MX2007004900A - Rna constructs - Google Patents

Abstract

The present invention concerns concatemer and/or stabilized RNA constructs capable of forming dsRNA, optionally comprising a sequence capable of protecting the dsRNA against RNA processing in a host cell. The invention also relates to methods of producing these constructs and to methods for using these constructs. The constructs according to the present invention are particularly useful in plant pest control.

Description

RNA CONSTRUCTS FIELD OF THE INVENTION The present invention relates to the field of gene silencing mediated by a double-stranded RNA (dsRNA). More particularly, the present invention relates to genetic constructs designed to be more effective in the silencing of dsRNA by (i) targeting multiple target sequences and / or by (ii) expressing dsRNA that is protected against RNA processing. These constructs are especially useful in the control of plant pests mediated by dsRNA. BACKGROUND OF THE INVENTION Many constructs of dsRNA have been described in the art. A classical dsRNA is produced from a DNA construct comprising two convergent promoters flanking the complementary sequence of the target sequence which requires down-regulation (see, for example, WO00 / 01846). As the gene silencing technology mediated by dsRNA advanced, new constructs were designed to improve the dsRNA for several purposes. In order to produce the dsRNA more efficiently, a structure of stem-loop-stem or "hair pin" was developed. In accordance with what is described for example in WO0099 / 53050, this hair pin allows the formation of dsRNA from a single RNA transcript. The RNA transcript comprises the sense version and the antisense version of the complementary sequence, separated by a non-complementary loop structure that allows the RNA transcript to bend back and the base pair in a stem portion of dsRNA . In order to produce more effective dsRNA in the silencing of genes, multiple copies of the sequence complementary to the target sequence were incorporated into a construct and converted into a dsRNA. WO99 / 49029 describes in more detail a non-synthetic gene comprising multiple structural gene sequences, wherein each structural gene sequence is substantially identical to the target gene. WO2004 / 001013 describes constructs specially designed to be used in clinical applications for the prevention or treatment of diseases or infections without generation of adverse side effects caused by dsRNA-induced toxicity. It has been reported that a certain dsRNA can induce an interferon response that can cause cell death (Jaramillo et al., Cancer., Cancer Invest., 13: 327-338, 1995). These constructs are characterized by portions that are sensitive to RNA processing in order to improve the formation of short interfering RNAs (siRNAs) that mediate gene silencing while avoiding the dsRNA toxicity caused by long dsRNA (more than 30 pairs of bases) . Short interfering RNAs (siRNAs) mediate the dissociation of target RNAs from a single specific chain. These siRNAs usually have a length of approximately 21 nt, suggesting that the expression of siRNA in the host organism causes an efficient and specific down regulation of gene expression, resulting in a functional deactivation of the target genes. The silencing of dsRNA gene finds applications in many different areas, such as gene silencing mediated by dsRNA in plants. The gene silencing mediated by dsRNA also finds applications in the field of pest control affecting plants (WO 00/01864). In general terms, the harmful organism that affects plants is eradicated through the absorption of dsRNA capable of silencing the expression of a target gene, said expression being necessary for the viability, growth and / or development of the pest. The contacting of organisms that affect plants with dsRNA can occur in several ways, an example is the production of dsRNA within the plant cell affected by the pest. One problem with the expression of dsRNA in plants is that it can be processed by the RNA processing machinery of the plant cell (Susi et al., 2004. PMB 54: 157-174, Baulcombe, 2004, Nature 431: 356-363). COMPENDIUM OF THE INVENTION While the formation of short interfering RNAs (siRNAs) of approximately 21 nt is desirable for gene silencing, it has now been found by the present inventors that the minimum length of dsRNA must be at least 80-100 nt with the object of being efficiently absorbed by the body to be fought. There are indications that, in invertebrates such as free living nematode C. elegans or the parasitic nematode of Meloidogyne incognita plants, these longer fragments are more effective in silencing genes, possibly due to a more efficient absorption of these genes. Long dsRNA by the invertebrate. The present invention solves this problem by a supply of dsRNA constructs that are efficient in gene silencing mediated by dsRNA, while retaining a sufficient length. In addition, the present invention offers concatamer dsRNA design, allowing combining several short fragments in a longer dsRNA construct and allowing to increase the effectiveness of the control of the viability, growth and / or development of the harmful organism. Alternatively or additionally the present invention offers stabilized dsRNA constructs that protect the dsRNA against RNA processing in the host cell. The constructs described herein and suitable for efficient pest control mediated by dsRNA are designed to meet some of the following requirements (1) the dsRNA construct has good stability in the host cell that produces the dsRNA; (2) the dsRNA is absorbed by the harmful organisms; (3) dsRNA has good stability in harmful organisms; and / or (4) the dsRNA is effective in the harmful organism to control its viability, growth and / or development. These designs and constructs of dsRNA have one or more of the following advantages: (1) The concatemer and / or stabilized constructs of the present invention allow the incorporation of multiple dsRNA fragments to target multiple target sequences with target genes simultaneously. These multiple target sequences or target genes may come from the same harmful species or different harmful species. These multiple target sequence or multiple target genes may be orthologous or homologous or may not be related. Alternatively, the concatemer and / or stabilized constructs allow the incorporation of multiple dsRNA fragments directed against a part or against several parts of a target gene; (2) the constructs of the present invention allow the development of dsRNA whose length and / or size and / or shape is compatible with sufficient absorption by a harmful organism; (3) Unlike the dsRNA constructs of the prior art which have been designed to be rapidly processed into smaller fragments, it is now one of the purposes of the present invention to design more stable dsRNA in the host organism or in the host cell (for example in the plant and / or in the plague of the plant). This is achieved by incorporating into the dsRNA a sequence capable of protecting the dsRNA against the processing of the dsRNA; (4) The constructs of the present invention have the advantage of being stable in the host organism where the dsRNA construct is produced. For example, when expressed in a plant cell, the dsRNA construct in accordance with that provided by the present invention is protected against RNA processing in the plant. In this way, the dRNA is less divided by the machinery of the host organism and can be absorbed in a more intact form (eg, larger) by the organism harmful to the plant when it is fed into or from the plant. The present invention further relates to the DNA construct encoding the dsRNA constructs according to the present invention for expressing vectors comprising such DNA constructs, and to host cells comprising such dsRNA, DNA or expression vectors. The present invention also encompasses methods for producing dsRNA constructs of this type, methods for the production of DNA expression constructs, methods for the production of host cells as methods for using those constructs in gene silencing, methods for producing transgenic organisms and methods to control pests. DETAILED DESCRIPTION OF THE INVENTION Concatemer Constructions In accordance with the first embodiment, the present invention relates to an isolated (eg, substantially pure) double stranded ribonucleic acid (dsRNA) effective in silencing the gene by RNAi, wherein the dsRNA (portion or fragment) comprises multiple fragments of dsRNA, each fragment comprising complementary fused strands, one of which is complementary to at least a part of the nucleotide sequence of a target sequence to be silenced or a target gene of interest; said dsRNA is capable of forming a portion or fragment of double-stranded RNA. A concatemer construct according to the present invention comprises multiple fragments of dsRNA within a stem of dsRNA. Said concatemer construct can be used "per se", hereinafter referred to as "a concatemer construct per se" or it can be used as a stem of dsRNA in the stabilized RNA constructs described herein. Accordingly, the RNA constructs of the present invention comprising multiple fragments of dsRNA in a stem of dsRNA are also generally known as "concatemers". As a list of non-limiting examples of "concatemers", the present invention offers a clover leaf concatamer, a silent bell concatemer, a hair pin concatemer, a stem concatemer of dsRNA. All these concatemers may optionally be stabilized with a blockage in accordance with what is described herein and may optionally be equipped with a linker in accordance with what is described herein. The present invention therefore relates to concatemerized and / or stabilized RNA constructs comprising double-stranded RNA (also referred to as a dsRNA molecule) comprising fused complementary strands, one of which has a nucleotide sequence that is complementary of at least a part of a target nucleotide sequence of a target gene of a deleterious species. In one embodiment, multiple RNA fragments are present which are complementary to different sequences (eg, different sequences) in a target gene. In another embodiment, the present invention also relates to constructs of concatemer and / or stabilized RNA in accordance with that described above, comprising multiple RNA fragments that are complementary to different (eg, different) target gene sequences. In one embodiment, the dsRNA fragments are separated by a linker sequence or by a block. Preferably, the linker sequence is double-stranded and the chains are complementary, thus also forming a double-stranded region. The sequence of both may comprise a short random nucleotide sequence that is not complementary to the target sequences. The term "multiple" in the context of the present invention refers to at least two, at least three, at least four, at least five, at least six, etc. and up to at least 10, 15, 20 or at least 30. The present invention therefore relates to isolated dsRNA or to dsRNA construct isolated from according to that described herein, wherein said dsRNA comprises at least one repeat of a fragment of dsRNA. As used herein, a "repeat" refers to two copies of the same dsRNA fragment. In another embodiment, the present invention relates to isolated dsRNA or a dsRNA construct isolated in accordance with that described herein, wherein said dsRNA comprises at least one repeat of a series of dsRNA fragments. Thus, in accordance with what is described herein, a repeat refers to two copies of a series of dsRNA fragments. The present invention also relates to an isolated dsRNA according to that described above wherein said dsRNA comprises at least two or three copies, preferably at least four, five or six copies, more preferably at least seven, eight, nine, ten or more copies of a dsRNA fragment or a series of dsRNA fragments. In other words, said multiple fragments of dsRNA are repeats of a single fragment of dsRNA or of a series of fragments of dsRNA. It should be clear that the term "multiple dsRNA" also encompasses dsRNAs that comprise copies of one or more fragments of dsRNA and that further comprise other fragments of dsRNA that are different from the repeated, copied or multimerized dsRNA fragments. Accordingly, the invention also relates to an isolated dsRNA comprising one or more repeats of dsRNA fragments and further comprising at least one fragment of dsRNA that is distinct from the repeated fragment (s). The term "complementary" as used herein refers to DNA-DNA and RNA-RNA complementarity as well as DNA-RNA complementarity. Analogously, the term "RNA equivalent" refers to the fact that in the DNA sequence (s), the "T" base may be replaced by the corresponding "U" base normally present in ribonucleic acids. A "complementary region" as used herein refers to a region that is complementary to at least a part of a nucleotide sequence of a target gene. The "complementarity" when used within the context of the present invention for a dsRNA refers to having a substantial sequence identity with one of the chains of the target sequence. In the embodiment of the present invention, the complementary region will generally comprise a nucleotide sequence having a sequence identity greater than about 75% with the corresponding sequence of the target gene; however, a higher homology can produce a more efficient modulation of the expression of the target gene. Preferably, the sequence identity is about 80%, 85%, 90%, 95%, and even more preferably, greater than about 99%. In the context of the present invention, the expression "more than approximately" has the same meaning as "at least". Preferably the complementary region is a fragment that is not harmful to organisms other than the target organism (s). Preferably, the fragment has no more than 20 contiguous nucleotides in common with a sequence of an organism other than the target organism. For example, when the target organism is a pathogen of the plants, such as a plant parasitic nematode or an insect, the fragment has no more than 20 contiguous nucleotides in common with the nucleotide sequence of a plant or a mammal ( in particular, a human being). The terms "double-stranded RNA (dsRNA)" and "RNA capable of forming a dsRNA" are used here interchangeably. The term "dsRNA construct" as used herein encompasses all constructs capable of forming double-stranded RNA, such as for example any of the concatamer or stabilized constructs described herein. As described further, the dsRNA or dsRNA construct may comprise other sequences that are not complementary to a target sequence or gene but that have other functions. The terms "double-charged RNA fragment" or "double-stranded RNA region" refer to a small double-stranded RNA entity that corresponds to (a part of) the target gene. As used herein, the expression "corresponds to" means "complementary to". In one embodiment, in the dsRNA of the present invention, said multiple fragments of dsRNA are not separated by a non-complementary region. That means that no non-hybridizing RNA region is present between the separated dsRNA fragments. In accordance with other embodiments, in the dsRNA of the invention the fragments of the dsRNA are not separated by a spacer or a blocking sequence in accordance with what is further described. In concatemer constructs, the length of each of the dsRNA fragments can be at least 17 base pairs, 18 base pairs, 19 base pairs, 20 base pairs, 21 base pairs, 22 base pairs , 23 base pairs, 24 base pairs, 25 base pairs or more, for example approximately 30 base pairs, approximately 40 base pairs, approximately 50 base pairs, approximately 60 base pairs, approximately 70 base pairs , approximately 80 base pairs, approximately 90 base pairs, approximately 100 base pairs, approximately 110 base pairs, or approximately 120 base pairs. Preferred dsRNA fragments in a concatemer construct have a length between 17 and 2000 base pairs, preferably between 21 and 1000 or 500 or 250 base pairs, preferably between 40 and 150 base pairs, more preferably between 50 and 120 base pairs or any number between these values. A "white gene" as used herein refers to a gene that must be silenced in the target species. A target gene encompasses a promoter region, a 5 'untranslated region, a coding sequence in which introns may be present, and a 3' untranslated region. The target gene can be selected from the genome of any target species in accordance with what is described herein. According to one embodiment, the target sequence is selected within the genome of an organism, said organism is different from the organism in which the dsRNA is expressed. That means that the dsRNA is expressed in the cell or organism and is subsequently transferred or absorbed by another cell or organism comprising the target gene. In accordance with a specific embodiment of the present invention, the dsRNA is expressed in a plant or in a plant cell and the target gene is selected from the genome of a bacterium, a virus, a virion, an invertebrate, more particularly a plant-damaging species, such as a virion, a virus, a nematode, a fungus or an insect. A "transfer" of the dsRNA from the plant to the pest means that the dsRNA is produced in the plant cell and is absorbed, relocated or put in contact with the harmful organism. A nematode that parasitizes plants or an insect, for example, can absorb the dsRNA produced in the plant by feeding on the cytoplasm of the plant cell. A fungal cell that is in contact with the dsRNA can be a pathogenic fungal cell of the plant in a life stage outside a plant cell, for example, in the form of a hyphae, germinative tube, appressorium, conidium (asexual spore), ascocarp, cleistotecium, or ascospora (sexual spore outside the plant). Alternatively, the fungal cell in contact with the dsRNA is a pathogenic fungal cell for the plant in a life stage within a plant cell, for example, a pathogenic form, for example a penetration spindle, a hyphae, a spore or a haustorio. According to other embodiments of the invention, it may be sufficient to contact the pest or plague species cell with the dsRNA, in which case the dsRNA transfer means contacting a composition comprising the dsRNA or dsRNA construct. According to another embodiment, the dsRNA is expressed in a bacterial or fungal cell and the bacterial or fungal cell is absorbed or eaten by the harmful species. According to another embodiment, the dsRNA is isolated from the bacterial or fungal cell or purified from the bacterial or fungal cell expressing the dsRNA, and the dsRNA is provided as a pesticide or in a pesticidal formulation to the harmful species. Particular suitable white genes are genes involved in an essential biological pathway of the target species, which means that the target gene is an essential gene for the target species and that the silencing of the target gene has an adverse effect on viability, growth and / or development of the white species. Suitable target genes include genes associated with infection, spread or pathogenesis of the deleterious species in the host organism. Choice of target gene (s) to be targeted by a concatemer construct The choice of target gene (s) to be targeted by a single concatemer construct depends on the choice of the target gene to be silenced in the target organism or in target organisms in order to achieve the desired effect of pest control. For the concatemers designed below, the target gene (s) was (were) selected from one or more of the following gene categories: 1. "Essential" genes include genes that are vital to one or more target organisms and result in a lethal or severe phenotype (eg, movement, feeding, paralysis, fluid absorption, fertility) when they are silenced. The choice of a strong lethal white gene results in a potent RNAi effect. In the concatamer constructs of the invention, multiple dsRNA fragments that target the same very effective lethal genes or different highly effective lethal genes were combined to further increase the potency, efficacy or speed of the dsRNA in pest control. 2. "Pathogenicity genes" are genes involved in the pathogenicity or infectivity of the pest. The approach of these genes can reduce the pathogenicity or the infectivity of the pest thus protecting the infected organism against an infestation by the harmful species. 3. "Weak genes" encompass white genes with a particularly interesting function but which are in fact weak genotypic when silenced independently. Focusing a particular but weak white gene results in a specific RNAi effect, which means that the mode of action is very focused and controlled. For example, interesting but weak genes could be genes that are very specific for species or even genes restricted to species but that do not result in an effective RNAi effect when they are focused separately. In the concatamer constructs of the invention, multiple dsRNA fragments that target a single weak gene or different weak genes were combined to obtain a strong RNA effect. 4. "Pest-specific" genes encompass genes that do not have a substantial homologous counterpart in non-deleterious organisms as can be determined through homology searches, such as BLAST searches. The choice of a specific target gene for pest results in a specific RNAi effect for species with no effect or substantially no adverse effect on non-target organisms. 5. "Conserved genes" encompass conserved genes (at the amino acid level) between the target organism and the non-target organism (s). Certain white genes can have a very effective RNAi effect, but they can be very conserved between organisms. To reduce possible effects on non-target species, such effective but conserved genes were analyzed and target sequences of the variable regions of these conserved genes were chosen to be targeted by the dsRNA fragments in the concatemer constructs of the invention exemplified herein. Here, conservation is evaluated at the level of the nucleic acid sequence. Such variable regions therefore comprise the less conserved sections of the target gene (s). 6. Genes of "conserved pathway" encompass genes involved in the same biological pathway or cellular process, or encompass genes that have the same functionality in different species. to. Preferred examples of such "conserved pathway" target genes are genes involved in vital cellular pathways or functions, said pathways or functions are sensitive to RNAi, for example, without being limited to these examples, endocytosis, the cytoskeleton, intracellular and intercellular transport, binding calcium, import and export of nucleus, nucleic acid binding, peptidase-signal protein binding, proteasome, protein translation, vesicle transport, neurotransmission, aqueous balance, ionic equilibrium, gene transcription, splicing, mitosis, meyosis, organization , stability or integrity of chromosome, micro RNAs, siRNAs, post-translational protein modifications, transport of electrodes, metabolism (anabolism or catabolism), apoptosis, membrane integrity, and cell adhesion. b. In one embodiment, the concatemer constructs according to the present invention target multiple genes from the same biological pathway, resulting in a specific and potent RNAi effect and more efficient control of pests. c. Alternatively, the concatamer constructs according to the present invention focus on multiple genes from different biological pathways, resulting in a broad cellular RNAi effect and more efficient control of pests. d. Alternatively, a combination of b) and e). Choice of target sequence (s) focused by the dsRNA fragments in the concatemer construct. Once a target gene has been selected (or once multiple target genes have been selected), one or more particular target sequences to be targeted through the dsRNA fragment of the concatemer construct are selected from this target gene (these target genes). In the concatemer constructs of the present invention, the selection of said target sequences was made based on one or more of the following selection criteria: 1. The target sequence targeted by a fragment of dsRNA in the concatemer construct does not have a substantial nucleotide sequence homology with non-target organisms. A preferred criterion is that the target sequence does not have substantial homology to human sequences and / or does not have substantial homology to sequences of host plants and organisms living in symbiosis with the plant (e.g., symbiotic bacteria with the plant). A non-limiting list of host plants in accordance with the present invention comprises, for example, corn, cotton, tomato, potato, banana, sugarcane, sunflower, alfalfa, wheat, rice, sorghum, millet, and soybean. 2. The target sequence targeted by a fragment of dsRNA in the concatemer construct is selected from within a region of the target gene that contains the best siRNA predicted, such prediction can be made for example in accordance with the "Tuschl rules" (Yuan et al. al. "siRNA Selection Server: an automated siRNA oligonucleotide prediction server", W130-134, Nucleic acid research, 2004, vol 32, Web Server issue). Basically, this criterion includes the determination of the percentage content of GC versus the percentage content of AT of the DNA. Preferably, the target sequences targeted by the dsRNA fragments of the concatamer constructs of the present invention have a GC content within a range of about 40% to about 60%, preferably have a GC content of about 50%. Alternative predictions for selecting siRNA sequences can be found at the following points: Saetrom snd Snove 2004 ("A comparison of siRNA efficacy predictors", Biochem. Biophys., Common Res. Vol 321 (1): 247-253); Chalk et al.2004 ("Improved and automated prediction of effective siRNA.", Biochem, Byophys, Res. Commun. 319 (1): 264-74); Levenkova et al. 2004 ("Gene specific siRNA selector.", Bioinformatics, 20 (3): 430-2); Reynolds et al. 2004 ("Rational siRNA design for RNA interference.", Nat. Biotechnol.22 (3): 326-30); Henschel et 1. 2004 ("DEQOR: a web based tool for the design and quality control of siRNAs.", Nucleic Acids Res. (Web Server issue): W113-20). 3. The target sequence targeted by the dsRNA fragment in the concatemer construct is in a conserved region (at the nucleotide acid level) of the target gene. Such conserved regions are determined by comparing the sequences of homologous genes from the same species and / or from different species. As such, multiple members of gene families can be downregulated in one species or in multiple species. 4. Alternatively, the target sequence targeted by the dsRNA fragment in the concatemer construct is a non-conserved region of the target gene (for the reasons explained hereinabove). Ways to combine multiple fragments of dsRNA in a concatemer construct: All the alternatives provided above for target gene selection and target sequence selection can easily be combined with each other. The corresponding dsRNA fragments (or regions) that target such target genes and target sequences can be combined in various ways in the concatemer construct. In the concatemer constructs of the invention, one or more of the following forms of combining dsRNA fragments were used (see also figure 1 and 20): 1. When multiple dsRNA fragments that target a single target gene are combined, they can be combined in the original order (that is, the order in which the fragments appear in the target gene) in the concatemer construct, 2. Alternatively, the original order of the fragments can be ignored in such a way that they are scrambled and combined randomly or deliberately in any order in the concatemer construct, 3. Alternatively, a single fragment can be repeated several times, for example from 1 to 10. times, for example 1,2,3,4,5,6,7,8,9 or 10 times in the concatemer construct, or 4. The fragments of dsRNA (which target a single target gene or different target genes) may combine in sense orientation or antisense. The possibility of combining fragments of dsRNA in the concatemer construct is especially helpful to avoid a coincidental non-target splice in the conjunction of the multiple dsRNA fragments in the concatemer construct. For example, when two fragments of dsRNA without homology with non-target organism in 20 consecutive nucleotides are combined, a new sequence that may have homology to a non-target organism in a range of 20 consecutive nucleotides may arise at the conjunction. In such a case, the design of concatemers according to that described here allows one of the dsRNA fragments to be converted in another orientation (for example, converting from sense to antisense) and / or allowing changing the order of the fragments (for example, converting from AB to BA in the concatemer construct) to overcome this problem. Furthermore, it is advantageous that in the nucleotide sequence of the final concatemer construct no splice acceptor sites or splice donors are created. It is also recommended that the nucleotide sequence of the final concatemer construct does not contain a large open reading frame (ORF). This possibility of combining fragments of dsRNA in the concatemer construct is also useful for cloning purposes since the separated fragments can be randomly linked to each other. The dsRNA constructs of the invention can be formed from a single RNA polynucleotide molecule that includes regions of self-complementarity, such that when it is bent it can form a structure that includes one or more double-stranded portions effective in silencing gene by RNAi. The constructs can also be formed from two or more separate strands of polynucleotides that together form a double-stranded, folded or assembled structure that includes at least a double-stranded portion effective to silence a gene by RNAi. The RNA constructs may include, when bent or assembled, both double-stranded regions and single-stranded regions, as illustrated in the accompanying Figures. The RNA constructs can include non-natural bases and / or bonds of non-natural structures. The dsRNA or the dsRNA constructs comprising multiple fragments of dsRNA can be known here generally as concatemers. The actual fragment that is double-stranded is also known as "portion". Said portion contains one or more fragments of dsRNA. The concatemer and / or stabilized constructs and methods of the present invention are particularly useful for combining multiple target sequences simultaneously. These multiple sequences can originate from a white gene. Alternatively, multiple white sequences can originate from multiple target genes. These multiple white genes can originate from the same harmful species. Alternatively, these multiple target genes may originate from different harmful species of the same order or from different orders. These multiple target genes may be related, for example, they may be homologous or orthologous, or they may not be related. Accordingly, a concatemer dsRNA construct of the present invention, for example, in the form of a concatemer stalk, a concatemer hair pin or a concatemer cloverleaf can simultaneously focus multiple sequences originating from the same species harmful, or may simultaneously target multiple target genes of the same pest species, or may simultaneously target multiple target genes from multiple pest spacings of the same order or from different orders. The present invention therefore encompasses a dsRNA or dsRNA construct comprising at least two fragments of dsRNA, wherein each fragment of dsRNA comprises a strand that is complementary to at least a part of the nucleotide sequence of a different target sequence (per different examples). In one embodiment, said different target sequences originate from a single (or same) target gene. In another embodiment, said different target sequences originate from different (for example, different) target genes. In accordance with a particular embodiment of the present invention, the concatemer targets multiple target genes that originate from multiple species. For example, a concatemer can focus multiple genes of multiple plant pest organisms, and by expression of the concatamer in the plant, the plant acquires resistance against multiple plant pests simultaneously. Similarly, a plant or a surface or substance susceptible to infestation by a pest may be sprayed with a composition (or the like) comprising the concatamers of dsRNA, thereby protecting the plant or the surface or substance against infestation by multiple pests. For example, the plant acquires resistance against nematodes and insects or against nematodes, insects and / or fungi. Likewise, the concatemer construct allows the plant to acquire resistance against multiple nematodes of a different genus, of a different family, of a different order or of a different kind and / or against insects of a different genus, a different family or a different order and / or against fungi of a different gender, of a different family or of a different order. In another particular embodiment of the present invention, the concatemer targets multiple target genes that originate from different species of the same order. For example, a concatemer that targets genes from different bacterial, viral, fungal, insect or nematode species can be used as a broad-spectrum killer of bacteria, viruses, fungi, insects or as a broad-spectrum killer of nematodes. The combination of dsRNA fragment that targets multiple target sequences of different pest species in a concatamer construct according to the present invention is favorable to extend the pest species spectrum of the RNAi effect of the dsRNA molecules. In another particular embodiment of the present invention, the concatemer targets multiple target genes that originate from the same organism, for example, from the same pest species. Such a construct offers the advantage that several weak white genes from the same organism can be silenced together to efficiently control the pest organism, while the silencing of one or several of the weak white genes separately is not effective in controlling the pest. Likewise, several strong white genes from the same organism can be silenced simultaneously in order to further improve the effectiveness of the pest control or to prevent the occurrence of resistance of the harmful organism by mutation. The present invention therefore encompasses an isolated dsRNA or dsRNA construct isolated in accordance with that described above., wherein said different target genes originate from a single target species (or pest), or where said different target genes originate from different target species (or pests); for example, species of plague that belong to the same genera, families, orders or even rows (phyla) (in one modality) or to different genera, families, orders or even rows (phyla) (in other modalities). The dsRNA constructs described here and which target multiple target genes are characterized by the accumulation of multiple RNAi capacity, resulting in synergistic effects and the ability to activate multiple RNAi effects in the target cell or in the target organism. Figure 3 shows the different types of dsRNA core of the present invention, which are part of the concatamer and / or dsRNA construct stabilized according to that described herein. In the core of type A dsRNA, the single repeat gene fragment may be complementary to a target gene sequence or a non-target gene sequence. In the nucleus of type B dsRNA, multiple gene fragments may be present in sense orientation or antisense orientation and may originate from a single target gene or from different target genes, eg from the same species or of different species. The nucleus of type B dsRNA therefore represents a basic concatamer in stem format. In the core of type C dsRNA, the sense or antisense chain comprises, for example, 5 to 7 mutations in each fragment of -21 base pairs. These mutations can be, for example, mutations of C to T. The antisense chain or sense chain does not comprise mutations and is 100% complementary to the target gene mRNA. This type of construct will provide protection against the transcriptional gene silencing of the transgene. In this type of construct, fragments of single or multiple genes may be included. Stabilized Constructs In accordance with another embodiment of the present invention, there is provided a substantially pure ribonucleic acid (RNA) construct capable of forming a double stranded RNA (dsRNA) portion in RNAi gene silencing, said RNA construct comprising at least one sequence capable of protecting the dsRNA (portion) against RNA processing. More specifically, the invention relates to an isolated RNA construct comprising at least one fragment of dsRNA, wherein dsRNA comprises fused complementary strands, one of which is complementary to at least a part of the nucleotide sequence of a target sequence. , said RNA construct further comprises at least one sequence that protects the dsRNA against RNA processing. Also encompassed are RNA constructs comprising any of the dsRNA molecules (concatemers) described above, said RNA construct further comprising at least one sequence that protects the dsRNA (or portion of dsRNA) against RNA processing. The "protection against RNA processing" is preventing or hindering or inhibiting the processing of RNA. In accordance with one embodiment of the present invention, the constructs are protected in the host cell, particularly in a plant cell and / or in a plant pest species. When a stabilized or protected construct is described, the term nucleus refers to the portion of dsRNA, said nucleus may comprise at least one fragment of dsRNA or may comprise multiple fragments of dsRNA, eg, a concatemer, in accordance with that described with details above. The present invention also relates to isolated RNA constructs wherein said at least one sequence (capable of) protecting the dsRNA against dsRNA processing is selected from a GC-rich coupling, a short non-complementary loop between 4 and 100 nucleotides (for example 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 nucleotides) a blockage of mismatch and a structure of binding RNA a protein In one embodiment of the invention, a sequence capable of protecting the portion of dsRNA against RNA processing is also known as a "blockage". Examples of blocks according to the present invention are given below: 1. A "GC-rich" coupling (see Figure 2a) is a segment of nucleotides with multiple G residues (contiguous) that are coupled with a complementary strand comprising multiple C residues (contiguous). The base pair composition of the GC-rich coupling can vary and the length of the GC-rich coupling can vary from about 5 base pairs to about 100 base pairs. 2. A "non-complementary loop" (see Figure 2B) that can protect RNA against RNA processing has a length, for example, between about 3 nt and about 100 nt, preferably less than 9 nt and more preferably about 4 nt or about 5 nt. The sequence can be selected randomly or it can be homologous to specific sequences such as for example miRNAs (conserved). 3. A "mismatch block" (see Figure 2C) is a dsRNA where some nucleotides are not forming base pairs. In a blockade of mismatch there is a fair enough number of correspondences included in the dsRNA to allow an appropriate dsRNA pairing (preferably approximately 67% to 74% of the bases are coupled). Correspondence errors consist mainly of insertions and deletions in one chain in relation to the other. Viroids (eg, Pospiviroidae, Avsunviroidae, Hepadnavirus family, human hepatitis delta virus, potato spindle tuber viroid, avocado sunspot viroid or citridcos exocortex viroid) serve as excellent examples in nature for design lack of correspondence blockages that encourage the processing of dsRNA in host species. An example of a blockage of mismatch is a block comprising a sequence in accordance with that described in Chang et al. (J. Virol. 2003 Nov; 77 (22): 11910-7), said document is incorporated herein by reference. These sequences are derived from potato spindle tuber viroid RNAs (PSTVd), avocado sunspot viroid (ASBVd) or human hepatitis delta virus (HDV), have predicted base and intramolecular pairing of 70 %, 67% and 74%, respectively, and are resistant to the activity of dicer. These sequences are shown in Figures 4 of Chang et al. And they can be used as blocks in the constructs of the present invention, each separately or in combination with each other. Accordingly, the present invention also encompasses dsRNA constructs suitable for silencing RNA, said constructs comprising as a sequence capable of protecting the dsRNA against RNA processing, the HDV sequence mentioned above, PSTVd sequence, ASBVd sequence or HDV combinations. - PSTVd- ASBVd or HDV- ASBVd- PSTVd. Examples of such a blockage of unique correspondence are provided in Figure 2C, as well as an example of a composite mismatch block. Another example of a mismatch blocking is dsRNA complementary to a target sequence of a target species, comprising approximately 70% intramolecular base pairing. For example, the antisense strand does not comprise mutations and is 100% complementary to the target sequence while the sense strand comprises approximately 30% of mutations causing mismatches in the dsRNA. 4. Another type of blockage is RNA structures of protein binding. These are RNA sequences recognized and linked by proteins, preferably by proteins endogenous to the host cell wherein the dsRNA construct according to the present invention is expressed.

