation can be used to obtain lines resistant to pathogen virus, by introducing transgenes encoding for viral sequences, in order to achieve the expression inhibition of the virus itself (Flavell et al., 1994) .
  • Analogous applications are suitable for animals, wherein some indications suggest that silencing can inhibit the suitable expression of introduced transgenes (Garrick et al., 1998). On the contrary, there are instances wherein it is desirable not to have or to reduce the gene silencing, i.e. where a transgene is to be over-expressed. It is known that the co-suppression is strictly correlated both with the presence of an high copy number of the transgene, and with a transgene high expression. This correlation can hamper the production of transgenic organisms which express a transgene at high levels, because more high is the expression and/or the copy number, more probable is to evoke silencing responses.
  • Neurospora qde-2 gene essential for silencing mechanism, can allow the isolation of mutant lines in other organisms, mutated in genes homologous to qde-2. For example by means of amplifications using degenerated primers, designed from the most conserved regions of qde-2 gene, mutant lines in homologous genes can be identified, by analysis of insertion mutant gene banks, already available for many plant species. Both in fungi and animals such mutants can be obtained, following the identification of the homologous gene, by means of "gene disruption" techniques using homologous recombination.
  • qde-2 or homologous genes can be introduced into suitable expression vectors to express them in an anti-sense orientation in order to inhibit the expression of resident endogenous genes.
  • portions of qde-2 or of homologous genes can be over-expressed, in order to obtain a negative dominant effect and thus blocking the function of qde-2 endogenous genes .
  • the authors of the present invention have cloned and characterised the Neurospora crassa qde-2 gene .
  • the sequence analysis of the qde-2 gene detected a region having a significant homology with the sequence of a C. elegans gene, rde-1 , involved in the dsRNA mediated interference (Tabara et al., 1999).
  • the authors of the invention for the first time have demonstrated that the transgene induced post- transcriptional gene silencing and the dsRNA interference share common genetic mechanisms. This supports the hypothesis that the sequence specific gene silencing phenomena evolved from an ancestral mechanism aimed to protect the genome against transposons. Furthermore, the results of the authors suggest that dsRNA molecules are involved in the post-transcriptional gene silencing in fungi.
  • dsRNA molecules could be produced directly from integrated trangenes as a result of the presence of inverted repeats or as an out come of transcription from convergent inverted promoters.
  • single stranded aberrant RNA may be used as a template by an RNA-dependent RNA polymerase (such as QDE-1 protein) able to produce dsRNAs .
  • homology is intended as similarity, i.e. number of identical residues + number of conserved residues with respect to the total residues of the considered sequence.
  • an isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and in comprising a domain responsible for dsRNA interference, wherein the domain is at least 25% homologous with the amino acid sequence from aa . 373 to aa . 910 of sequence in fig. 1 (SEQ ID No. 2) .
  • the domain is at least 30% homologous with the amino acid sequence from aa. 373 to aa. 910 of sequence in fig. 1 (SEQ ID No. 2). More preferably the domain is at least 38% homologous with the amino acid sequence from aa. 373 to aa. 910 of sequence in fig. 1 (SEQ ID No. 2) .
  • the domain comprises the amino acid sequence from aa . 373 to aa. 910 of sequence in fig. 1 (SEQ ID No. 2).
  • the isolated nucleic acid molecule encodes for a protein having the amino acid sequence of fig. 1 (SEQ ID No. 2) or functional portions thereof. Even more preferably the isolated nucleic acid molecule has the sequence of fig. 1 (SEQ ID No. 1) or its complementary sequence.
  • a further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in bacteria, the isolated nucleic acid molecule of the invention.
  • a promoter which directs the expression in bacteria
  • any plasmid suitable for a correct and effective expression of the protein of the expression in bacteria can be used and it is within the scope of the invention.
  • a further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in plants or in specific plant organs, the isolated nucleic acid molecule of the invention, both in a sense and anti-sense orientation.
  • a promoter which directs the expression in plants or in specific plant organs, the isolated nucleic acid molecule of the invention, both in a sense and anti-sense orientation.
  • a further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in fungi, the isolated nucleic acid molecule of the invention, both in a sense and anti- sense orientation.
  • a promoter which directs the expression in fungi, the isolated nucleic acid molecule of the invention, both in a sense and anti- sense orientation.
  • a further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in animals, the isolated nucleic acid molecule of the invention, both in a sense and anti- sense orientation.
  • a promoter which directs the expression in animals, the isolated nucleic acid molecule of the invention, both in a sense and anti- sense orientation.
  • a further object of the invention is a prokaryotic organism transformed by using the expression vector active in bacteria of the invention.
  • a further object of the invention is a plant or a specific plant organ transformed by using the expression vector active in plants of the invention.
  • a further object of the invention is a plant mutated at the isolated nucleic acid molecule of the invention having a reduced or inhibited silencing activity.
  • a further object of the invention is a fungus transformed with the expression vector of the invention active in fungi.
  • a further object of the invention is a fungus mutated at the isolated nucleic acid molecule of the invention and having reduced or inhibited silencing activity.
  • a further object of the invention is a non-human animal transformed with the expression vector of the invention active in animals.
  • a further object of the invention is a non-human animal mutated at the isolated nucleic acid molecule of the invention and having a reduced or inhibited silencing activity.