When these blocks are occupied by the binding protein, they protect the portion of dsRNA against RNA processing. Examples of such "protein binding RNA blocks" are IRES; 5 'regions of the virus genome; I WILL GO; binding domain of plant dsRNA (e.g., domain type Hyl-1); endogenous dsRNA binding proteins (or domain) (eg, transcription factors, translation factors, ribosome components, SRP, PTB domains, etc.) provided that they are transgenically expressed in a way that does not interfere with the protein function of wild type; and others . An "IRES" is an internal ribosome entry site. A general representation of dsRNA constructs comprising IRES is provided in Figure 2E. Sequences represented by SEQ ID Nos: 1 to 7 represent IRES sequence of CrPV-type viruses. HTH sequences of the grasshopper-type parasite virus IRES (CRPV type) are a suitable example of an IRES. The attached nucleotides are derived from the following viral genbank nucleotide sequences: PSIV: AB006531, nt 6005-6204; HiPV: AB017037, nt 6286-6484; DVC: AF014388, nt 6078-6278; RhPV: AF022937, nt 6935-7121; TrV: AF178440, nt 5925-6123; CrPV: AF218039, nt 6029-6228; BQCV: AF183905, nt 5647-5848 (Kanamori and Nakashima, RNA 2001 7 (2): 266-74). The identification header is collected as follows: < genbank access numbers > _ < start position > _ < Suspension position > _ < species name > . Another suitable IRES sequence can be found by a person with knowledge in the field. Preferably, sequence IRES are recognizable by ribosomes of different organisms, preferably are recognizable by ribosomes of a plant or a plant pest species. Examples of plant IRES sequence are IRES sequence of Arabidoposis thaliana, Cuscuta japonica, Fuñaria hydrometrica, Nicotiana tabacum, Oryza sativa, Triticum aestivum or Zea mays in accordance with that described in document O03 / 020928, said document including the sequences of IRES is incorporated herein by reference as if it were fully reproduced. The IRES sequences are incorporated into the constructs of the present invention in case of constructs in accordance with that represented by SEQ ID Nos: 18 to 21. Another example of a 5 'region of a virus or a fragment thereof, useful, a Blocking in the constructs of the present invention is described and illustrated in Miller et al. (1998. J. Mol. Biol. 284 (3): 591-608). Other examples of IRES sequence that are encompassed by the present invention are described and illustrated, for example, in Spahn et al. (2004. Cell 20 118 (4): 465-475). In addition, 3 'regions of viruses or fragments thereof can also be used as a blockade.

An "IRE" is an Iron Regulatory ent. An IRE suitable as a closure in the constructs of the present invention is the IRE ent derived from the homologous derived from the soybean NRAMP homologue GmDMTl in accordance with that described in Kaiser et al. (Plant J. 2003, 35 (3), 295-304). This document is incorporated herein by reference and the sequence of the IRE is represented by SEQ ID NO: 8. Other examples of protein binding RNA blockages are RNA sequence recognized by RNA binding proteins according to what is described for example in Lorkovic and Barta (Nucleic Acids Res. 2002 Feb 1; 30 (3): 623-35). RNA binding proteins from the flowering Arabidopsis thaliana plant that have an RNA recognition motif (RRM) or a K homology domain (KH) are described. The corresponding RNA sequences recognized by their proteins can be cloned by techniques well known to a person skilled in the art, for example, through the Un-Hybrid technique. Figure 4 shows a preferred construct according to the present invention. According to a specific embodiment, the present invention relates to an RNA construct isolated in accordance with that described above, comprising at least one protective sequence sted from the internal ribosome entry sites (IRESes) of the encephalomyocarditis virus ( EMCV), and the upward current of liras (UNR). In one embodiment, a sequence comprising at least a portion of the sequences CV.IRES is presented in SEQ ID NO: 13. Constructs comprising at least part of the sequence of EMCV-IRES are represented by SEQ ID Nos: 18 and 19. In another embodiment, a sequence comprising at least a portion of the sequence of UNR-IRES is presented in SEQ ID NO: 14. Constructs comprising at least a portion of the sequence UNR-IRES are represented by SEQ ID Nos: 20 and 21. The IRES sequence of the EMCV viral genome is represented in Genbank accession number NC_001479; the IRES sequence of the human UNR genome is represented in Genbank accession number NM_001007553. Accordingly, the invention relates to the use of the complete IRES sequence or a functional fragment thereof in the RNA construct and comprising fragments of dsRNA in accordance with that described above. Inventions of the scope of the present invention include that any of the above-mentioned cores can be combined with any other to form a composite block. Specific examples of such compositions are the closed GC coupling or a closed correspondence blockage in accordance with that depicted in the figures.

The length of a block can vary from about 3 base pairs to about 10,000 base pairs, in the step of double chain locks or from 3 nt to about 10,000 nt in the case of single chain blocks. The blocks may have an additional advantage in the sense of causing a steric hindrance to the RNA processing machinery of the host cell. The location of the blocks in the constructs of the present invention may be a terminal position at the end of the dsrNA or it could be found integrated somewhere (inside) in the dsRNA. Accordingly, the position of the number of the blocks may vary. Preferably, 2 or 4 blocks are present at the end (the border) of the dsRNA portion, in the case of a payo (or concatemer) RNA nucleus. Preferably, a block or a combination of blocks is present as a fourth stem in the case of a multi-stalk "trefoil leaf" dsRNA core type (see, for example, Figure 5, constructs 1 and 2). Another mechanism to protect the dsRNA against RNA processing is to integrate the effective dsRNA fragment into the silencing of the gene in a larger RNA structure that occurs naturally and that is normally not processed or that has reduced processing in its natural environment. Examples of such natural unprocessed RNAs are miRNA, Trna, RNA ribosome, components of the spliceosome or other non-coding RNASs transcribed from RNA polymerase promoters I, II, or III. Accordingly, within the framework of the present invention are natural unprocessed RNAs comprising a dsRNA fragment complementary to a target sequence, eg, a target plant pest sequence, and which can silence the explosion of a target gene. Advantageously, these constructs can provide a camouflage for the fragment of dsRNA capable of silencing genes and which contributes to the stability of this fragment of dsRNA in a host cell. This approach can be combined with any type of dsRNA blocking exemplified herein and / or with any other sequence capable of protecting the dsRNA against RNA processing as presented herein as an example and / or with any linker in accordance with the example presented herein.

Another mechanism to protect the dsRNA counting RNA processing according to the present invention is what is known as a "Triple RNA" construct. Triple RNA comprises 3 parallel strands of RNA which are encoded by two separate strands of RNA where: The first strand of RNA comprises from 5 'to 3': (a) a strand of sense RNA core corresponding to a target sequence (nucleus), followed by (b) a second region of sense RNA (B) followed by (c) a long non-complementary loop, said loop a. is greater than the length of the RNA of the nucleus, the region of RNA (B) of the nucleus and the region of RNA (A) together. b. optionally comprises a block in accordance with what is described above, such as IRES, c. is followed by (b) a third sense RNA region (A) and wherein the second RNA strand comprises from 5 'to 3' (a) a region (A) of antisense RNA complementary to the region (A) of sense RNA, (b) A core chain of antisense RNA corresponding to the target sequence and complementary to the sense core RNA, (c) A region (B) of antisense RNA complementary to the region (B) of sense RNA. Another mechanism to protect dsRNA against RNA processing is to integrate the dsRNA nucleus into a viroid type dsRNA structure in accordance with what is described and illustrated for example in Navarro and Flores (2000 EMBO Journal 19 (11) p 2662. The dsRNA can be incorporated within the viroid as such or in the mutated viroid to avoid internal dissociation (for example, by ribozymes) or to avoid translation Mutations may be based on information from Dais et al. (1991, NAR 19 (8)) p 1893) These types of constructs can be transported to chloroplasts where they can receive additional protection against dsRNA processing.Another mechanism to protect dsRNA against processing is to signal the dsRNA to an intracellular compartment of the host cell.For example, the dsRNA may be compartmentalized in an intermediate host cell before being transferred to the target host cell.In particular, the dsRNA construct could be compartmentalized in a plant cell, for example, can be located in the chloroplast, mitochondria or plastid, before being transferred to the pest species of the plant, for example, the nematode or insect detrimental to the plant. Compartmentalization can occur in several ways, for example through the use of viroid structures or through the use of signal sequences, for example chloroplast, mitochondrial or plastid signal sequences. These organelles are prokaryotic in origin and can offer a protective environment away from the plant's RNA processing machinery. Another mechanism to protect the dsRNA against RNA processing is to express the sense and antisense separately and focus them towards different locations within the host that express sense chains and antisense chains. In this embodiment, the sense and antisense mRNA fragments corresponding to a gene selected from a particular pest species are cloned back from different promoters that drive the expression (i) of separate plant tissues (ii) within the same cell but in separate cell compartments. These promoters are specitissues or organelles and allow strong simultaneous expression in different cell compartments or in adjacent tissues. For example, sense and antisense chains can be targeted to different plant tissues, to different cell types, or to different subcellular organelles or different subcellular locations. For example, in a sheet, the sense chain may be expressed in the rib cells while the antisense chain is expressed in the palisaded tissue. The advantage of this technique is that the sense and antisense chains are never together in the plant cell, and therefore no degradation or self silencing or interference of RNA within the plant by Dicer can occur. When harmful organisms feed on the plant, the chains are released and mixed allowing fusion of dsRNA in the intestinal lumen and base pairing between sense and antisense chains can occur to form long dsRNA. Subsequently, this dsRNA can be efficiently absorbed which causes the desired RNAi response, causing the degradation of the white mRNA in the pest and the death of said pest. This approach can be accompanied by the feeding of the harmful species with two bacterial mushrooms, for example present in one composition, one strain that produces the sense chain and the other strain that produces the antisense chain. According to another embodiment, the present invention encompasses any of the dsRNA molecules or RNA constructs described herein capable of forming a portion of dsRNA in gene silencing, which further comprises at least one linker, for example said linker is selected from a conditionally self-isolating RNA sequence, such as a pH responsive linker or a hydrophobic sensitive linker, and an intron. In the presence of a blockage in accordance with what is described herein, the function of the linker can be to release the blockage before silencing the gene, causing RNA processing of the dsRNA construct by the intermediate host cell or by the host white cell. In the absence of a blockage, for example within the concatemer construct itself, the function of the linker may be to decouple the multiple fragments of dsRNA and divide the long dsRNA into pieces effective in gene silencing. Advantageously, in this situation, the linker sequence can promote the division of the long dsRNA into pieces under particular circumstances, resulting in the release of separated dsRNA fragments under these circumstances and causing a more efficient gene silencing by these smaller dsRNA fragments. Different types of linkers for dsRNA constructs are provided by the present invention. The "conditionally self-isolating linkers" are RNA sequences capable of being processed under certain conditions. 1. An example of suitable conditionally self-isolating linkers is an RNA sequence that is self-dissociating under low pH conditions. Suitable examples of such RNA sequences are described by Jayasena and Gold (Proc Nati Acad Sci U S A. 1997 Sep 30; 94 (20): 10612-7), said document is incorporated herein by reference. They are synthetic sequences obtained through the cloning of randomized sequences and retrieved through a SELEX protocol (systematic evolution of ligands by exponential enrichment, Gold et al., 1995. Ann. Rev. Biochem. 64: 763-797). 2. Other examples of suitable conditionally self-isolating linkers are RNA sequences that are self-dissociating under high pH conditions. Suitable examples of such RNA sequences are described by Borda et al. (Nucleic Acids Res. 2003 May 15; 31 (10): 2595-600), said document is incorporated herein by reference. A suitable linker sequence originates from the catalytic core of the hammerhead ribozyme HH16. According to a particular embodiment of the present invention, the aforementioned pH-dependent self-isolating linkers are used in constructs designed to be produced in harmful organism control plants. Here, the linkers can be used to disconnect the blocks of a stabilized construct or to disconnect the multiple fragments of dsRNA from a concatemer construct in the harmful organism. According to a particular embodiment, the detrimental species has an intestinal system, such as, for example, nematodes and insects, and the linker is self-dissociating in the intestine of such harmful species, for example, a species detrimental to a plant. The pH in the intestine is variable within a range of extremely acid to extremely basic. Harmful species of particular insects of interest for the application of this technique are stem borers or, for example, the tobacco budworm. 3. Alternatively, the linkers are self-dissociating in the endosomes. This can be beneficial when the constructs of the present invention are absorbed by the harmful organisms through endocytosis or trancytosis, and are therefore compartmentalized in the endosomes of the deleterious species. Endosomes can have a low pH environment causing dissociation of the linker. 4. Other examples of suitable conditionally self-isolating linkers are self-drying RNA sequences under hydrophobic conditions. Suitable examples are RNA sequences described by Riepe et al. (FEBS Lett, 1999 Aug 27; 457 (2): 193-9), said document is incorporated herein by reference. A highly specific auto-dissociation reaction occurs in the hydrophobic interior of a micelle. These RNA sequences are derived from ribozymes of hammerhead and hairpin. The linkers mentioned above which are self-dissociating under hydrophobic conditions are especially useful in dsRNA constructs of the present invention when they are used to be transferred from one cell to another through the transit in a cell wall, for example when passing through the cell wall of the cell. an organism harmful to a plant. Organisms harmful to particular plants of interest for the application of this technique are fungi that parasitize plants or viruses or bacteria that parasitize plants. An intron can also be used as a linker. An "intron" as used herein can also be used as a linker. An "intron" as used herein may be any non-coding RNA sequence of a messenger RNA. Particular sequences of introns suitable for the constructs of the present invention (1) are rich in U (35-45%); (2) have an average length of 100 base pairs (varying between about 50 base pairs and about 500 base pairs), said base pairs may be randomly selected or may be based on known introns sequences; (3) start at the 5 'end with -AG: GT- or CG: GT and / or (4) have at their 3' -AG end: GC or -AG: AA. In accordance with the present invention, a linker sequence may be present between the dsRNA fragment or not. For example, when present, the linker may comprise a short sequence of random nucleotides that is not complementary to target sequences but that is the result of cloning. In other embodiments, for example, when the dsRNA comprising the dsRNA fragments is chemically synthesized, the dsRNA fragments may be directly adjacent to each other without the presence of non-target sequences. A non-complementary RNA sequence per se, within a range of about 1 base pair to about 10000 base pairs, for example, of at least 10, 20, 30, 50, 60, 70, 80, 90, 100, 120, 500, 1000, 1500, 2000, 3000, 10000 base pairs, or any intermediate number, can also be used as a linker. The linker can be located at the edge of the dsRNA construct. Alternatively, the linker can be located between the different fragments of dsRNA integrated into the dsRNA. In addition, as shown in Figure 6, multiple linkers and multiple locks may be located at the edge of the dsRNA construct or within said construct. According to a particular embodiment, the linker is located adjacent to or close to a blocking sequence, more preferably a linker is located adjacent or close to each blocking sequence. A feature of the concatemer and / or stabilized constructs of the present invention is that with a stabilized concatemer and / or construct, multiple types of dsRNA nuclei can be combined and / or multiple types of linkers can be combined. For example, in a trefoil leaf structure any one or several of the 4 stems of dsRNA may comprise a GC coupling or a mismatch block and in addition any one or more of the 4 dsRNA may comprise a non-complementary loop capable of protecting the RNA construct against RNA processing. This also applies to the silent bell structure according to the present invention wherein at least one stem edge of dsRNA comprises a non-complementary loop that is capable of protecting the RNA construct against the processing of RNMA (see Figure 7). SEQ ID Nos: 9 to 12 represent different DNA sequences used in the examples described herein. These sequences represent a silent bell construct with sense and antisense fragments against beta-tubulin target genes that originate in M. incognita, C. elegants, grasshoppers and Magnaporthe grisea. These constructs also comprise a pH responsive linker (underlined) and a short loop (in frame). The silent bell RNA construct of the present invention may also comprise, on at least one of the edges of the stem of dsRNA, a GC coupling or a mismatch block. Additional examples of dsRNA constructs comprising linkers and protein binding RNA sequences are shown in Figure 8. According to another embodiment, an interstage base pairing module may be included within the construct of the present invention. These interstage base pairing modules may be included within the construct of the present invention. These interstage base pairing modules contribute to the stability of the dsRNA in the host cell and allow complex dsRNA constructs to fold compactly.

According to another embodiment, within the constructs of the present invention, a portion capable of delivering the dsRNA to the deleterious species may be included. Such constructs are described in the applicant's patent application which is incorporated herein in its entirety. In one embodiment, the dsRNA construct described herein further comprises at least one aptamer. The term "aptamer" or "aptamer sequence" or "aptamer domain" are used herein as synonyms and are well-known terms of a person skilled in the art. These terms refer to synthetic nucleic acid ligands capable of specifically binding to a wide variety of target molecules such as for example proteins or metabolites. As used herein, aptamers are oligonucleotide sequences with the ability to recognize virtually any class of target molecules with high affinity and specificity. In a preferred embodiment, the aptamer binds specifically to a structure in a plant tissue or to a structure in the pest species. According to one embodiment, the invention offers dsRNA constructs comprising aptamers that target dsRNA with a high binding site affinity in the deleterious species. They can be located in the intestinal epithelial cells of pests when they are eating, in other cells in the body of the pest when eating or even on surfaces of cells that interact or for example fungi that feed on plant tissue. In certain embodiments of the present invention, the dsRNA construct can therefore comprise an aptamer that allows endocytosis in the intestinal cell of a harmful organism, for example an enterosite. In another example, the aptamer allows (or promotes or enables) transitosis from the lumen of the intestine into the coelum fluid or hemolymph of the harmful organism. In other embodiments of the present invention the dsRNA construct may comprise an aptamer that allows endocytosis in a tissue cell of the detrimental organism, such as, but not limited to, a muscle cell, a gonad cell, a nerve cell. In another example, an aptamer allows (or promotes or enables) transitosis from an endothelial cell that lines an organ to the lumen of said organ of the deleterious organism. In other embodiments of the present invention the dsRNA construct comprises at least two aptamers, for example an aptamer that allows (or promotes or enables) transitosis from the intestinal cell of a harmful organism to the coelum fluid or the haemolymph of the harmful organism, and another aptamer that allows (or promotes or enables) endocytosis in a tissue cell of the harmful organism. Alternatively, the dsRNA can be co-expressed with an RNA delivery molecule consisting of different modules. Said delivery molecule may consist, for example, of a polypeptide sequence comprising (i) at least one RNA binding domain, (ii) at least one approach polypeptide capable of binding to an endocytosis receptor molecule and / or cellular transitosis and (iii) optionally at least one peptide linker and / or at least one purification tag. Said delivery promoter molecule is used to facilitate the adsorption and correct delivery of double-stranded RNA to a suitable target site in a deleterious organism that feeds on the plant for the purpose of RNA interference. The terms "RNA delivery module", "RNA delivery molecule" and "RNA delivery vehicle" are used here synonymously and refer to the multidomain or multimodular protein that binds to the silencing molecule mediated by dsRNA. In one embodiment of the present invention, the RNA delivery molecule consisting of different modules comprises at least one RNA binding module, at least one focusing module that can be endosynced and / or transitored or that can be attached to a molecule of endocytosis receptor and / or cellular transitosis, optionally at least one linker for linking the dsRNA binding molecule with the focusing molecule, and optionally a module comprising a purification tag.