  • a further object of the invention refers to a protein characterized in having a silencing activity and in comprising a domain responsible for dsRNA interference, wherein the domain is at least 25% homologous with the amino acid sequence from aa. 373 to aa. 910 in fig. 1 (SEQ ID No. 2) .
  • the domain is at least 30% homologous with the amino acid sequence from aa. 373 to aa . 910 in fig. 1 (SEQ ID No. 2) .
  • the domain is at least 38% homologous with the amino acid sequence from aa. 373 to aa. 910 in fig. 1 (SEQ ID No. 2) .
  • the domain comprises the amino acid sequence from aa.
  • the isolated nucleic acid molecule encodes for a protein having the amino acid sequence of fig._ 1 (SEQ ID No. 2) or functional portions thereof.
  • I-t is within the scope of the present invention the use of the isolated nucleic acid molecule of the invention to modulate gene silencing in plants, animals and fungi .
  • Figure 1 The isolated nucleic acid molecule of the
  • Figure 2 It is schematically represented the pMXY2 plasmid insertion site, in the 80 mutant, used for insertional mutagenesis and consequent polimorphism of the restriction fragments by mean of DNA southern blot of a WT strain and of 80 and 820 mutant strains by using the entire restored flanking region as probe.
  • the 820 mutant has a complete deletion of the qde-2 gene.
  • Figure 3 Multiple alignment, at the conserved region, among qde-2 and other proteins belonging to ago- elF2C family: A. thaliana ago-1 ; rabbit elF2C; C. elegans rde-1 . Identical amino acids are shown in bold. MATERIALS AND METHODS E. coli strains
  • E. coli strain HB101 (F ⁇ , hsdS20(rb “ , mb “ ) , supE44, recA13, aral4, proA2, rspL20 (str r ) , xyl-5) was used for cloning.
  • Neurospora crassa following strains supplied by Fungal Genetic Stock Center (FGSC, Dpt . Of Microbiology, University of Kansas Medical Ctr. Kansas City, KA) were used:
  • the qde mutants were obtained by UV mutagenesis.
  • the transforming strain (6xw) silenced at the albino-1 gene was used, qde mutants were selected for their ability to recover a wild type unsilenced phenotype and then classified in three different complementation groups.
  • al -2 gene quelling frequency all of qde used mutants are defective for the general silencing mechanism.
  • the plasmid contains the Bml gene (allele responsible of the benilate drug resistance) , that was used as selective marker after transformation.
  • the genomic DNA containing the qde-2 gene was isolated from a N. Crassa gene library in cosmids . (Cabibbo et al., 1991). N. crassa transformation
  • Chromosomal DNA was prepared as disclosed by Irelan et al., 1993. 5 ⁇ g of genomic DNA were digested and blotted as reported in Maniatis et al. DNA probes were: a) as to the al -1 gene the probe is represented by a Xbal-Clal restriction fragment of pX16 (Cogoni et al., 1996); b) as to the Bml gene the probe is represented by the 2.6Kb Sail fragment of pMXY2.
  • Northern Blot Analysis N. crassa total RNA was extracted according to the protocol described by Cogoni et al . , 1996. The mycelium was grown for two days at 30°C, then powdered in liquid nitrogen before RNA extraction.
  • RNA For Northern analysis 10 ⁇ g of RNA were formaldehyde denatured, electrophoresed on a 1% agarose, 7% formaldehyde gel, and blotted over Hybond N (Amersham) membranes. Hybridization was carried out in 50% formamide in the presence of P labeled DNA probe 1.5x10 cpm/ml .
  • RESULTS Isolation of silencing mutant by insertional mutagenesis
  • the 6XW strain shows an albino phenotype due to the lack of carotenoid biosynthesis, as results by the silencing of the albino 1 gene expression (Schmidhauser et al., 1990).
  • a mutation interfering with the silencing machinery is easily detectable by producing a wild type phenotype (bright orange) of the carotenoid biosynthesis.
  • complementation assays it was possible to establish that qde mutants belong to three complementation groups, indicating the presence of three genetic loci involved in the Neurospora silencing mechanism.
  • an insertional mutagenesis was carried out with the 6XW strain, previously used for UV mutagenesis.
  • the insertional mutagenesis was carried out by transforming the 6XW strain with a plasmid, taking advantage of the fact that, after the transformation, plasmids are randomly inserted in the Neurospora crassa genome.
  • the mutagenesis was carried out transforming the 6XW silenced strain with pMXY2 (see Materials and Methods) which contains the benilate resistance as selective marker.
  • Transformed strains able to grow in the presence of benilate containing medium and showing a wild type phenotype for the carotenoid biosynthesis were selected.
  • a benilate resistant strain (80) was isolated, which showed the bright orange phenotype expected for a qde gene mutation.
  • the genomic DNA of the strain 80 was extracted and digested with Smal and Hindlll restriction enzymes. After blotting, DNA was hybridized with a probe corresponding to the coding sequence of al -1 .
  • the Smal site is present only once in the al -1 transgene containing plasmid and the digestion by using said enzyme produces a 5.5Kb fragment corresponding to tandem arrayed al-1 transgenes, while a
  • 3.1Kb fragment is expected from the resident al-1 locus.