One module of the RNA delivery molecule is an RNA binding domain. An "RNA binding domain", as used herein, can bind generic double-stranded RNA or specifically, generic or single-stranded RNA specifically. The RNA binding molecule can bind dsRNA and / or ssRNA in a structure-specific manner. Preferred examples of RNA binding proteins include, but are not limited to, protein HK022 NUN of colífago, protein Lie T of Bacillus subtilis, or coat protein MS2 of bacteriophage or essential parts or homologs thereof. A second module of the RNA delivery molecule comprises a focusing module. The terms "focus module" and "focus protein" are used here as synonyms and both refer to a protein, or an essential part or a homologue thereof that can focus the RNA delivery molecule towards a focus site in a living harmful organism. The focusing module preferably comprises a protein that can be endocytocidal and / or transitored in a cell of the harmful organism, or a protein capable of binding with an endocytosis receptor molecule and / or transitosis present in a cell or tissue of the harmful organism , or any combination of the above.

Stalk-loop-stem structures An example of a dsRNA or an RNA capable of forming a dsRNA is a hairpin construct. A hairpin structure or "stem-loop-stem" structure is a nucleic acid molecule, preferably an RNA nucleic acid, comprising in the order of 5 'to 3', a first strand, a loop, and a second chain, wherein said first chain and said second chain hybridize to each other under physiological conditions and said loop connects said first chain with said second chain to form at least one region of double-stranded RNA. When different stem-loop-stem structures are present in a dsRNA molecule, the connection between the stem-loop-stem structures can be of several forms. For example, they may be chemically crosslinked to form an RNA complex. Alternatively, the multiple stem-loop-stem structures are genetically linked together with a grease nipple as mentioned above. In a preferred embodiment, from 2 to 20 stem-loop-stem structures may be linked together in a "sphere" structure. In a more preferred embodiment, 4 stem-loop-stem structures are linked together in a trefoil leaf structure, where the 5 'and 3' border of the RNA construct forms a fourth portion of the stem of dsRNA. In another embodiment, the trefoil leaf structures of the present invention may comprise at least one GC coupling or a mismatch block or other type of blockage in accordance with what is described herein. The concatemer and / or stabilized constructs according to the present invention are especially useful for the control of pests affecting plants, more particularly in pests that affect plants selective for the absorption of dsRNA. For example, nematodes are selective in terms of the length of the dsRNA to be absorbed. It has been shown that fragments of 100 base pairs are not absorbed as efficiently as fragments of 200 to 500 base pairs. Likewise fungi and insects can be selective in the absorption of dsRNA. Taking into account the selective uptake of dsRNA by some deleterious organisms, the entire length of the dsRNA constructs described herein, when bent or assembled, is generally within a range of 17 to 20,000 base pairs, preferably between 21 and 1000 Base pairs. More preferably, the length is at least 17 base pairs, 18 base pairs, 19 base pairs, 20 base pairs, 21 base pairs, 50 base pairs, 80 base pairs, 100 base pairs, 150 base pairs, 200 base pairs, 250 base pairs, 300 base pairs, 350 base pairs, 400 base pairs, 450 base pairs, 500 base pairs, 550 base pairs, 600 base pairs, 650 base pairs, or 700 base pairs, 900 base pairs, bases, 1000 base pairs, 1100 base pairs, 1200 base pairs, 1300 base pairs, 1400 base pairs or 1500 base pairs. Preferably, the length is approximately 50 base pairs, 80 base pairs, 100 base pairs, 150 base pairs, 200 base pairs, 250 base pairs, 300 base pairs, 350 base pairs, 400 pairs of bases, 450 base pairs, or 500 base pairs. Even more preferably, the total length of any of the stabilized concatamers and / or dsRNA constructs described herein is 150 base pairs, 250 base pairs, or 350 base pairs. The present invention therefore relates to any of the isolated O RNA dsRNA constructs described herein wherein the dsRNA portion has a length between about 17 and 2000 base pairs, preferably between about 50 and 1000 base pairs, with greater preference between approximately 80 and 500 base pairs. White Species and Pests White "species" as used in the present invention can refer to any species. Suitable white species are selected from the group comprising virions, viruses, bacteria, yeasts, fungi, insects, protozoa, metazoans (including nematodes), algae, plants, animals (including mammals, including humans). The most suitable white species for the methods of the present invention are the white species that are harmful organisms, more particularly pests that affect the plants, such as nematodes, insects, fungi, bacteria and viruses. According to a specific embodiment, the invention relates to any of the isolated dsRNA or RNA constructs described, wherein the target sequence or the target gene is from an organism detrimental to plants (ie, target species). The term "nematode" as used herein encompasses species of the order Nematoda. Many species of nematodes are parasitic and cause health problems to humans and animals (eg species of the orders Ascaradida, Oxyurida, Strongylida, Stronglyloides and Trichocephalida), as well as to plants and fungi (eg species of Aphelenchida orders , Tylenchida and others). Preferably, the term "nematode" as used herein refers to nematodes parasitizing plants and nematodes that live in the soil. Nematodes parasitizing plants include, but are not limited to, ectoparasites such as, for example, Xiphinema spp., Longdorus spp., And Trichodorus spp., Semiparasites such as Tylenchulus spp., Migratory endoparasites such as Pratylenchus spp., Radopholus spp., and Scutellonerna. Ssp; sedentary parasites such as Heterodera spp., Globodera spp., and Meloidogyne spp., and endoparasites of stem and leaf such as Ditylenchus spp., Aphelenchoides spp., and Hirschmaniella spp. Preferably, the term "nematode" as used herein refers to soil nematodes that parasitize the roots such as, for example, nematode-forming cyst of the genera Heterodera and Globodera and root-knot nematodes of the Meloidogyne genus. The RNA constructs of the present invention are essentially suitable for controlling harmful species such as Meloidogyne incognita, Heterodera glycines (soy cyst nematode) and Globodera rostochiensis (potato cyst nematode). However, the use of the dsRNA constructs according to the present invention is not restricted in any way to these genera and species but extends in the same way to other nematodes. The term "mushrooms" as used herein includes all species of the Fungi order. According to a preferred embodiment of the present invention, the target gene originates from a fungus that parasitizes plants such as Magnaporthe oryzae (rice blasto, formerly Maggnaporthe grisae, anamorph Pyricularia oryzae Cav. And Pyricularia grisae); Rhizoctonia ssp., Especially Rhizoctonia solani and Rhizoctonia oryzae; Gibberella fujikuroi; Sclerotinium spp .;Helminthosporium sigmoideum; Pythium spp .; Alternate spp., Especially Alternaría solaní; Fusaríum spp., Especially Fusaríum solaní and Fusaríum germínearum; Acremoníella spp .; Leptosphaeria salvíníí; Puccinia spp., Particularly Puccinia recóndita and Puccinia striiformis; Septoria nodorum; Pyrenophora teres; Rhincosporium secalis; Erysiphe spp., Particularly Erysiphe graminis; Cladosporium spp., Pyrenophora spp., Tilletia spp., Phytophthora spp., Particularly Phytophthora infestans; Plasmopara viticola; Uncinula necator, Botrytis cinerea; Guiguardia bidwellii; C. viticulture; Venturia inaequalis; Erwinia armylovora; Podosphaera leucotricha; Venturia pirina; Phakospora sp (soybean rust), Ustilago maydis (black corn rust). The term "insect" as used herein includes all species of insects. According to a preferred embodiment of the invention, insects are insects that damage plants. Important insects that constitute pests for plants that must be controlled by the methods of the present invention comprise, among other insects of the coleoptera order, selected for example from the non-limiting list of Lissorhopterus oryzophílus, Echinocnemus squamos, Oulema oryzae, Diabrotica spp. (Diabrotica virgifera virgifera, Daibrotica undecimpunctata howardi, Diabrotica barberi), Chaetocnema pulicaria, Sitophilus zeamais, Anthonomus granáis, Epilachna varivestis, Cerotoma trifurcata, Leptinotarsa decemlineata. Alternatively, the insects that constitute pests for the plants that must be controlled by the methods of the present invention belong to the order of Homoptera. More particularly, insects of the homoptera order are selected from the non-limiting list of Nilaparvata lugens, Laodelphax striatellus, Sogatella furcifera, Nephotettix virescens, Rhopalosiphum maidis, Aphis spp. (Aphis gossypii, Aphis glyeines), Empoasea spp. (Empoasca fabae, Empoasca solana), Bemisia tabaci, Myzus persicae, Macrosiphum euphorbiae. Insects that constitute pests for plants that must be controlled by the methods of the present invention may also belong to the order of Leptidoptera, selected for example from the non-limiting list of Heliothis spp. , Helicoverpa spp. , Spodoptera spp. , Ostrinia spp. , Pectinophora spp, Agrotis spp. , Scirphophaga spp. , Cnaphalocrocis spp. , Sesamia spp, Chilo spp., Anticarsia spp., Pseudoplusia spp., Epinotia spp. , and Rachiplusia spp. , preferably Heliothis virescens, Helicoverpa zea, Helicoverpa armigera, Helicoverpa punctera, Ostrinia nubilafis, Spodoptera frugiperda, Agrotis ipsilon, Pectinophora gossypiella, Scirphophaga ineertulas, Cnaphalocrocis medinalis, Sesamia inferens, Chilopartellus, Anticarsia gemmatalis, Pseudoplusia includens, Epinotia aporema and Rachiplusia nu. The RNA constructs of the present invention are particularly suitable for controlling harmful species such as the brown rice grasshopper (Nilaparvata lugens), the rice stem borer [chilo suppressalis) and the Colorado potato beetle (Liptinotarsa delineata). The "bacteria" that damage the plants and that can be controlled with the constructs and methods of the present invention are for example Agrojbacterium ssp .; Arachnia ssp .; Clavibaeter ssp .; Corynebacterium ssp .; Erwinia ssp .; usobacterium ssp .; Hafnia ssp .; Pseudomonas ssp .; Spiroplasma ssp .; Streptomyces ssp .; Xanthomonas ssp .; Xylella ssp. and Xylophilus ssp. The "viruses" that damage the plants and can be controlled with the constructs and methods of the present invention are for example the African cassava mosaic virus; alfalfa mosaic virus; pattern virus of American plum lines; latent virus of the Andean potato; mottled virus of the Andean potato; chlorotic apple leaf spot virus; apple mosaic virus; apple stem router virus; mosaic virus of arabis virus of arracacha B, oca strain; Asparagus virus 2; viroid of the Australian vine; viroid sun spots of avocado; soft mosaic virus of barley; mosaic virus from barley strips; yellow dwarf barley virus; yellow mosaic virus of barley; bean common mosaic virus; bean golden mosaic virus; bean leaf rolling virus; speckled bean pod; bean yellow mosaic virus; beard mosaic virus with beard; rolled beet virus; leaf beet wound virus; Beetle mosaic virus; beet necrotic yellow vein virus; pseudo yellow beetle virus; yellow beetle virus; bean yellow dwarf virus; mottled belladonna virus; latent blackberry virus; rust (and analogues); speckled leaf blueberry virus; bean wilt virus; bromovirus; swollen cocoa bud virus; cocoa yellow mosaic virus; X virus of the cactus; viroide of cadan-cadang; cryptic carnation virus; carnation-labeled ring virus; latent carnation virus; mottled carnation virus; carnation necrotic spot virus; Carnation ring spot virus; carnation vein mottled virus; common cassava mosaic virus; cauliflower mosaic virus; leafy virus of the cherry tree; rough cherry leaf virus (American); rugose cherry virus; Chrysanthemum B virus; viroid of chrysanthemum dwarfism; citrus viroid exocortis; Roughness virus of the daughter of citrus fruits; citrus mosoie virus; Citric sadness virus (European isolates); citrus tristeza virus (non-European isolates); citrus variegation virus; citrus rust; citrus viroid; yellow vein virus of clover; group of mild mosaic virus of dactylis glomerata; dactylis glomerata ray virus; soft mottled chickpea virus; cucumber mosaic virus; yellow cucumber virus; cucumovirus satellites, cymbidium mosaic virus; dahlia mosaic virus; dalanga mosaic virus; diantovirus; true bean mosaic virus; Elder carlavirus; euphorbia mosaic virus; tomato virus from florida; dormant Algerian vine virus; latent bulgarian virus of the vine; fan leaf virus of the vine; golden mycoplasma flavescent of the vine; virus associated with winding of vine leaf (I to V); grapevine tunisin ring spot virus; grapevine virus A; viroids with yellow spots on the vine (I and II); vine chromium mosaic virus; latent virus of heracleus; Hypertex mosaic virus; latent virus of the jungle mother; latent hop virus (American); latent hops virus; hop mosaic virus; viroids of hops dwarfism; hop virus A; Hoppy virus C; hydrangea ring spot virus; iliavirus; soft mosaic virus of the iris; porro yellow ray virus; leprosis; infectious yellow lettuce virus; lettuce mosaic virus; Lilac chlorotic leaf spot virus; speckled lilac ring virus; asymptomatic lily virus; luteovirus satellites; corn dwarf mosaic virus; corn strips virus; marafivirus; necrotic melon spot virus; latent ring spot virus of mirobolano; latent narcissus virus; narcissus mosaic virus; Narcissus tip necrosis virus; narcissus yellow stripe virus; oat golden strips virus; oat mosaic virus; odontoglossus ring spot virus; olive latent ring stain virus; Onion yellow dwarf virus; papaya mosaic virus; papaya ring spot virus; yellow spot virus of chibyria; early pea darkening virus; pea mosaic virus; mosaic virus carried in pea seed; peach mosaic virus (American); pear mycoplasma; pelargonio leaf bending virus; mild pepper virus; plant reovirus; pattern virus of plum lines (American); Pox virus of the plum; mosaic virus of good night flower; poplar mosaic virus; potato mosaic virus aucuba; potato ring black spot virus; potato leaf roll virus; potato leaf roll virus (non-European isolates); potato top virus; potato tuber viroid; potato virus A; potato A virus (non-European isolates); potato virus M; potato virus M (non-European isolates); S potato virus; potato virus S (non-European isolates); potato virus T; potato X virus; potato virus X (non-European isolates); potato virus Y; potato virus Y (non-European isolates); potato yellow dwarf virus; potato yellow mosaic virus; plum dwarf virus; plum necrotic ring spot virus; raspberry dwarf virus; folded raspberry leaf virus; raspberry leaf virus (American); raspberry ring spot virus; raspberry vein chlorosis virus; purple clover mottled virus; purple clover vein mosaic virus; lancéola mosaic virus; group of rice strand viruses; rubus yellow virus; v of sago cocoa; satellites (other than those mentioned); Satsuma dwarf virus; v latent step; sharka virus; Sovmovirus; chenopodium mosaic virus, yellow vein virus of the cerraja; latent spinach virus; bent pumpkin leaf virus; mycoplasma of stolbur; strawberry virus; latent C virus of the strawberry; latent ring stain virus of strawberry; mild yellow strawberry edge virus; strawberry vein virus; yellow beet virus; potato leaf virus; tobacco virus; tobacco mosaic virus; tobacco necrosis virus; tobacco virus; tobacco ring spot virus; tobacco stripe virus; Tobacco dwarfing virus; viroide of apic tomato dwarfism; Tomato virus; black tomato ring virus; thyroid tip of tomato; tomato dwarf virus; Tomato mosaic virus; viroid of male tomato plant; tomato ring spot virus; tomato spotted wilt virus; yellow leaf tomato virus; tuna mosaic virus; tulip virus; satellites of turnip virus; turnip virus; turnip mosaic virus; turnip yellow mosaic virus; thymoviruses; mottled tobacco virus; other viroids; mosaic virus of watermelon 2; wheat dwarf virus; wheat mosaic virus carried in soil; mosaic virus from wheat strips; yellow wheat mosaic virus; white clover mosaic virus; sweet potato mosaic virus; squash yellow spot virus; and yellow pumpkin mosaic virus; Recombinant DNA Constructs In accordance with a further aspect of the present invention, there is provided an isolated nucleic acid (deoxyribonucleic acid (DNA)) encoding any of the dsRNA or dsRNA constructs described herein. In addition, the present invention also provides recombinant DNA constructs, for example expression constructs, comprising said nucleic acid or said nucleic acids. Expression constructs, also encompassed by the term "recombinant DNA construct", facilitate the introduction into the cell of a plant and / or facilitate the expression and / or facilitate the maintenance of a nucleotide sequence encoding a dsRNA construct in accordance with with the present invention. Accordingly, a recombinant DNA construct (e.g., an expression construct) is provided, comprising a nucleic acid encoding a dsRNA or RNA construct in accordance with that described herein, operably linked to one or more control sequences capable of of driving the expression of the nucleic acid above and, optionally, a transcription termination sequence. Preferably, the control sequence is selected from the group comprising constitutive promoters or tissue-specific promoters in accordance with that described herein. Accordingly, the present invention also relates to a transgene encoding any of the double-stranded RNA or double-stranded RNA constructs described herein, placed under the control of a strong constitutive promoter, such as, for example, any of those selected within the scope of the invention. group consisting of CaMV35S promoter, doubled CaMV35S promoter, ubiquitin promoter, actin promoter, rubisco promoter, or G0S2 promoter, 34S promoter of figwort mosaic virus (FV). The expression constructs may be inserted into a plasmid or vector, which may be commercially available. In accordance with one embodiment of the present invention, the expression construct is a plant expression vector, suitable for transformation into plants and suitable for maintenance and expression of an RNA construct according to the present invention in a transformed plant cell. . The term "control sequence" as used herein should be taken in a broad context and refers to nucleic acid regulatory sequences that can boost and / or regulate the expression of the sequences to which they are linked and / or operably linked. The term mentioned above encompasses promoters and nucleic acids or synthetic fusion molecules or derivatives thereof which activate or increase the expression of a nucleic acid, which are known as activators or enhancers. The term "operably linked" as used herein refers to a functional link between the promoter sequence and the gene of interest such that the promoter sequence can initiate transcription of the dsRNA construct. In accordance with one embodiment of the present invention, the control sequence is operable in a plant; preferably the control sequence is derived from a plant sequence. The term "control sequence" encompasses a promoter or a sequence capable of activating or increasing the expression of a nucleic acid molecule in a cell, tissue or organ.

By way of example, a sequence of transgene nucleotides encoding the double-stranded RNA or the RNA construct can be placed under the control of a specific promoter for development or growth or inducible stage that allows the activation of dsRNA transcription by the addition of the inducer for an inducible promoter or when the particular stage of growth or development is reached. In addition, when using the methods of the present invention for the development of transgenic plants resistant to pests, it may be beneficial to place the nucleic acid encoding the double-stranded RNA according to the present invention under the control of a tissue-specific promoter. . In order to improve the transfer of the dsRNA from the plant cell to the pest, the plants could preferably express the dsRNA in a plant part accessed or bathed first by the plant pest. In case of a pathogenic plague for a plant, preferred tissues to express the dsRNA are the roots, leaves and stem. In case of pests that suck pathogenic plants, the dsRNA can be expressed in the phloem under the control of a promoter that directs the expressed dsRNA towards the phloem. Accordingly, the methods of the present invention, a preferred promoter for plant tissue can be used, such as for example a root-specific promoter, a leaf-specific promoter, or a stem-specific promoter. Suitable examples of a specific promoter for root are PsMTA (Fordam-Skelton, A.P., et al., 1997 Plant Molecular Biology 34: 659-668.) And the Class III promoter chitinase. Examples of specific promoters for photosynthetic or specific tissues for leaf and stem that are also photoactivated are promoters of two chlorophyll binding proteins (cabl and cab2) from beet (Stahl DJ, et al., 2004 BMC Biotechnology 2004 4:31 ), ribulose diphosphate carboxylase (Rubisco), encoded by rbcS (Nomura M. et al., 2000 Plant Mol. Biol. 44: 99-106), subunits A (gapA) and B (gapB) of glyceroldeido-3-phosphate dehydrogenase Chloroplast (Conley TR et al., 1994 Mol Cell, Biol. 19: 2525-33, Know HB et al., 1994 Plant Physiol. 105: 357-67), promoter of the Solanum tuberosum gene encoding the leaf-specific protein and stem (ST-LS1) (Zaidi MA et al., 2005 Transgenic Res. 14: 289-98), stem-regulated genes, defensible, such as for example JAS promoters (patent publication no. 2005003412 / US-A1), specific promoters for flowers such as for example chalcone synthase promoter (Faktor O. et al., 1996 Plant Mol. Biol. 32: 849), and specific promoters for fruit such as for example strawberry RJ39 (WO 98 31812). In addition, the present invention relates to a recombinant DNA construct wherein said regulatory sequence is selected from the group comprising tissue-specific promoters such as, for example, any promoter selected from the group consisting of promoters specific for roots of genes encoding PsMTA. Class III chitinase, specific promoters for photosynthetic tissues such as, for example, the promoters of cabl and cab2 proteins, rbcS, gapA, gapB and ST-LS1, JAS promoters, chalcone synthase promoter and strawberry RJ39 promoter. In other embodiments of the present invention, other promoters useful for the expression of dsRNA are used which include, without being limited to these examples, promoters for an RNA Poli, an RNA PoIII, an RNA PoIIII, T7 RNA polymerase or SP6 RNA polymerase. According to a specific embodiment, the nucleic acid is cloned between two regulatory sequences that are in the opposite direction relative to each other, said regulatory sequences are operably linked to said nucleic acid and auxiliary regulatory sequences independently selected within the group consisting of Poly RNA. , an RNA PoIII, an RNA PoIIII T7 RNA polymerase or SP6 RNA polymerase. These promoters are typically used for the in vitro production of dsRNA, said dsRNA is then included in an antipesticide agent, such as, for example, in a liquid, dew or antipesticide powder. Accordingly, the present invention also encompasses a method for generating any of the double stranded RNA or RNA construct of the invention. This method comprises the steps of: a. contacting an isolated nucleic acid in a recombinant DNA construct of the invention with cell-free components; or b. introducing (for example, by transformation, transfection or injection) an isolated nucleic acid or a recombinant DNA construct of the invention into a cell, under conditions that allow the transcription of said nucleic acid or recombinant DNA construct to produce the dsRNA or construct of RNA. Accordingly, the present invention also encompasses a cell, e.g., a host cell, comprising any of the dsRNA molecules, RNA constructs, nucleotide sequence or recombinant DNA constructs described herein. The invention further encompasses prokaryotic cells (such as, but not limited to, gram-positive and gram-negative bacterial cells) and eukaryotic cells (such as, but not limited to, these examples, yeast cells or plant cells). Preferably, said cell is a bacterial cell or a plant cell. The present invention also encompasses a transgenic plant, reproductive or propagating material for a transgenic plant comprising said plant cell.

Optionally, one or more transcription termination sequences may also be incorporated into the expression construct. The term "transcription termination sequence" encompasses a control sequence at the end of a transcriptional unit that signals 3 'processing and polyadenylation of a primary transcript and transcription termination. Additional regulatory elements such as transcriptional or transnational enhancers can be incorporated into the expression construct. The expression constructs of the present invention may also include an origin of replication that is required for the maintenance and / or replication of a specific cell type. An example is when an expression construct must be maintained in a bacterial cell as an episomal genetic element (e.g., plasmid or cosmid molecule) in a cell. Preferred origins of replication include, but are not limited to, these examples, fl-ori and colEl ori. The expression construct may optionally comprise a selectable marker gene. As used herein, the term "selectable marker gene" includes any gene that grants a phenotype in a cell where it is expressed in order to facilitate the identification and / or selection of cells that are transfected or transformed with an expression construct. of the invention. Suitable markers are markers that confer resistance to antibiotics or herbicides or visual markers. Examples of selectable markers include neomycin phosphotransferase (nptll), hygromycin phosphotransferase (hpt) or Basta. Additional examples of suitable selectable markers include genes for resistance against ampicillin (Amp '), tetracycline (Te'), kanamycin (Kan '), phosphinothricin, and chloramphenicol (CAT). Other suitable marker genes provide a metabolic trait, for example manA. Genes of visual markers can also be used and include, for example, beta-glucuronidase (GUS), luciferase and Green Fluorescent Protein (GFP, for its acronym in English). Transgenic Cells and Plants The present invention also relates to a plant comprising at least one dsRNA, at least one RNA construct, at least one nucleic acid or at least one recombinant DNA construct or plant cell described herein. The invention also relates to a seed, or to a plant cell comprising any of the nucleotide sequences or recombinant DNA construct encoding any of the dsRNA or RNA construct described herein. Plants that have been stably transformed with a transgene encoding the dsRNA can be supplied as seed, reproductive material, propagation material or cell culture material that does not actively express the dsRNA but has the ability to do so. The term "plant" as used herein encompasses a plant cell, a plant tissue (including callus) part of the plant, whole plant, ancestors and progeny. A plant part can be any part or organ of the plant and includes, for example, a seed, fruit, stem, leaf, shoot, flora, anther, root or tuber. The term "plant" also includes suspension cultures, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, and micro spores. The plant as used here refers to all plants including algae, ferns and trees. In a preferred embodiment, the plant belongs to the super family of Viridiplantae, with additional preference the plant being a monocot or a dicot. According to one embodiment of the present invention, the plant is susceptible to infestation by a pest of the plant, for example, a pathogenic nematode for plant, fungus or insect. Particular plants useful in the methods of the present invention are crop plants which include for example monocots such as sugarcane and cereals (including wheat), oats, barley, sorghum, rye, millet, corn, rice, eragostris or digitaria) and dicotyledons such as potatoes, plantains, tomatoes, grapes, apples, pears, soybeans, sugarcane, alfalfa, rapeseed and cotton. Particular trees that can be used in the methods of the present invention are pine, eucalyptus and poplar. The "administration" of a DNA to a cell can be achieved through several forms, each well known to a person skilled in the art. Example of useful techniques are shots, ballistics, electroporation, transection and transformation. For particular embodiments of the present invention, wherein the cell is a plant cell, general techniques for the expression of exogenous double-stranded RNA in plants for the purposes of RNAi are known in the art (see Baulcombe D, 2004, Nature. 431 (7006): 356-63 RNA silencing in plants, the contents of which are incorporated herein by reference). More particularly, methods for the expression of double-stranded RNA in plants for the purposes of down-regulating gene expression in plant pests such as for example nematodes or insects are also known in the art. Similar methods can be applied in an analogous manner for the purpose of expressing double-stranded RNA in plants for the purposes of down-regulating the expression of a target gene in a plant-pathogenic fungus. In order to achieve this effect it is only necessary that the plant expresses "transcribes" the double-stranded RNA in a part of the plant that comes into direct contact with the fungus, in such a way that the double-stranded RNA can be absorbed by the mushroom. Depending on the nature of the fungus and its relationship to the host plant, the expression of the dsRNA could occur within a cell or tissue of a plant in which the fungus is also present during its life cycle, or the RNA can be secreted in a space between cells, such as the apoplast, which is occupied by the fungus during its life cycle. In addition, the dsRNA can be located in the cell of the plant, for example, in the cytosol, or in the plant cell organelles such as for example chloroplast, mitochondria, vacuole or endoplastic reticulum. Alternatively, the dsRNA can be secreted by the plant cell and by the plant to the outside of the plant. As such, the dsRNA can form a protective layer on the surface of the plant. The present invention therefore relates to a method for the production of a transgenic cell or of a transgenic organism, said method comprises the step of administering a recombinant RNA construct described herein to said cell or organism. Preferably, said cell is a plant cell or said organism is a plant. The invention further relates to any transgenic cell or transgenic organism obtainable through the method described above, preferably said transgenic cell or transgenic organism is a plant cell or a plant organism.

The methods of the present invention for the production of a transgenic organism may further comprise the steps of culturing the transgenic cell under conditions that promote growth and development. When the transgenic organism is a plant, these methods can also include the steps of regenerating a plant from a plant tissue, allowing growth to reach maturity and reproduction. Alternatively, the transgenic plant tissue can take other forms or can be part of another plant, examples are chimeric plants and grafts (for example a transformed rhizome grafted onto an untransformed sapling). Compositions According to one embodiment, the invention relates to a composition comprising at least one dsRNA or an RNA construct described herein and a physiologically or agronomically acceptable excipient or diluent carrier. The invention also encompasses the use of said composition in the form of a pesticide for a plant or for the propagation or reproduction material of a plant. According to another embodiment, the invention relates to a composition comprising at least one nucleic acid or recombinant DNA construct described herein, and a physiologically or agronomically acceptable excipient or diluent carrier.

A composition may contain additional components that serve to stabilize the dsRNA and / or prevent degradation of the dsRNA during prolonged storage of the composition. The composition may also contain components that increase or promote the absorption of the dsRNA by the pest organism. It includes, for example, chemical agents that generally promote the absorption of RNA in cells, for example, lipofectamine, etc., and enzymes or chemical agents capable of directing the fungal cell wall, for example, chitinase. The composition may be in any physical form suitable for application to the pest, to substrates, to cells (e.g., plant cells), or to an organism infected by a pest species or susceptible to being infected by a pest species. It is contemplated that the "composition" of the present invention may be supplied as a "kit of parts" comprising the double-stranded RNA in a container and a diluent or carrier suitable for the RNA in a separate container. The invention also relates to the supply of double stranded RNA alone without additional components. In these embodiments, the dsRNA can be delivered in a concentrated form, such as a concentrated aqueous solution. It can also be supplied in frozen form or in lyophilized form. The latter form may be more stable for long-term storage and may be thawed and / or reconstituted with a suitable diluent immediately before use. The present invention also relates to the medical use of any of the double-stranded RNAs, double-stranded RNA constructs, nucleotide sequences, recombinant DNA constructs or compositions described herein. In particular, the present invention relates to pesticidal compositions developed to be used in agriculture or horticulture. Those pesticidal compositions can be prepared in a manner known per se. For example, the active compounds can be converted into customary formulations, such as, for example, solutions, emulsions, wettable powders, granules which can be dispersed in water, suspensions, powders, dusts, foaming agents, pastes, soluble powders. , granules, suspo-emulsion concentrates, microcapsules, fumigants, natural and synthetic materials impregnated with active compound and very thin capsules and polymeric substances. In addition, the pesticidal compositions according to the present invention may comprise a synergist. The dsRNA or dsRNA construct according to the present invention, as such or in its formulations, they can also be used in a mixture with known fungicides, bactericides, acaricides, nematicides or insecticides in order to extend, for example, the spectrum of activity or to prevent the development of resistance. In many cases, this results in synergistic effect, ie, the activity of the mixture exceeds the activity of the individual components. In addition, the active components according to the present invention, as such or in their formulations or mixtures mentioned above can also be used in a mixture with other known active compounds, such as for example herbicides, fertilizers and / or growth regulators. The present invention also relates to fibrous pesticide compositions and their use as a pesticide, wherein the fibrous composition comprises a non-woven fiber and an effective amount of at least one of the dsRNAs or dsRNA constructs described herein, covalently fixed or stably adsorbed in the fiber. In one embodiment, the fibrous composition comprises at least two dsRNAs or dsRNA constructs in accordance with that described herein. In a further particular embodiment, the fiber is biodegradable and the adsorbed dsRNA or dsRNA construct adsorbed in accordance with that described herein can be released slowly in a localized area of the environment to control pests in this area for a period of time. The present invention also encompasses solid formulations of slow release pesticidal compound in accordance with what is described herein, and its use as a pesticide. The formulations release the compound in accordance with what is described herein (a) in the environment (soil, aqueous medium, plant) in a controlled and slow manner (complete release is effected within several days to a few months). To prepare the slow release formulations, all the components can be either melted together directly in the form of a physical substance or mixed with a preformed polymer melt and then extruded. The present invention also relates to diatomaceous earth surfactant compositions for pesticidal use in the form of dispersible dry granules comprising at least one dsRNA or dsRNA construct compound or at least two dsRNAs or dsRNA construct compounds in accordance as described herein, the granules comprise, in addition to the diatomaceous earth, a surfactant composition designed to provide binding, rewetting and disintegrating properties to the granules. By diatomaceous earth we understand a silica material characterized by a large surface area per unit volume. A diatomaceous earth is a material that occurs naturally and consists mainly of accumulated husks or frustules of intricately structured amorphous hydrous silica secreted by diatomaceous. The dry granules that can be dispersed can be prepared through standard granulation process in tray, or through homogeneous extrusion process. It is to be noted that granules which are prepared in the absence of a pesticide by extrusion process can be subsequently spiked with dsRNA (s) or dsRNA construct (s) to adhere them onto the granules. The present invention also offers solid, water-insoluble lipoespheres and their use as a pesticide, wherein said lipospheres are formed from a solid hydrophobic core having a layer of a phospholipid integrated in the surface of the core, containing at least one dsRNA or construct. of dsRNA in accordance with that described herein in the nucleus, in the phospholipid, adhered to the phospholipid, or a combination thereof. In one embodiment said lipoesphere comprises at least two dsRNA or dsRNA construct in accordance with that described herein. The pesticidal compound containing lipospheres has several advantages including stability, low reagent cost, ease of manufacture, high dispersion capacity in an aqueous medium, a release rate for the entrapping compound that is controlled by the phospholipid coating and the vehicle.