  • the number of al -1 transgenic copies present in the 80 strain is comparable to that present in the silenced 6XW strain.
  • the strain 80 is mutated in qde-2 gene
  • the strain 80 was assayed in a heterokaryon assay with a wild type strain and with M7 , M20 ( qde-1 ) M10, Mil ( qde-2) , Mil , M18 ( qde-3) mutants and with a wild strain (Cogoni and Macino, 1997) . As shown in Table 1 the al -1 gene silencing is restored producing an albino phenotype in all of heterocaryons but M10 and Mil. This behavior is consistent with the presence of a qde-2 gene recessive mutation in the strain 80.
  • WT heterokaryon wit a wilei type phenotype for carotenoid accumulation
  • AL heterokaryon with an albino phenotype wherein the al -1 gene silencing is restored.
  • the fragment from pQcl plasmid was used to probe a Neurospora crassa genomic library in cosmids .
  • the 20C1 cosmid was used to subclone a 5.7 Kb BaitiHI-BamHI fragment.
  • the 938 aa sequence (SEQ ID No. 2) was used to search in database of amino acid sequences, by using the BLASTP algorithm. As showed in fig. 3, the search identified significant homologies with argonaute-1 gene [with expected values (E value) of 2e-57] of A . Thal iana (mutants of this gene show developmental anomalies) ; rde- 1 gene [with expected values (E value) of le-23] of C elegans, involved in gene silencing phenomena induced by double stranded RNA; elF2C gene [with expected values (E value) of 5e-60] of rabbit isolated as an element belonging to transcription beginning complex. Plant expression vector
  • the qde-2 gene was inserted, in a sense orientation, into a vector containing a plant expression "cassette", including the 35S promoter and the PI-II "terminator" sequences.
  • the vector also includes the Streptomyces hygroscopicus bar gene, which confers the phosphinotricine herbicide resistance to transformed plants .
  • qde-2 was inserted in an anti-sense orientation with respect to the 35S promoter.
  • the obtained vectors can be utilized to over- express the qde-2 gene in plants, or to repress the gene expression of resident genes, which are homologous to qde-2.
  • the qde-2 gene was inserted in a vector containing a fungal specific expression "cassette", comprising the A . nidulans trpC gene promoter and terminator, both in a sense and an anti-sense orientation.
  • the vector contains the bacterial hph gene, which confers the hygro icine drug resistance.
  • the sense plasmid can be used to over express the qde-2 gene, whereas the anti- 5. sense plasmid is used to repress the expression of qde-2 homologous genes in various fungine species.
  • Mammalian expression vector Mammalian expression vector
  • the qde-2 gene was inserted in a vector containing a mammalian specific expression "cassette", including the 0 cytomegalovirus (CMV) promoter and SV40 termination and polyadenylation sequences both in a sense and anti-sense orientation.
  • the vector includes also the neomicine phototransferase gene, as marker for mammalian cell selection.
  • the sense plasmid can be used to over express 5 the qde-2 gene, whereas the anti-sense plasmid can be used to repress the expression of qde-2 homologous genes in various mammalian species.

Abstract

An isolated nucleic acid molecule encoding for a protein characterized in that it has a silencing activity and comprises a domain responsible for dsRNA interference is disclosed; furthermore expression vectors suitable for the expression of said sequence in bacteria, plants, animals and fungi are disclosed; the invention refers also to organisms transformed by such vectors.

Description

I SOLATION AND CHARACTERI ZATION OF A N. CRASSA S ILENCING GENE
AND USES THEREOF
The present invention relates to the isolation and characterization of a Neurospora crassa gene encoding for an essential activity in the co-suppression process and to uses and applications thereof in vegetal, animal and fungine fields. The production of transgenic organisms is of large utility both in basic and applied biological research. The transgenic DNA is usually integrated in the genome and transferred as a Mendelian character. However, in various instances, the transgene introduction induces gene silencing phenomena (Flavell, R.B. 1994), i.e. the repression of the expression of the transgene itself and/or of one or more endogenous homologous genes.
The gene silencing (suppression of gene expression) can act at two levels: transcriptional (trans- inactivation) where transgenes contain sequences homologous to the silenced gene promoter (Vaucheret, 1993); and post-transcriptional (co-suppression) which requires ho ologies between coding regions (Flavell, 1994; Stam et al . , 1997; Baulcombe, 1996). Generally the silencing induced by a transgene requires an almost complete sequence homology (from 70% to 100%) between transgene and silenced gene sequences (Elkind, 1990) .
In the Neurospora crassa filamentous fungus, during the vegetative phase, the presence of transgenes induces a post-transcriptional gene silencing phenomenon, named "quelling" (Cogoni et al . , 1996). By using the al -1 gene (albino 1) (Schmidhauser et al., 1990) as silencing visual marker, many features of the phenomenon have been discovered (Cogoni et al . ,
1996) . Particularly the al -1 gene "quelling" in Neurospora is- characterized in that: 1) the- gene silencing is reversible further to the loss of transgene copies; 2) the reduction of mRNA basal level results from a post-transcriptional effect; 3) transgenes containing at least a region of 132 base pairs which is identical to the region encoding for the target gene are sufficient to induce the "quelling"; 4) the duplication of promoter sequences is ineffective to induce the silencing; 5) the "quelling" exhibits a dominant behavior in eterocarions containing both transgenic and untransformed nuclei, indicating the involvement of a trans-acting diffusible molecule among the nuclei; 6) the expression of an aberrant RNA transcribed by the transgenic locus is strictly correlated to silencing, suggesting that the "quelling" can be induced and/or mediated by a transgenic RNA molecule.
Therefore homologies between Neurospora silencing and plant co-suppression can be pointed out. The gene silencing in Neurospora is reversible, as result of transgenic copies instability during mitotic phase; in plants also the co-suppression reversion is associated with the reduction of transgene copy number, resulting from intra-chromosomal recombination during mitosis or meiosis (Mittelstein Scheid et al., 1994; Stam et al . ,
1997) . Thus both in plants and in Neurospora the transgene presence is required to maintain the silencing.
As in Neurospora , a decrease of the mRNA basal level of the silenced gene results from a post-transcriptional mechanism (Dehio and Schell 1994; van Blokand et al., 1994; de Carvalho et al., 1995). Furthermore to induce the "quelling", transgenes must contain a portion of the silencing target gene coding sequence, being the promoter region ineffective. In plants coding regions with no promoter sequences can induce silencing (van Blokand et al., 1994) and, as in the "quelling", promoters or functionally active gene products are not required for the co-suppression. One of the similarities between "quelling" and co- suppression in plants is that both mechanisms are mediated by diffusion factors. In Neurospora eterokaryotic strains, nuclei wherein the albino-1 gene is silenced are able to induce the al -1 gene silencing of the other not transformed nuclei, all sharing the same cytoplasmic environment (Cogoni et al., 1996). In plants the presence of a diffusion factor results from the fact that the co-suppression is effective in inhibiting the replication of Tobacco Etch Virus (TEV) , a RNA virus with an exclusively cytoplasmic cycle. The occurrence of highly diffusible factors, which are effective to mediate the co-suppression, has been demonstrated using the grafting technique in tobacco (Palaqui et al . , 1997), showing that silenced tobacco plants are able to transfer the silencing to non-silenced plants through grafting.
The fact that "quelling" and co-suppression share all these features suggests that mechanisms involved in post-transcriptional gene silencing in plants and in fungi can be evolved by an ancestral common mechanism. Recently gene inactivation phenomena resulting from transgene introduction have been disclosed in animals. In Drosophila melanogaster the location of a transgene close to heterochromatic centers results in a variegate expression (Wallrath and Elgin, 1995; Pirrotta, V.,
1997) . - Similar expression profiles have been observed when the reference transgene is within tandem arrayed transposons, indicating that tandem repeats are effective to induce the chromatin condensation. (Dorer and Henikoff, 1994). Again in Drosophila Pal-Bhadra et al . (1997) have observed that the transgene introduction can lead to gene inactivation phenomena, similar to the co- suppression.
Gene silencing phenomena resulting from transegene sequence repeats have been disclosed recently in mammalians .
Garrick et al. (1998) produced mouse transgenic lines wherein 100 transgenic copies are present in a unique locus and are repeats-arrayed in direct tandem. The transgene expression has been disclosed to be inversely proportional to the number of occurring copies, indicating that silencing phenomena dependent on repeat copies are present also in mammalians.
It has been recently found that double stranded RNA molecules can induce a sequence-specific silencing in several organisms (Fire A., 1999). The mechanism known as dsRNAi (double stranded RNA interference) acts at a post- transcriptional level by inducing sequence-specific degradation of homologous mRNAs (Montgomery, Xu and Fire,
1998) . Urider this aspect, dsRNAi and quelling in Neurospora are similar mechanisms, both of them acting at a post-transcriptional level. In addition, both RNA- induced silencing and DNA-induced silencing can be transmitted from cell to cell. Therefore the identification of Neurospora genes which are involved in the silencing is the first step to modulate the same process in plants, animals and fungi. The silencing modulation is of great relevance when transgenic organisms able to express the desired phenotype are produced.
The authors of the present invention have already isolated Neurospora crassa strains mutated at essential functions for gene silencing (Cogoni and Macino, 1997); 15 independent isolated mutants define three complementation groups, thus identifying the qde-1 , qde-2 and qde-3 genes { qde stands for "quelling"-deficient) , whose products are essential to the silencing machinery. qde genes are essential to the Neurospora silencing, as suggested by the fact that silencing of three independent genes { al -1, al-2 and qa -2) is impaired by qde mutations (Cogoni and Macino, 1997).
The authors of the present invention have already identified qde-3 gene (PCT WO 00/327885) and qde-1 gene (PCT WO 00/50581) .
The authors of the invention have identified and cloned now one out of Neurospora qde genes, the qde-2 gene, thus identifying one of required factors for silencing. By considering the similarity between "quelling" and co-suppression, genes orthologous to the isolated gene are involved in co-suppression and more generally in gene silencing in other organisms, like plants, fungi and animals.
The present invention can be applied with reference to two general scopes: 1) silencing potentiation as a tool for inactivating more effectively and durably a desired gene, .and 2) silencing suppression to obtain a better expression of the introduced transgenes.