The invention further relates to pesticidal formulations in the form of microcapsules having a capsule wall made of a urea / dialdehyde precondensate and comprising at least one compound in accordance with that described herein. In a specific embodiment, the composition can be a coating that can be applied on a substrate in order to protect the substrate against infestation by a species of pest and / or to prevent, stop or reduce the growth of pest on the substrate and prevent consequently the damage caused by the pest species. In this embodiment, the composition can be used to protect any substrate or material susceptible to infestation or damage caused by a species of pest, for example food and other perishable materials, and substrates such as wood. An example of such a plague species are fungi. Preferred white fungal species for this modality include, but are not limited to, these examples: Stachibotrys spp. , Alternate spp. , or Cladosporium spp. The nature of the excipients and the physical form of the composition may vary according to the nature of the substrate to be treated. For example, the composition can be a liquid applied by brush or spray on the material or substrate to be protected either printed on the material or substrate to be treated, or a coating that is applied on the material or substrate to be treated. Methods The present invention further encompasses a method for the treatment and / or prevention of fungal infestation in a substrate, comprising the application of an effective amount of any of the compositions described herein on said substrate. The present invention also relates to methods for treating and / or preventing the growth of plague and / or infestation by plague of a plant or propagation material or reproduction of a plant, comprising the application of an effective amount of a chain RNA double, an RNA construct or a composition according to what is described herein to a plant or to a propagating or reproducing material of a plant. The present invention also relates to methods for the treatment and / or prevention of pest infestation in a substrate, comprising the application of an effective amount of double-stranded RNA, an RNA construct, or a composition in accordance with described here on said substrate. In another embodiment, the invention relates to a method for controlling the growth of a pest in a cell or an organism or to prevent pest infestation of a cell or an organism susceptible to infection by said pest species, which comprises laying in contact of said pest species with any of the double-stranded RNAs or dsRNA constructs described herein, whereby the double-stranded RNA or double-stranded RNA construct is absorbed by said pest species and thus controlled the growth or the infestation is avoided. In another embodiment, the invention relates to a method for downregulating the expression of at least one target gene in a pest species, comprising contacting said pest species with any of the double-stranded RNAs or dsRNA constructs described herein, whereby the double-stranded RNA or double-stranded RNA construct is absorbed by the pest species and therefore the expression of the target gene of the pest or of the target genes is downregulated in a downward manner. said plague. As illustrated in the examples, bacteria can be engineered to produce any of the dsRNA or dsRNA constructs of the present invention. These bacteria can be eaten by the pest species. When absorbed, the dsRNA can initiate an RNAi response, causing the degradation of the white mRNA and weakening or killing the pest that feeds. Therefore, in a more specific embodiment said double-stranded RNA or double-stranded RNA construct is expressed by a prokaryotic such as a bacterium., or a eukaryotic, such as a yeast, host cell or host organism. Some bacteria have a very close interaction with the host plant, such as symbiotic Rhizobium with legumes (for example Soya). Such recombinant bacteria could be mixed with the seeds (i.e., coated) and used as soil improvers. Alternatively, bacteria that produce dsRNA can be raised directly on crops such as Bacillus thuringiensis species. Possible applications include intensive greenhouse crops, for example crops that are less interesting from a GMO perspective as well as crops in a wider field such as soybean. This approach has several advantages such as: since the problem of possible division by a plant host organism is not present, it allows the delivery of large dsRNA fragments in the intestinal lumen of the pest that is feeding; the use of bacteria as insecticides does not include the generation of transgenic crops especially for certain crops where transgenic variants are difficult to obtain; there is a broad and flexible application in the sense that different crops can be treated simultaneously in the same field and / or pests can be focused simultaneously as for example by the combination of different bacteria that produce different dsRNAs. According to another specific embodiment, the invention encompasses GMO approaches and therefore refers to a method with that described herein wherein said double-stranded RNA is expressed by said cell or organism infected with said pest species or susceptible to being infested by said pest species as for example said cell is a plant cell or said organism is a plant. The invention further relates to any of the methods described above wherein said double-stranded RNA or double-stranded RNA construct is expressed from at least one recombinant DNA construct in accordance with that described. In additional embodiments of the invention, the dsRNA or dsRNA construct is expressed from two (or more) DNA constructs and the fused transcripts form double-stranded RNA or the double-stranded RNA construct. The invention further relates to the method for the production of a plant resistant to a pathogenic pest for the plant comprising: a) transforming a plant cell with a recombinant DNA construct of any of claims 19 to 21, b) regenerating a plant from the transformed plant cell; c) cultivating the transformed plant under conditions suitable for expression of recombinant DNA construct, said cultivated transformed plant is resistant to said pest compared to an untransformed plant. In another embodiment, the present embodiment encompasses plants that comprise more than one dsRNA, a recombinant dsRNA construct, each comprising or encoding a single dsRNA fragment; said plants can be obtained by crossing at least two transgenic plants. Said recombinant DNA constructs may comprise distinct regulatory sequences. Said recombinant DNA constructs can have a different origin (ie, come from different plasmids or different vectors or different expression vectors). The present invention also encompasses methods for the production of transgenic plants wherein the recombinant DNA construct comprises between the left border and the right border of, for example, the plant expression sequences, rather than a dsRNA or dsRNA construct comprising multiple fragments of dsRNA, said fragments of dsRNA may be the same or may be different; or wherein each of the dsRNA or dsRNA construct within a recombinant dsRNA construct comprises the same dsRNA fragment.

The invention further relates to a method for increasing the yield of a plant, said method comprising reproducing a plant of any of the nucleotide sequences or recombinant DNA constructs of the invention in a format that can be expressed. The invention also relates to the use of a double-stranded RNA, a double-stranded RNA construct, a nucleotide sequence, a recombinant RNA construct, a cell, or a composition described herein, for the treatment of plant infection by plague. According to another embodiment, the invention relates to a kit comprising any of the double-stranded RNAs, double-stranded RNA construct, nucleotide sequence, recombinant DNA construct, cells or compositions described herein for treating plant infection by pest. . The kit can be supplied with instructions suitable for use. The instructions can be printed in an appropriate package where the other components are supplied or can be provided as a separate entity, or they can be in the form of a sheet or a brochure for example. The instructions can be rolled or folded for example when they are stored and can then be developed or unfolded to give indications of use of two remaining components of the kit. In a specific embodiment, the method of the present invention can also be used as a tool for experimental research, particularly in the field of functional genomics. The down-regulated regulation of pest genes by RNAi can be used in in vitro or in vivo assays in order to study gene function, in an approach analogous to the approach that has been described in the art for the nematode worm C. elegans and also Drosophila melanogaster. Assays based on the focused down-regulation of specific pest genes leading to a measurable phenotype can also form the basis of composite screens for novel anti-aphroplastic agents. DESCRIPTION OF THE FIGURES The present invention will now be described with reference to the following Figures in which: Figure 1 shows examples of concatemer constructs with optimal selection of target gene, selection of target sequence, and combination of dsRNA fragments in concatamer construct according to that described herein. Figure 2 shows the different types of blocking according to the present invention. Figure 3 shows the types of different dsRNA nuclei of the present invention that are part of the concatemer and / or dsRNA constructs stabilized in accordance with what is described herein. Figure 4 shows a preferred construct according to the present invention. Figure 5: In nuclei of dsRNA type 1 and 2, which are known as "clover leaf" type deRNA nuclei, each stem can comprise a combination of the dsRNA nuclei types A, B or C of the Figure 3. Multiple stems can be introduced with or without the linker and / or a lock in position Y. The stems can be branched or unbranched. These branched and unbranched stems can be combined within a construct according to the present invention. The linker or a block in position Y contains a short loop at its end. In position X, the dsRNA of core 1 or 2 may contain a stem, a linker and / or a block. When location X is located, a GC-rich coupling or a mismatch block also forms a stem of dsRNA, optionally coupled with additional blocks. A stem of dsRNA in position X is constituted by a 5 'fragment that finds its complementary sequence at the 3 or 4 end of the RNA chain. This type of nucleus of dsRNA molecules can form "closed" three-dimensional structures of star or spherical type that offer an additional level of protection against RNA processing. In the nucleus of type 3 dsRNA, the lock at position Y is preferably a short loop and the linker at position X is preferably an intron. The dsRNA construct starts and ends preferably with a Z-linker / blocking combination, which is found at the edges of the construct. Figure 6 shows a schematic presentation of the general building blocks used in the stabilized dsRNA constructs of the present invention. In each of the constructs A, B, C or D, different combinations of dsRNA nuclei (eg, concatemers) are possible, different combinations of linker sequences are possible, different blocking combinations are possible and the number of different blocks construction may vary. In addition, construct D, in different combinations of internal linker and / or blocking blocks are possible. Figure 7 shows a "silent bell" construct according to the present invention, comprising fragments of sense and antisense fragments of the target gene, F39H11.5 of C. elegans and two short loops to protect the construct against RNA processing . Figure 8 shows examples of hair pins wherein linkers in accordance with the present invention are combined with locks that are RNA structures of a protein. Figure 9 shows the beta-tubulin sequence of Meloidogyne (SEQ ID NO 43) with annotation of the primers used for the production of three fragments of dsRNA of different lengths, specifically 105, 158 or 508 base pairs. Figure 10 shows the number of J2 Meloidogyne larvae incognito in motion (counted 2, 3, 4, 6 and 22 hours after agar plate placement) after feeding overnight with double-stranded beta-tubulin RNA different lengths: 1) without dsRNA; 2) dsRNA of 105 base pairs; 3) dsRNA of 258 base pairs; 4) dsRNA of 508 base pairs. Figures 11 and 12 show the results of protection against RNAse III division by IRES sequences according to Example 2.1. Figures 13 to 20 represent constructs of concatemers according to that described in Example 3 and Table 3. Figure 21 shows the construction of concatemers comprising from 1 to 6 repeating units of dsRNA fragments of rps-4 of 80 base pairs (in accordance with what is described in Example 3.1). Figure 22 shows the RNAi efficiency of repeating units 1 to 6 of dsRNA fragments of 80 base pairs of rps-4 of Figure 21. Figure 23 shows the stage of development of larvae for repeat 3 to 6 of Figures 21 and 22.

Figure 24 shows the construction of concatemers comprising 6 + 0.5 + 1, 4 + 2.3 + 3.2 + 4 rps-4 and unc-22 repeating units of dsRNA fragments of 80 base pairs (in accordance with what is described in example 3.2). Figure 25 shows the RNAi efficiency of the repeated units of Figure 24. Figures 26 and 27 show the lethal character by deactivation of sub-lethal genes sym-1 and sym-5 (in accordance with that described in Example 4 ). Figure 28 shows the effect of co-deactivation of sub-lethal genes sym-1 and sym-5 using fragments of dsRNA separately, mixed or single constructs (in accordance with that described in Example 5). Figure 29 represents a list of example sequences of the invention. EXAMPLES The invention will be further understood with reference to the following non-limiting examples. Example 1: The efficiency of dsRNA in nematodes depends on the length of short interfering RNAs (siRNAs) mediate the dissociation of specific single-stranded target RNAs. These siRNAs usually have a length of approximately 21 nt, suggesting that siRNA expression in the host organism causes an efficient and specific down regulation of gene expression, resulting in the functional inactivation of the focused genes. However, there are indications that in the case of invertebrates (for example, independent nematodes C. elegans and parasitic nematodes of plants Meloidogyne incognita), the minimum length of invertebrate fed dsRNA must be at least 80-100 nt to be effective because possibly to a more efficient absorption of these fragments of long dsRNA or invertebrates. Similar results were now observed for the plant parasitic nematode Meloidogyne incognita (SEQ ID NO: 43). Fragments of dsRNA from the genes of beta-tubulin of M. incognita with different lengths (105 base pairs, 258 pairs of base pairs and 508 base pairs) were produced in vitro (T7 Ribomax Express RNAi System, Promega) using the specific primers as shown in Figure 9 and Table 1. Table 1: General information on different fragments of beta-tubulin from M. incognita and the primers used to isolate them FW primer RV primer Fragment length GAU140 GAU143 105 base pairs GAU140 GAU142 258 base pairs GAU140 GAU141 508 base pairs An in vitro drinking test was used to test the efficacy of these beta-tubulin dsRNAS in J2 Meloidogyne incognita. J2 is the second stage of juvenile larval ineffective nematode. J2s was stimulated to be fed from a liquid medium containing M9 buffer, PEG and 5 mg / ml dsRNA from the different constructs of beta-tubulin or free FITC (0.1 mg / ml). J2 were incubated at 26 ° C. Intake of dsRNA was checked by visualization of FITC uptake via fluorescence microscopy. The down regulation of the endogenous target genes was reviewed by quantitative PCR or by monitoring phenotypic effects (lethality / morbidity) of the J2 larvae. The down-regulation of the endogenous beta-tubulin gene caused a phenotypic effect of reduced mobility of J2 larvae (Figure 10). This reduced mobility was observed for the J2 larvae that ingested the dsRNA of 258 base pairs and 508 base pairs. This effect can be observed in the case of J2 larvae that ingested the dsRNA of 105 base pairs. Example 2: Protection of dsRNA 2.1 Protection against cleavage by RNAse III by IRES sequences In this example, a fragment of dsRNA was weakened at both sites by a blocking sequence that exhibited extensive secondary structures. Secondary structures at the ends delay the processing of dsRNA by two enzymes RNase III, human Dicer and E.coli RNase II. As protective blocking sequences, the internal ribozoma entry sites (IRESes) of encephalomyocarditis virus (EMCV) and upstream of N-ras (UNR) were used. IRESes form complex secondary structures with multiple stem-loop regions to which proteins can bind such as for example ribosomes and the polypyrimidine tract binding protein (PTB). In a plant cell, EMCV IRES can protect from dsRNA linked against division by its secondary structuring as well as cellular binding factors, thus avoiding sterically the access of Dicer to dsRNA. The IRES sequences used in this example were a 559 nt fragment upstream of the EMCV viral polyprotein coding sequence (Genbank accession number NC_001479, nucleotides 279-836) with additional nucleotide A in position 776 (SEQ ID NO 13) , and a 342 nt fragment upstream of the human UNR protein coding sequence (Genbank accession number NM_001007553, nucleotides 69-410; SEQ ID NO 14). The dsRNA fragment used in this example is a 505 base pair fragment of the C. elegans rps-4 cDNA (Genbank accession number NM_068702, nucleotides 122-62, SEQ ID NO 15). The IRES sequences were amplified with PCR primers that carried the appropriate restriction sites at the ends and cloned into vector containing two T7 promoter sites that failed a multiple cloning site. The rps-4 fragment was amplified by polymerase chain reaction and cloned in the TOPO-TA® vector (Invitrogen). From there, it was cloned in both orientations in the plasmids containing IRES using the Eco RI sites from the TOPO-TA® vector. Plasmids were isolated using QIAprep® Spin Miniprep Kit (Qiagen). To prepare annealing from the plasmids containing IRES, plasmids were linearized after IRES and a polymerase chain reaction (PCR) was performed with a direct T7 primer and a T7 forward primer and a reverse primer specific for IRES. RNA was prepared through in vitro transcription using the T7 RiboMAX ™ Express RNAi System (Promega). The sequence of the resulting sense and antisense strands of the dsRNA constructs is given in SEQ ID Nos: 16 to 21. Upon fusion, the double-stranded rps-4 RNA is flagged by an IRES sequence in every 3 ' Control dsRNA from rps-4 was prepared from a hardened PCR derivative in which case one of the PCR primers was extended with a T7 promoter site. It shows that the IRES sequences protect the dsRNA against cleavage, unbound dsRNA and linked IRES were incubated with two RNase III enzymes commercially available in accordance with the manufacturers protocol. In the first experiment, 400 ng of unbound or linked dsRNA was incubated with IRES at a temperature of 37 ° C with a unit of recombinant human Dicer enzyme (Stratagene). The reaction was stopped after 0, 1, 2 or 3 hours, carried out on a 20% polyacrylamide gel and stained with ethidium bromide. For comparison 25, 50, 75, 100 and 150 ng with 21-mer of double chain (siRNA) not related was loaded in the same gel. The divided product migrated just above the siRNA marker. At each of the incubation time points of 1, 2 and 3 hours, less product was formed divided in the reactions with the dsRNA bound with IRES compared to what was observed with the unbound dsRNA (see Figure 11). The bands that migrate up in the gel represent the unprocessed dsRNA by processing intermediates (a batch of the dsRNA bound with IRES did not enter the wells and adhered in the groove, a significant fraction of this will have been washed during the dyeing of the gel). At each time point, more intermediate processing products were found with the unbound dsRNA compared to dsRNA bound to IRES, as it was detached from the gel DNA trace of the high molecular weight band that migrated in the gel. The EMCVA IRES and UNR IRES also protected against processing by another RNase III enzyme isolated from E. coli. 3 pmol of unbound or IRES-linked dsRNA were incubated at 37 ° C with 0.4 unit of ShortCut ™ enzyme from recombinant E. coli RNase III (New England Biolabs Inc.). A reaction buffer containing manganese was used to promote the processing of dsRNA in a heterogeneous mixture of siRNAs of 18-25 base pairs. The reaction was stopped after 20 minutes by instantaneous freezing in liquid nitrogen, carried out on a 20% polyacrylamide gel and stained with ethyl bromide. For comparison, 25, 75 and 150 ng of an unrelated 21-mer siRNA was loaded onto the same gel. In parallel, the same set of reactions was carried out in the presence of 60 pmol of recombinant human PTB-GST fusion protein that was expressed in bacteria and purified on GST column (PTB as Genbank sequence NP_114368.1, fusion in the sixth amino acid). All conditions were tested in two independent reactions. As was the case with human Dicer, dsRNA bound with IRES was less processed by bacterial RNase III than unbound dsRNA (see Figure 12, compare lanes Rl and R2 with Ul, U2, El and E2). In reactions with unbound dsRNA, almost all of the long dsRNA is processed into the final product or intermediate products of low molecular weight processing. In the reactions with dsRNA bound to IRES, most of the initial material remained unprocessed (bands in the groove and the upper part of the gel) and also intermediate processing processes of high molecular weights were present. In addition, the presence of PTB protects this RNA linked to IRES even more against RNAA processing in accordance with what was observed with the low levels of final product and high levels of intermediate processing products. In the association of dsRNA bound to the URN IRES, also partially processed bands of molecular weight indicate an increased resistance to RNA processing (see Figure 12 compare lanes Ul and U2 with U3 and U4, and lanes El and E2 with E3 And E4). 2.2 Construction of dsRNA with linker and blocking sequence (s) that protect dsRNA against RNA processing. Beta-tubulin white genes of different white species are isolated through information by RT PCR with degenerative primers developed based on the sequence of known beta-tubulin genes. For example, suitable fragments of known beta-tubulin, suitable fragments of the Meligogyne beta-tubulin gene incognita to be used in the constructors of the present invention are represented in Figure 9 and by SEQ ID NO: 43. Additionally, genes are isolated "stoppage of a cell" (OCS) target such as, for example, C. elegans OCS white gene F39H11.5. The sequence of F39H11.5 is located in genbank (access number Z81079, version 1, gi number 1627924, region 841-1770 in the complementary chain) as well as the gene of C. elegans sup-35 (access number genbank mRNA number NM_067031) is used as the target gene and the fragment for the silencing constructs of dsRNA is between nucleotide 396 and nucleotide 999. The length of the dsRNA constructs tested from approximately 300 base pairs. Simple stem-core dsRNA constructs, which focusing on a simple white sequence are tested as well as concatemers, which target multiple target sequences. In the case of concatemer, the length of each dsRNA fragment is approximately 80 base pairs or approximately 25 base pairs. In another construct, the total length of the concatemer dsRNA is about 250 base pairs or about 300 base pairs and each dsRNA fragment is about 20 or about 25 base pairs, or about 50 or about 60 or about 70 pairs of base. Blockages are present on both stem edges of dsRNA. The blocks are non-complementary loops of 5 base pairs. Table 2. General information about stabilized constructs. Different constructs of the present invention are tested in four different species, among the species of plant pests: Meloidogyne incognita, Caenorhabditis elegans, Grasshopper for example Nilaparvata lugens and Magnaporthe grísea.