The isolated qde-2 gene can be introduced alone or with qde-1 and/or qde-3 genes in plants, animals or fungi, in order to inactivate the expression of selected genes. The aim is to activate a sequence-specific silencing mechanism both in deficient organisms and in organisms wherein the same is not very efficient. The gene silencing can be induced also by introducing specific double stranded DNA or RNA sequences, homologous to the gene to be inactivated.
As to the silencing potentiation, the over- expression of one or more genes controlling the phenomenon can lead to higher efficiency and/or stability thereof. Therefore the introduction of qde-2 gene or of homologous genes thereof in organisms can constitute a tool to repress more effectively gene functions. Particularly this approach is specially useful in plants wherein the co-suppression is usually used for the "knock-out" of gene functions. In plants again the gene silencing potentiation can be used to obtain lines resistant to pathogen virus, by introducing transgenes encoding for viral sequences, in order to achieve the expression inhibition of the virus itself (Flavell et al., 1994) .
Analogous applications are suitable for animals, wherein some indications suggest that silencing can inhibit the suitable expression of introduced transgenes (Garrick et al., 1998). On the contrary, there are instances wherein it is desirable not to have or to reduce the gene silencing, i.e. where a transgene is to be over-expressed. It is known that the co-suppression is strictly correlated both with the presence of an high copy number of the transgene, and with a transgene high expression. This correlation can hamper the production of transgenic organisms which express a transgene at high levels, because more high is the expression and/or the copy number, more probable is to evoke silencing responses. As above mentioned, analogous mechanisms of gene inactivation, dependent on a high copy number, have been disclosed in animals. In these circumstances plant or animal lines, totally or partially ineffective for silencing, constitute an ideal recipient wherein the desired gene can be over-expressed. The invention can be applied within this scope using different approaches: A) Identification and production of mutant lines in genes homologous to qde-2 gene, in plants, animals and fungi .
The identification of Neurospora qde-2 gene, essential for silencing mechanism, can allow the isolation of mutant lines in other organisms, mutated in genes homologous to qde-2. For example by means of amplifications using degenerated primers, designed from the most conserved regions of qde-2 gene, mutant lines in homologous genes can be identified, by analysis of insertion mutant gene banks, already available for many plant species. Both in fungi and animals such mutants can be obtained, following the identification of the homologous gene, by means of "gene disruption" techniques using homologous recombination. B) Reduction of qde-2 gene expression
Other strategies for the production of silencing- deficient lines comprise the use of Neurospora qde-2 gene or homologous genes thereof, qde-2 or homologous genes can be introduced into suitable expression vectors to express them in an anti-sense orientation in order to inhibit the expression of resident endogenous genes. Alternatively portions of qde-2 or of homologous genes can be over-expressed, in order to obtain a negative dominant effect and thus blocking the function of qde-2 endogenous genes .
The authors of the present invention have cloned and characterised the Neurospora crassa qde-2 gene . The sequence analysis of the qde-2 gene detected a region having a significant homology with the sequence of a C. elegans gene, rde-1 , involved in the dsRNA mediated interference (Tabara et al., 1999). The authors of the invention for the first time have demonstrated that the transgene induced post- transcriptional gene silencing and the dsRNA interference share common genetic mechanisms. This supports the hypothesis that the sequence specific gene silencing phenomena evolved from an ancestral mechanism aimed to protect the genome against transposons. Furthermore, the results of the authors suggest that dsRNA molecules are involved in the post-transcriptional gene silencing in fungi. dsRNA molecules could be produced directly from integrated trangenes as a result of the presence of inverted repeats or as an out come of transcription from convergent inverted promoters. Alternatively, single stranded aberrant RNA may be used as a template by an RNA-dependent RNA polymerase (such as QDE-1 protein) able to produce dsRNAs .
Within the scope of the invention the term homology is intended as similarity, i.e. number of identical residues + number of conserved residues with respect to the total residues of the considered sequence.
Therefore it is an object of the present invention an isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and in comprising a domain responsible for dsRNA interference, wherein the domain is at least 25% homologous with the amino acid sequence from aa . 373 to aa . 910 of sequence in fig. 1 (SEQ ID No. 2) . Preferably the domain is at least 30% homologous with the amino acid sequence from aa. 373 to aa. 910 of sequence in fig. 1 (SEQ ID No. 2). More preferably the domain is at least 38% homologous with the amino acid sequence from aa. 373 to aa. 910 of sequence in fig. 1 (SEQ ID No. 2) . Most preferably the domain comprises the amino acid sequence from aa . 373 to aa. 910 of sequence in fig. 1 (SEQ ID No. 2). According to a particular embodiment the isolated nucleic acid molecule encodes for a protein having the amino acid sequence of fig. 1 (SEQ ID No. 2) or functional portions thereof. Even more preferably the isolated nucleic acid molecule has the sequence of fig. 1 (SEQ ID No. 1) or its complementary sequence.
A further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in bacteria, the isolated nucleic acid molecule of the invention. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the expression in bacteria can be used and it is within the scope of the invention.
A further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in plants or in specific plant organs, the isolated nucleic acid molecule of the invention, both in a sense and anti-sense orientation. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the invention in plants or in specific plant organs can be used and it is within the scope of the invention.
A further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in fungi, the isolated nucleic acid molecule of the invention, both in a sense and anti- sense orientation. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the inventive protein in fungi can be used and it is within the scope of the invention.
A further object of the invention is an expression vector comprising, under the control of a promoter which directs the expression in animals, the isolated nucleic acid molecule of the invention, both in a sense and anti- sense orientation. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the invention in animals can be used and it is within the scope of the invention.
A further object of the invention is a prokaryotic organism transformed by using the expression vector active in bacteria of the invention.
A further object of the invention is a plant or a specific plant organ transformed by using the expression vector active in plants of the invention. A further object of the invention is a plant mutated at the isolated nucleic acid molecule of the invention having a reduced or inhibited silencing activity. A further object of the invention is a fungus transformed with the expression vector of the invention active in fungi.
A further object of the invention is a fungus mutated at the isolated nucleic acid molecule of the invention and having reduced or inhibited silencing activity.
A further object of the invention is a non-human animal transformed with the expression vector of the invention active in animals. A further object of the invention is a non-human animal mutated at the isolated nucleic acid molecule of the invention and having a reduced or inhibited silencing activity.
A further object of the invention refers to a protein characterized in having a silencing activity and in comprising a domain responsible for dsRNA interference, wherein the domain is at least 25% homologous with the amino acid sequence from aa. 373 to aa. 910 in fig. 1 (SEQ ID No. 2) . Preferably the domain is at least 30% homologous with the amino acid sequence from aa. 373 to aa . 910 in fig. 1 (SEQ ID No. 2) . More preferably the domain is at least 38% homologous with the amino acid sequence from aa. 373 to aa. 910 in fig. 1 (SEQ ID No. 2) . Most preferably the domain comprises the amino acid sequence from aa. 373 to aa. 910 in fig. 1 (SEQ ID No. 2) . According to a particular embodiment the isolated nucleic acid molecule encodes for a protein having the amino acid sequence of fig._ 1 (SEQ ID No. 2) or functional portions thereof.
I-t is within the scope of the present invention the use of the isolated nucleic acid molecule of the invention to modulate gene silencing in plants, animals and fungi .
The present invention now will be described by way of non limiting examples with reference to the following figures: Figure 1: The isolated nucleic acid molecule of the
5.7 Kb fragment containing the qde-2 gene and flanking sequences (SEQ ID No.l). The amino acid sequence (SEQ ID No. 2) is shown above the nucleotide sequence.
Figure 2: It is schematically represented the pMXY2 plasmid insertion site, in the 80 mutant, used for insertional mutagenesis and consequent polimorphism of the restriction fragments by mean of DNA southern blot of a WT strain and of 80 and 820 mutant strains by using the entire restored flanking region as probe. The 820 mutant has a complete deletion of the qde-2 gene.
Figure 3: Multiple alignment, at the conserved region, among qde-2 and other proteins belonging to ago- elF2C family: A. thaliana ago-1 ; rabbit elF2C; C. elegans rde-1 . Identical amino acids are shown in bold. MATERIALS AND METHODS E. coli strains
E. coli strain HB101 (F~, hsdS20(rb", mb") , supE44, recA13, aral4, proA2, rspL20 (strr) , xyl-5) was used for cloning. Neurospora crassa strains and growing conditions
Neurospora crassa following strains, supplied by Fungal Genetic Stock Center (FGSC, Dpt . Of Microbiology, University of Kansas Medical Ctr. Kansas City, KA) were used:
- Wild" type (FGSC 987);
- qa-2/aro9 (FGSC 3957A) , (FGSC 3958a). The 6XW strain (Cogoni et al . , 1996) was obtained upon transformation of the FGCS 3958a strain with pXl6 plasmid (Cogoni et al., 1996). This plasmid contains the qa -2 gene used as selective marker and the al -1 coding sequence . The mutant strains M7, M20 ( qde-1 ) ; M10, Mil (qde-
2) ; Mil , M18 ( qde-3) are described in Cogoni and Macino, 1997.
The qde mutants were obtained by UV mutagenesis. As recipient the transforming strain (6xw) silenced at the albino-1 gene was used, qde mutants were selected for their ability to recover a wild type unsilenced phenotype and then classified in three different complementation groups. By analyzing the al -2 gene quelling frequency all of qde used mutants are defective for the general silencing mechanism.
Complementation assays with not forced heterocaryons were carried out according to Davis and DeSerres, 1970. Plasmids and libraries The plasmid pMXY2, disclosed in Campbell et al.
1994, used for insertional mutagenesis was obtained from Fungal Genetic Stock Center (FGSC, Dpt . Of Microbiology, University of Kansas Medical Ctr. Kansas City, KA) . The plasmid contains the Bml gene (allele responsible of the benilate drug resistance) , that was used as selective marker after transformation. The genomic DNA containing the qde-2 gene was isolated from a N. Crassa gene library in cosmids . (Cabibbo et al., 1991). N. crassa transformation
Spheroplasts were prepared according to the Akins and Lambowitz (1985) protocol. Southern Blot Analysis
Chromosomal DNA was prepared as disclosed by Irelan et al., 1993. 5 μg of genomic DNA were digested and blotted as reported in Maniatis et al. DNA probes were: a) as to the al -1 gene the probe is represented by a Xbal-Clal restriction fragment of pX16 (Cogoni et al., 1996); b) as to the Bml gene the probe is represented by the 2.6Kb Sail fragment of pMXY2. Northern Blot Analysis N. crassa total RNA was extracted according to the protocol described by Cogoni et al . , 1996. The mycelium was grown for two days at 30°C, then powdered in liquid nitrogen before RNA extraction. For Northern analysis 10 μg of RNA were formaldehyde denatured, electrophoresed on a 1% agarose, 7% formaldehyde gel, and blotted over Hybond N (Amersham) membranes. Hybridization was carried out in 50% formamide in the presence of P labeled DNA probe 1.5x10 cpm/ml . RESULTS Isolation of silencing mutant by insertional mutagenesis
Previously a Neurospora strain (6XW) wherein the albino-1 resident gene was steadily silenced was used for
UV mutagenisis that brought to the isolation of qde
("quelling" deficient) mutants in N. crassa induced gene silencing (Cogoni and Mancino 1997).