M. C. Grasshopper M. Incognita elegans grísea Gene White beta-beta-beta-beta-tubulin tubulin tubulin tubulin sup 35 and / or OCS Nucleus dsRNA cA, Cb cA, Cb cA, Cb cA, Cb Block loop loop loop loop short short short short pairs of 5 pairs of 5 pairs of 5 base pairs of bases of bases of bases Type of sensitive sensitive sensitive sensitive Linker to pH at pH at pH Absorption Test Assay of soaked spray In vitro absorption consumption in liquid sheet of liquid In plant roots in x plant roots in hair, whole, hair plant callus callus, whole whole plant The specific constructs used in these examples are also represented here in SEQ ID Nos: 9 to 12. Example 3: Design and cloning of efficient concatamer constructs of dsRNA for pest control Constructs of concatemers were designed to understand different combinations of dsRNA fragments that target different white genes; because they focus a different white sequence on such white genes, which focus on sequences that have the same different lengths; or that repeat the same sequence several times. The dsRNA concatemer constructs of the present invention have a total length of less than 700 base pairs and are preferably within a range of about 250 base pairs to about 500 base pairs. Preferably, the length of the concatamer construct of dsRNA is such that the corresponding ssRNA can efficiently form a hairpin dsRNA. The construct format of the concatemer construct of the present invention may be a dsRNA per se or it may be a dsRNA of hair pin. A dsRNA per se or a hair pin can be prepared through in vitro transcription or through recombinant expression systems. Table 3: the following concatemer constructs were cloned. Schematic presentations are provided in the figures. As used herein "Freefrag" refers to a fragment of dsRNA without substantial homology of nucleotide sequences with non-target organism. Name Gen Description Figure and / or blank ** SEQ ID NO Cl A 1x80 base pairs, Figure 21, 22, selected in SEQ ID NO: 27 content of GC * C2 A 2x80 base pairs, Figure 21, 22, selected in SEQ ID NO: 26 content of GC * C3 A 3x80 base pairs, Figure 21, 22, 23, selected in SEQ ID No: 25 content of GC * C4 A 4x80 base pairs, Figure 21, 22, selected in SEQ ID No: 24 content of GC * C5 A 5x80 base pairs, Figure 21, 22, selected in SEQ ID No: 23 content of GC * C6 A 6x80 base pairs, Figure 21, 22, 23 selected in SEQ ID No: 22 and 28 GC content * C7 B 1x80 base pairs, selected in GC content * C8 B 2x80 base pairs, selected in GC content * C9 B 3x80 base pairs, selected in GC content * CIO B 4x80 base pairs, selected in content of GC * Cll B 5x80 base pairs, selected on GC content * C12 B 6x80 base pairs, selected on GC content * C13 4x40 base pairs Figure 13 of conserved region GC content * C14 5x50 base pairs Figure 13 of conserved region C15 Freefrag in order Figure 13 biological C16 1 Freefrag scrambled Figure 13 C17 2 6x60 base pairs Figure 14 of conserved region C18 6x60 base pairs Figure 14 of non-conserved region C19 6x60 base pairs Figure 16 of conserved region C20 6x60 base pairs Figure 16 of non-conserved region C21 1x80 base pairs Figure 24, 25, of A, 5x80 pairs of SEQ ID NO: 29 bases of C, selected in GC content * C22 2x80 base pairs Figure 24, 25, of A, 4x80 pairs of SEQ ID NO: 30 bases of C, selected in content of GC * C23 3x80 base pairs Figure 24, 25, of A, 3x80 pairs of SEQ ID NO: 31 bases of C, selected on GC content * C24 4x80 base pairs Figure 24, 25, of A, 2x80 pairs of SEQ ID NO: 32 C bases, selected in GC content * C25 A-C 5x80 base pairs of A, 1x80 base pairs of C, selected in GC content * C26 B-C 1x80 base pairs of B, 5x80 base pairs of C, selected in GC content * C27 B-C 2x80 base pairs of B, 4x80 base pairs of C, selected in GC content * C28 B-C 3x80 base pairs of B, 3x80 base pairs of C, selected in GC content * C29 BC 4x80 base pairs of B, 2x80 base pairs of C, selected on GC content * C30 BC 5x80 base pairs of B, 1x80 base pairs of C, selected on GC content * C31 D 909 base pairs Figure 26, 27 D, selected in content of GC * C32 E 829 base pairs Figure 26, 27 E, selected in content of GC * C33 DC 50 base pairs D, selected in content of GC * C34 DC approximately 150 Figure 26 , 27 base pairs of D, SEQ ID Nos: 35 152 base pairs and 36 of C, selected in content of GC * C35 OF 50 base pairs D, 50 base pairs of E, selected in GC content * C36 OF approximately 150 Figure 28 base pairs of D, SEQ ID Nos: 39 about 150 and 42 of base pairs of E, selected at GC * C37 DE content of 2x50 base pairs of D, 2x50 base pairs of E, selected in content of GC * C38 DE 3x50 base pairs of D, 3x50 base pairs of E, selected in co GC content * C39 EC 50 base pairs of E, 50 base pairs of C, selected at GC content * C40 EC approximately 150 Figure 28, base pairs of E, SEQ ID Nos: 39 153 base pairs of C , And 38 selected in GC content * C41 17-18 White genes in the same Figure 17 via: protein translation path, selected fragments in GC content * + freefrag C41 19-20 White genes in the same Figure 17 21-22 via, for example, the proteasome pathway, selected fragments in GC content + freefrag C42 17-22 White gene combination Figure 18 23-24 from different pathways, for example protein translation, proteasome, transcription, pathways of nucleic acid binding and protein binding, selected fragments in GC content * + freefrag C43 3-1 Essential genes: Figure 15 4-5 70 base pairs each, 6-7 selected in content 8 of CG * C44 3-1 Essential genes: Figure 19 4-5 selection in content of 6-7 GC * + freefrag 8-C45 Specific genes for Figure 15 11-12 insects: 70 pairs of 13-14 bases each, 15-16 selected in content C46 9-10 of GC * 11-12 Specific genes for Figure 19 13-14 pests: selection in 15-16 content of GC * + freefrag * Fragments are selected in GC content between 40% and 60% ** Genes 1 to 25: target genes; gene A = rps-4 from C. elegans; gene B = rps-14 of C. elegans; C gene = unc-22 of C. elegans; gene D = sym-1 of C. elegans; gene E = sym-5 of C. elegants. 3. 1 The effectiveness of dsRNA in nematodes improves with increasing number of repeat units of a small fragment This example describes that a dsRNA fragment of 80 base pairs is sufficient to induce RNAi, and that the efficiency rises when this fragment is repeated several times in the same construct. a) Fragments of dsRNA The fragments of dsRNA used in this example contain from 1 to 6 repeat units of a fragment of 80 base pairs (SEQ ID NO: 50) of the gene rps-4 C. elegants (accession number Genbank NM_068702, nucleotides 474-553). A schematic representation of these constructs are shown in Figure 21, the sequences of fragments of dsRNA (sense strands) used are represented by SEQ ID Nos: 22 to 27 b) Methods Cloning: a DNA fragment was manufactured synthetically which contained 6 repeat units of rps- separated by restriction sites (see Figure 21). This fragment was first cloned into a vector such that it was flanked by two T7 promoter sites. Plasmids containing 5, 4, 3, 2 or 1 Repeated units respectively were derived from this plasmid by digestion with the appropriate restriction enzyme (s) and religation of the linearized plasmids. Preparation of RNA: Plasmids were isolated using the EndoFree® Plasmid Maxi Kit (Qiagen) and in two separate reactions digested with Eco RI and Hind III respectively. RNA was prepared by in vitro transcription using the T7 RiboMax ™ Express RNai System (Promega). The sequences of the resulting dsRNA (sense strands) used are represented by SEQ ID Nos: 22 to 27. Elegant RNAi: chic L larvae were allowed to ingest M9 buffer containing dsRNA for 24 hours at a temperature of 20 ° C and then transferred to regular NGM plates. The animals were examined after 3 days of growth at 20 ° C and for all the animals the development stage was determined, c) Results Exposure to dsRNA of rps-4 induced a delay in growth and suspended development in early larval stages for all constructs. RNAi efficiency increased with increasing numbers of repeat units of rps-4 present in the dsRNA fragment (see Figure 22). Efficacy was measured as the ability of dsRNA to prevent animals from becoming adults in 3 days. Furthermore, the higher the number of repeat units of rps-4 in the dsRNA fragment, the lower the concentration required to induce the same degree of growth inhibition. Increased efficacy was manifested not only in a greater number of animals with growth retardation or suspension of development, but also in a faster response (ie larvae suspended their development at earlier stages of development). Figure 23 shows that at the highest concentrations almost no larvae grew beyond the second larval stage (L2). In intermediate concentrations, some larvae have managed to grow until the third stage (L3) or fourth stage (L4) larval. The transition from "all adult" to "all L2" occurred more rapidly in the construct with 6 repeated units of rps-4 compared to the construct with 3 repeated units rps-4. 3. 2 The effectiveness of dsRNA in nematodes improves with increasing the number of repeated units of a small fragment This example is a variation of Example 3.1. In this example, however, the total fragment length is kept constant by replacing repeated units rps-4 with unc-22 repeating units. a) Fragments of dsRNA The fragments of dsRNA used in this example contain several numbers of the same fragment of 80 base pairs (SEQ ID NO 50) of the rps-4 gene of C. elegants described in Example 3.1 together with a variable number of a fragment of 80 base pairs of the gene unc-22 of C. elegants (accession number to Genbank NM_69872, nucleotides 9621-8700). The total number of repeat units in a dsRNA fragment always reaches a total of six, and therefore all molecules have the same length. The inactivation of unc-22 does not influence the growth in such a way that the effect of inhibition of growth is due to siRNAs specific for rps-4. Due to an additional base in one of the cloning primers, the Xba-Spe insert unc-22 in the multiple repeats contains 81 base pairs. The additional base pair is in position 1 b) Methods A DNA fragment was prepared synthetically which contained 6 repeat units of rps-4 separated by restriction sites (see Figure 21). This fragment was first cloned into a vector such that it was flanked by two T7 promoter sites. Subsequently, a repeating unit of rps-4 at a time was exchanged for a unc-22 repeat fragment that was amplified by polymerase chain reaction using primers with restriction sites flanking the unc-22 sequence (see Figure 24). Preparation of dsRNA and RNAi experiment were carried out in accordance with that described for Example 3.1. The sequence of the resulting dsRNA fragments (sense strands) is represented by SEQ ID Nos: 28 to 32. c) Results The efficiency of RNAi increased with increasing numbers of rps-4 repeats present in the dsRNA fragment ( see Figure 25). Fragments of dsRNA with 4 or more repeating units of rps-4 were equally active, but were more active than fragments with 2 or 3 repeat units. Since the uptake of dsRNA can be considered equal between these constructs, a likely explanation for the increased efficacy of fragments with 4 or more repeating units of rps-4 is that the division of these fragments results in more specific siRNAs for rps-4. Example 4: Induction of lethality by deactivation of multiple sublethal targets This example describes that the co-deactivation by RNAi of two genes with weak phenotypes per se, sym-1 and sym-5, results in a highly improved phenotype. a) Fragments of dsRNA For sym-1, a fragment of 829 base pairs corresponding to nucleotides 11972-2800 of Genbank sequence Z79594 was used. For sim-5, a fragment of 909 base pairs corresponding to nucleotides 8003-8911 of Genbank sequence Z79598 was used. The sequences of the dsRNA fragments (sense strand) used in this example are represented by SEQ ID Nos: 33 and 34. b) Feeding method: the fragments mentioned above were amplified with standard polymerase chain reaction primers PCR and cloned in the pGN49A vector (WO01 / 88121) between two T7 promoters and identical terminators driving their expression in the direction of sense and antisense when the T7 polymerase was expressed, which was induced by IPTG. The resulting plasmids were transformed into the bacterial strain AB301-105 (DE3). Larvae L. of elegant wild type were plated on NGM plates with IPTG seeded with transformed AB301-105 (DE3) bacteria, and examined after 3 days of growth at a temperature of 20 ° C. Injection: the fragments mentioned above they were amplified from wild-type genomic DNA using combinations of primers wherein a primer was extended with the T7 DNA polymerase promoter sequence. Polymerase chain reaction products were purified from the gel using the QIAquick® Gel Extraction Kit (Qiagen). RNA was prepared by in vitro transcription using the T7 RiboMAX ™ Express RNAi System (promega). Each fragment of dsRNA was injected at a rate of 0.7 xq / \ xl in both gonads of 12 gravid adults. The eggs laid in the period from 2 to 17 hours after the injection were separated and their development was examined after the incubation days at a temperature of 20 ° C. c) Results The deactivation effect of sym-1 and sym- 5 by RNAi was determined by feeding bacteria that express dsRNA to larvae in first stage (Ll) wild-type. LL larvae growing on bacteria that produce sym-1 dsRNA became all healthy adults within 3 days. Ll larvae that grew in bacteria that produce sym-5 dsRNA became all adult but approximately 30% of them presented a generally ill appearance. Nevertheless, almost all LL larvae that grew in the mixture of bacteria that produce sym-1 and sym-5 dsRNA presented a generally ill appearance in adult stage (see Figure 26).

To determine the effect of sym-1 and sym-5 on embryonic development, dsRNA was produced in vitro and injected into the gonad of healthy wild-type adults. When sym-1 dsRNA was injected alone, approximately 3% of the developing embryos died. When sym-5 dsRNA alone was injected, approximately 40% of developing embryos died. However, when sym-1 dsRNA and sym-5 dsRNA were mixed and when they were injected together, almost all the embryos died (see Figure 27). These results show that the co-deactivation of multiple genes with a mild phenotype per se can be beneficial to obtain a much stronger effect. Example 5: Induction of lethality by concatemers of sublethal targets This example describes the co-deactivation by RNAi of two genes by using a single construct containing fragments of each of the genes ("concatemer constructs"). a) Fragments of dsRNA The fragments sym-1 and sym-1, have a size within a range of 146 to 186 base pairs, and are subfragments of those used in example 4. These smaller fragments are used either separately either they are mixed or they are used in concatemers in the same RNA molecule. Since the concatemers have approximately twice the length of the individual fragments, the individual fragments are compensated for size by concatemerization with a 152-base pair fragment or 153 base pairs of the unrelated unc-22 gene. The following sequences were used in this example: Fragment Number of Nucleotides Genbank access gene sequence see Figure 29) sym-1 (a) * Z79594 12515 - 12677 SEQ ID NO 44 sym-1 (b) Z79594 12309 - 12494 SEQ ID NO 45 ssyymm - 55 ((aa )) ZZ7799559988 8675-8828 SEQ ID NO 46 sym-5 (b) Z79598 8514-661 SEQ ID NO 47 9072-9223 SEQ ID NO 48 unc-22 (a) NM_69872 (supplementary) 8609 - 8761 SEQ ID NO 49 unc 22 (b) NM_69872 (complementary) (* in fragment sym-1 (a), an "A" may be present in place of "T" in position 12630.) b) Methods Fragments were amplified with chain reaction of polymerase using primers with extensions of restriction sites and sequentially cloned in the Multiple Cloning Site of a plasmid cloning vector. DsRNA was prepared and injected in accordance with that described in Example 3.1 using primers with extensions of T7 promoters.