The 6XW strain shows an albino phenotype due to the lack of carotenoid biosynthesis, as results by the silencing of the albino 1 gene expression (Schmidhauser et al., 1990). A mutation interfering with the silencing machinery is easily detectable by producing a wild type phenotype (bright orange) of the carotenoid biosynthesis. By means of complementation assays it was possible to establish that qde mutants belong to three complementation groups, indicating the presence of three genetic loci involved in the Neurospora silencing mechanism. In order to isolate the qde genes an insertional mutagenesis was carried out with the 6XW strain, previously used for UV mutagenesis. The insertional mutagenesis was carried out by transforming the 6XW strain with a plasmid, taking advantage of the fact that, after the transformation, plasmids are randomly inserted in the Neurospora crassa genome. The mutagenesis was carried out transforming the 6XW silenced strain with pMXY2 (see Materials and Methods) which contains the benilate resistance as selective marker. Transformed strains able to grow in the presence of benilate containing medium and showing a wild type phenotype for the carotenoid biosynthesis were selected. Out of 50.000 isolated independent transformed strains, a benilate resistant strain (80) was isolated, which showed the bright orange phenotype expected for a qde gene mutation. In order to verify that the silencing release was effectively due to a gde gene mutation and not to the loss of al -1 transgene copies, the genomic DNA of the strain 80 was extracted and digested with Smal and Hindlll restriction enzymes. After blotting, DNA was hybridized with a probe corresponding to the coding sequence of al -1 . The Smal site is present only once in the al -1 transgene containing plasmid and the digestion by using said enzyme produces a 5.5Kb fragment corresponding to tandem arrayed al-1 transgenes, while a
3.1Kb fragment is expected from the resident al-1 locus.
The number of al -1 transgenic copies present in the 80 strain is comparable to that present in the silenced 6XW strain.
The strain 80 is mutated in qde-2 gene
The strain 80 was assayed in a heterokaryon assay with a wild type strain and with M7 , M20 ( qde-1 ) M10, Mil ( qde-2) , Mil , M18 ( qde-3) mutants and with a wild strain (Cogoni and Macino, 1997) . As shown in Table 1 the al -1 gene silencing is restored producing an albino phenotype in all of heterocaryons but M10 and Mil. This behavior is consistent with the presence of a qde-2 gene recessive mutation in the strain 80.
Table 1
Reciprocal heterokaryons among the mutant 80 and previously characterized qde mutants.
80 M7 M20 M10 Mil M17 M18
80 WT AL AL WT WT AL AL
M7 WT WT AL AL AL AL
M20 WT AL AL AL AL
M10 WT WT AL AL
Mil WT AL AL
M17 WT WT
M18 WT
WT = heterokaryon wit a wilei type phenotype for carotenoid accumulation;
AL = heterokaryon with an albino phenotype wherein the al -1 gene silencing is restored.
Recovery of sequences flanking the pMXY2 plasmid integration site In order to recover sequences flanking the integration site or sites the following methodology was carried out. The genomic DNA of strain 80 was digested with Aat II enzyme. Subsequently the genomic DNA was ligated and the product used to transform E. coli cells that was screened in an ampicillin-containing medium. PQcl plasmid was recovered and a DNA fragment containing sequences flanking the integration site was isolated from it by using Aat II and Cla I enzymes. Isolation of genomic clones, their subcloning and complementation of the qde-2 mutant
The fragment from pQcl plasmid was used to probe a Neurospora crassa genomic library in cosmids . Three cosmids 6G10, 20C1 and 23F2 containing about 35 Kb genomic DNA inserts, were isolated. Such cosmids were used in transformation experiments of Mil and 80 mutants. All of cosmids are able to restore the ai-1 gene silencing in the two mutants, determining the appearance of an albino phenotype. The 20C1 cosmid was used to subclone a 5.7 Kb BaitiHI-BamHI fragment. This subclone was used for transformation experiments and resulted to be able to complement the qde-2 phenotype, indicating that a qde-2 functional gene is present in this plasmid. Isolation and sequence of the qde-2 cDNA The sequence of BamHI-BamHI region allowed to deduce the amino acid sequence of the QDE-2 protein. The qde-2 gene encodes for a 938 aa. putative protein (104 KDa) . The genomic clone does not contain any introns since the reading frame does not contain any interruptions and intron acceptor and donor sequences were not identified (Fig. 1, Seq. ID No 1, 2) . The qde-2 gene comprises an homologous domain with encoding genes for proteins that are responsible for dsRNA interference
The 938 aa sequence (SEQ ID No. 2) was used to search in database of amino acid sequences, by using the BLASTP algorithm. As showed in fig. 3, the search identified significant homologies with argonaute-1 gene [with expected values (E value) of 2e-57] of A . Thal iana (mutants of this gene show developmental anomalies) ; rde- 1 gene [with expected values (E value) of le-23] of C elegans, involved in gene silencing phenomena induced by double stranded RNA; elF2C gene [with expected values (E value) of 5e-60] of rabbit isolated as an element belonging to transcription beginning complex. Plant expression vector
The qde-2 gene was inserted, in a sense orientation, into a vector containing a plant expression "cassette", including the 35S promoter and the PI-II "terminator" sequences. The vector also includes the Streptomyces hygroscopicus bar gene, which confers the phosphinotricine herbicide resistance to transformed plants . In an analogous vector to the above mentioned one, qde-2 was inserted in an anti-sense orientation with respect to the 35S promoter. The obtained vectors can be utilized to over- express the qde-2 gene in plants, or to repress the gene expression of resident genes, which are homologous to qde-2. Fungus expression vector The qde-2 gene was inserted in a vector containing a fungal specific expression "cassette", comprising the A . nidulans trpC gene promoter and terminator, both in a sense and an anti-sense orientation. In addition the vector contains the bacterial hph gene, which confers the hygro icine drug resistance. The sense plasmid can be used to over express the qde-2 gene, whereas the anti- 5. sense plasmid is used to repress the expression of qde-2 homologous genes in various fungine species. Mammalian expression vector
The qde-2 gene was inserted in a vector containing a mammalian specific expression "cassette", including the 0 cytomegalovirus (CMV) promoter and SV40 termination and polyadenylation sequences both in a sense and anti-sense orientation. The vector includes also the neomicine phototransferase gene, as marker for mammalian cell selection. The sense plasmid can be used to over express 5 the qde-2 gene, whereas the anti-sense plasmid can be used to repress the expression of qde-2 homologous genes in various mammalian species.
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Claims

Claims
1'. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and in comprising a domain responsible for dsRNA interference, wherein the domain is at least 25% homologous with the amino acid sequence from aa . 373 to aa. 910 of SEQ ID No. 2. 2. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 1, wherein the domain is at least 30% homologous with the amino acid sequence from aa . 373 to aa. 910 of SEQ ID No.
2.
3. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 2, wherein the domain is at least 38% homologous with the amino acid sequence from aa . 373 to aa. 910 of SEQ ID No. 2.
4. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 3, wherein the domain is the amino acid sequence from aa. 373 to aa . 910 of SEQ ID No. 2.
5. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 4, wherein said isolated nucleic acid molecule encodes for a protein having the amino acid sequence of SEQ ID No. 2, or functional portions thereof.
6. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 5, wherein said isolated nucleic acid molecule has the sequence of SEQ ID No. 1 or its complementary sequence.
7. Expression vector comprising, under the control of a promoter that directs the expression in bacteria, the isolated nucleic acid molecule according to any one of claims 1-6.
8. Expression vector comprising, under the control of a promoter that directs the expression in plants or in specific plant organs, the isolated nucleic acid molecule according to any one of claims 1-6, both in a sense and anti-sense orientation.
9. Expression vector comprising, under the control of a promoter that directs the expression in fungi, the isolated nucleic acid molecule according to any one of claims 1-6 both in a sense and anti-sense orientation.
10. Expression vector comprising, under the control of a promoter that directs the expression in animals, the isolated nucleic acid molecule according to any one of claims 1-6 both in a sense and anti-sense orientation.
11. Prokaryotic organism transformed by using the expression vector active in bacteria according to claim
7.
12. Plants or a specific plant organ transformed by using the expression vector active in plants according to claim 8.
13. Plant mutated at the isolated nucleic acid molecule according to any one of claims 1-6 having a reduced or inhibited silencing activity.
14. Fungus transformed by using the expression vector active in fungi according to claim 9.
15. Fungus mutated at the isolated nucleic acid molecule according to any one of claims 1-6 having a reduced or inhibited silencing activity.
16. Non-human animal transformed by using the expression vector active in animals according to claim 10.
17. Non-human animal mutated at the isolated nucleic acid molecule according to any one of claims 1-6 having a reduced or inhibited silencing activity.
18. Protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference wherein the domain is at least 25% homologous to the amino acid sequence from aa. 373 to aa. 910 of SEQ ID No. 2.
19. Protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 18 wherein the domain is at least 30% homologous to the amino acid sequence from aa. 373 to aa. 910 of SEQ ID No. 2.
20. Protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 19 wherein the domain is at least 38% homologous to the amino acid sequence from aa. 373 to aa . 910 of SEQ ID No. 2.
21. Protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 20 wherein the domain is the amino acid sequence from aa . 373 to aa . 910 of SEQ ID No. 2.
22. Protein characterized in having a silencing activity and comprising a domain responsible for dsRNA interference according to claim 21 comprising the amino acid sequence of SEQ ID No. 2 or functional portions thereof.
23. Use of the isolated nucleic acid molecule according to any one of claims 1-6 to modulate the gene silencing in plants, animals and fungi.
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