C) Results The fragments of sym-1 (Figure 28 A and B) and two fragments sym-5 (Figure 28 C and D) within a range of 146 to 186 base pairs did not induce a substantial embryonic lethality when they were injected separately. . Injection of a mixture of the sym-5 (b) fragments with any of the sym-1 fragments induced a substantial embryonic lethality, showing that the fragments used are active and confirming that the co-deactivation of multiple genes with a mild phenotype can induce a much stronger effect (Figure 28 E and F). Constructs of concatemers were made between the two fragments sym-1 and the two sym-5 (Figure 28 G, H, I and J) and tested in the same way. All four possible combinations induced embryonic lethality as concatemer, and the penetration was even stronger than in the case in which two dsRNA molecules were mixed (Figure 28 E and F). These results show that concatamer dsRNA molecules are effective in co-deactivating multiple genes. Example 6: In vitro tests for efficient uptake of dsRNA by plant parasitic nematodes and subsequent gene silencing The dsRNA constructs according to the present invention (for example the construct having a sequence represented by SEQ ID NO: 51) were cloned behind the T7 promoter both in the sense direction and in the antisense direction and were transcribed in vitro using the T7 Ribomax Express RNAi protocol (Promega). DsRNA was produced by mixing sense RNA and antisense RNA. These dsRNAs were used in the in vitro tests described below. With these in vitro tests, the performance of the constructs in accordance with the present invention was evaluated for efficient absorption, stability in the pest organism and efficiency in the silencing of the target gene. An in vitro liquid consumption test for C. elegans was used following the "soaking" protocol in accordance with that described by Tabara et al. (Science, 1998, 282 (5388): 430-431). An in vitro liquid consumption test for Meloidogyne incognita was carried out in accordance with that described in example 1 and is based on forced feeding. An in vitro assay for fungal dsRNA uptake was carried out in the following manner. The rice blasto fungus Magnaporthe grisea was "soaked" in medium containing double-stranded RNA (dsRNA) that targets the white fungal gene. More particularly, conidia (asexual spores) were generated by exposure of fungal micelles to light for 7-10 days. Conidia were harvested and resuspended in water at a density of 20,000 conidia / ml, and inoculated into 96 well plates (50 μg) hydrophilic or on the hydrophilic surface of an artificial membrane (20 μl). DsRNA transcribed in vitro was added in accordance with that described above to the spores at a final concentration within a range of 0.01-10 micrograms / ml in sterile water. After 16-30 hours of incubation at 28 ° C, the growth of the micelles in the wells was quantified by reading the optical density of the 96-well plates. The growth and phenotype of the micelles in the artificial membrane were also observed under a microscope. The germination of conidia on a hydrophilic surface mimics its germination within the leaf during the invasive growth of the fungus. Insect feeding test, such as the grasshopper Nilaparvata lugens and the Colorado potato beetle. { Leptinotarsa decimlineata), were based on artificial diet technique. This technique has been previously described by Couty A, Down RE, Gatehouse AM, Kaiser L, Pham-Delegue M and Poppy G in J Insect Physiol. 2001 Dec; 47 (12): 1357-1366) "Effects of artificial diet containing GNA and GNA-expressing potatoes on the development of the aphid parasitoid Aphidius ervi Haliday (Hymenopter: Aphidiidae)". That document is incorporated herein by reference. Example 7: In plant testing of dsRNA stability and efficient pest control The constructs of the present invention, ie, comprising SEQ ID NO: 51, were cloned back from the CaMV35S promoter, a root specific promoter or a promoter specific for feeding site (like tobRB7), present in a binary vector suitable for plant transformation. The binary vectors were transferred to Agrobacterium rhizogenes through triple parental coupling (for example, by E. coli HB101 containing the auxiliary plasmid pRK2013). The binary vectors were transferred from Esherichia coli to Agrobacterium turnefaciens. Subsequently, harvest plants (such as tomato, soybean, cotton, arabidopsis, rice, corn, potato or tobacco) were transformed with the constructs through Agrobacterium-mediated transformation technique well described in the art, for example described in " Transgenic Plants, Methods and Protocols, Methods in Molecular Biology, Volume 286, by Peña, Leandro "). As a negative control, Agrobacterium without binary vector was also used to transform the plants. Stability of the dsRNA constructs of the present invention in plant cells The stability of the dsRNA constructs expressed in accordance with the present invention was analyzed by quantitative real-time PCR polymerase chain reaction based on Taqman probes or by intercalating dyes (green SYBR), in accordance with the previously described.

The expressed dsRNA constructs were quantified in relation to a series of standard dilutions of the annealing. The results were normalized by using quantitative polymerase chain reaction data from a set of protective genes from the same samples (Vandesompele et al., Genome Biology 2002, 3: research003 .1-003 .11). The amount of the dsRNA constructs according to the present invention was compared to the present invention was compared to the amount of control dsRNA that does not comprise a block. Alternatively, the stability and shape of the dsRNA can be analyzed by Northern blot. Root transformation in tomato or cotton or potato hair The constructs of the present invention were introduced into cotyledons of tomato (for example Lycopersicum esculentum cv. Marmande), or tobacco or cotton (Gossypium hirsutum) through transformation with A rhizogenes. The transformed hair roots were subsequently tested for resistance to nematodes. The necessary number of independent transformed lines (for example 15) and replicas per line (for example 10) were inoculated with J2 larvae of Meloidogyne incognita. The phenotypic effects on gill formation and egg mass were measured and scored. Egg masses were laid to hatch and the parasite's fecundity was investigated. The pups were used to test the capacity of infection / viability of the second generation. An analogous test was carried out through which the hair roots were transformed with the dsRNA construct against a fungal white gene sequence and through which the roots in the hair were inoculated with a fungus. Transformation of whole plant Plant tissues (such as tomato tissue, for example) were transformed with A. tumefaciens with the constructs of the present invention and regenerated into whole plants. Whole transgenic plants were inoculated with the pest species and the phenotype of the plant and the inoculated pest species was monitored. Equivalents Those skilled in the art will recognize or be able to determine using a routine experiment many equivalents to the specific embodiments of the invention described herein. Such equivalents are contemplated within the following claims. All references disclosed herein are incorporated by reference in their entirety.

Claims (51)

1. CLAIMS 1. A double-stranded ribonucleic acid (dsRNA) isolated effective in gene silencing by RNAi, wherein the dsRNA comprises multiple fragments of dsRNA, each fragment comprises complementary fused strands, one of which is complementary to at least a part of the nucleotide sequence of a white sequence.
2. 2. An isolated dsRNA according to claim 1 wherein said multiple dsRNA fragments are not separated by a non-complementary region.
3. 3. An isolated dsRNA according to claim 1 or 2, wherein said dsRNA comprises (i) at least one repeat of a dsRNA fragment or (ii) at least one repeat of a series of dsRNA fragments.
4. 4. An isolated dsRNA according to any of claims 1 to 3, wherein said dsRNA comprises (i) at least two copies of a dsRNA fragment or (ii) at least two copies of a series of dsRNA fragments.
5. 5. An isolated dsRNA according to any of claims 1 to 4, wherein said multiple fragments of dsRNA are (i) repeats of a dsRNA fragment or (ii) repeats of a series of dsRNA fragments.
6. 6. An isolated dsRNA according to any of claims 3 to 5, further comprising at least one dsRNA fragment that is distinct from the other repeated fragments.
7. 7. An isolated dsRNA in accordance with the claims I, 2 or 6, comprising at least two fragments of dsRNA wherein each fragment of dsRNA comprises a chain that is complementary to at least a part of the nucleotide sequence of a different target sequence.
8. 8. An isolated dsRNA according to claim 7, wherein said different target sequences originate from a single target gene.
9. 9. An isolated dsRNA according to claim 7, wherein said different target sequences originate from different target genes.
10. 10. An isolated dsRNA in accordance with the claims 9. wherein said different white genes originate from a single white species.
11. II. An isolated dsRNA according to claim 9, wherein said different white genes originate from different target species.
12. 12. An isolated dsRNA according to claim 11, wherein said different white species belong to the same genus, family or order.
13. 13. An isolated dsRNA in accordance with the claims 11, wherein said different white species belong to a different genus, family, order or edge.
14. 14. An isolated RNA construct comprising a dsRNA according to any of claims 1 to 3, wherein the RNA construct comprises at least one sequence that protects the dsRNA against RNA processing.
15. 15. An isolated RNA construct comprising at least one fragment of dsRNA, wherein the dsRNA comprises fused complementary strands, one of which is complementary to at least a part of the nucleotide sequence of a target sequence, said RNA construct it further comprises at least one sequence that protects the dsRNA against RNA processing.
16. 16. An RNA construct isolated according to claim 14 or 15, wherein said at least one sequence that protects the RNA against RNA processing is selected from a GC-rich coupling, a non-complementary short loop of between 4 and 100 nucleotides, a blockage of mismatch and a protein binding RNA structure.
17. 17. An isolated RNA construct according to claim 14 or 15, wherein said at least one sequence that protects the dsRNA against RNA processing is selected from the internal ribosomal entry sites (IRESes) of the encephalomyocarditis virus ( EMCV) and upstream of N-ras (UNR).
18. 18. An RNA construct isolated according to any of claims 14 to 17, further comprising at least one linker.
19. 19. An isolated RNA construct according to claim 18, wherein said linker is selected from a conditionally self-isolating RNA sequence, such as a pH responsive linker or a hydrophobic sensitive linker, and an intron.
20. 20. An isolated dsRNA according to any one of claims 1 to 13 or RNA construct according to any of claims 14 to 19, wherein the target sequence or the target gene comes from an organism detrimental to plants.
21. 21. An isolated dsRNA according to any of claims 1 to 13 or RNA construct according to any of claims 14 to 20, wherein the portion of dsRNA has a length between about 80 base pairs and about 500 pairs of bases.
22. 22. An isolated nucleic acid (DNA deoxyribonucleic acid) encoding a dsRNA of any one of claims 1 to 13 or RNA construct of any of claims 14 to 21.
23. 23. A recombinant DNA construct comprising a nucleic acid of claim 22.
24. 24. A recombinant DNA construct according to claim 23, further comprising a regulatory sequence operably linked to said nucleic acid.
25. 25. A recombinant DNA construct according to claim 24, wherein said regulatory sequence is selected from the group containing constitutive promoters such as any promoter selected from the group comprising the CaMV35S promoter, bent CaMV35S promoter, ubiquitin promoter, promoter of actin, rubisco promoter, GOS2 promoter, escrofularia mosaic virus (FMV) 34S; or tissue-specific promoters such as for example any promoter selected from the group comprising root-specific promoters of genes encoding PsMTA class III chitinase, promoters specific for photosynthetic tissues, for example cabl and cab2 promoters, rbcS, gapA, gapB and proteins ST-LS1, JAS promoters, chalcone synthase promoter and RJ39 promoter of the strawberry.
26. 26. A recombinant DNA construct according to claim 23, wherein said nucleic acid is cloned between two regulatory sequences that are in the opposite direction relative to each other, said regulatory sequences operably linked to said nucleic acid and said independently selected regulatory sequences. within the group comprising RNA Poli, RNA PoIII, RNA PoIIII, T7 RNA polymerase or SP6 RNA polymerase.
27. 27. A host cell comprising a dsRNA of any one of claims 1 to 13, or an RNA construct of any of claims 14 to 21, a nucleic acid of claim 22, or a DNA construct of any of the claims 23 to 26.
28. 28. A host cell according to claim 27, which is selected from a bacterial cell, a yeast cell and a plant cell.
29. 29. A transgenic plant, reproductive or propagating material for a transgenic plant comprising a plant cell of claim 28.
30. 30. A plant further comprising a dsRNA of any of claims 1 to 13, at least one RNA construct of any of claims 14 to 21, at least one nucleic acid of claim 22 or at least one recombinant DNA construct of any of claims 23 to 26.
31. 31. A seed comprising at least nucleic acid of claim 22 or minus a recombinant DNA construct of any of claims 23 to 26.
32. 32. A method for the production of a transgenic cell or transgenic organism, comprising the step of administering a recombinant DNA-to-DNA construct of any one of claims 23 to 26 to said cell or organism.
33. 33. A method according to claim 32, wherein said cell is a plant cell and wherein said organism is a plant.
34. 34. A transgenic cell or a transgenic organism obtainable by a method according to claim 32 or 33.
35. 35. A transgenic cell or a transgenic organism according to claim 34, which is a plant cell or a plant .
36. 36. A composition comprising at least one dsRNA of any one of claims 1 to 13 or an RNA construct of any of claims 14 to 21 and a physiologically or agronomically acceptable excipient.
37. 37. A composition comprising at least one nucleic acid of claim 22 or a recombinant DNA construct of any of claims 23 to 26, and a physiologically or agronomically acceptable excipient.
38. 38. The use of a composition according to claim 36, in the form of a pesticide for a plant or for propagating or reproductive material of a plant.
39. 39. A method for the treatment and / or prevention of the growth of pests and / or infestation by pests of a plant or propagating or reproductive material of a plant, comprising the application of an effective amount of a double-stranded RNA of either of claims 1 to 13, an RNA construct of any of claims 14 to 21, or a composition according to claim 36 to a plant or propagating or reproductive material of a plant.
40. 40. A method for the treatment and / or prevention of pest infestation in a substrate, comprising the application of an effective amount of a double-stranded RNA of any of claims 1 to 13, an RNA construct of any of claims 14 to 21, or a composition according to claim 36 to said substrate.
41. 41. A method for controlling the growth of pests in a cell or in an organism or for preventing the infestation by pests of a cell or an organism susceptible to be infected by said pest species, comprising contacting said species of pests with a double-stranded RNA of any of claims 1 to 13 or a dsRNA construct of any of claims 14 to 21, or what the double-stranded RNA is absorbed by said species of pests and accordingly controls the growth of pests or prevents infestation by pests.
42. 42. A method for downregulating the expression of at least one target gene in a pest species, comprising contacting said pest species with a double-stranded RNA of any one of claims 1 to 13 or a RNA construct of any of claims 14 to 21, whereby the double-stranded RNA or RNA construct is absorbed by the pest species and therefore the expression of the target gene of the pest or white genes of the plague.
43. 43. A method according to claim 41 or 42, wherein said double-stranded RNA or RNA construct is expressed by a prokaryotic or eukaryotic host cell or by a host organism.
44. 44. A method according to claim 43, wherein said double-stranded RNA is expressed by said cell or organism infested by said pest species or susceptible to being infested by said pest species.
45. 45. A method according to claim 44, wherein said cell is a plant cell or wherein said organism is a plant.
46. 46. A method according to any of claims 39 to 45, wherein said double-stranded RNA or RNA construct is expressed from at least one recombinant DNA construct of any of claims 23 to 26.
47. 47. A method according to any of claims 39 to 45, wherein said double-stranded RNA or RNA construct is expressed from two recombinant DNA constructs of any of claims 23 to 26.
48. 48. A method for the production of a plant resistant to a pathogenic pest for the plant, comprising: a) transforming a plant cell with a recombinant DNA construct of any of claims 23 to 26, b) regenerating a plant from the transformed plant cell; and c) culturing the transformed plant under conditions suitable for the expression of the recombinant DNA construct, said cultured transformed plant therefore becomes resistant to said pest compared to an untransformed plant.
49. 49. A method for increasing the yield of a plant, comprising introducing into the plant at least one nucleic acid of claim 22 or a recombinant DNA construct of any of claims 23 to 26, in a format that can be voiced.
50. 50. The use of a double-stranded RNA of any of claims 1 to 13, an RNA construct of any of claims 14 to 21, or a nucleotide sequence of claim 22, or a recombinant DNA construct of either of claims 23 to 26, or a cell according to claim 27 or 28, or a composition according to claim 36 or 37 for the treatment of a pest infection or infestation of the plants.
51. 51. A kit comprising a double-stranded RNA of any of claims 1 to 13, an RNA construct of any of claims 14 to 21, or a nucleotide sequence of claim 22, or a recombinant DNA construct of any of claims 23 to 26, or a cell according to claim 27 or 28, or a composition according to claim 36 or 37 and instructions for the use of said double-stranded RNA, RNA construct, nucleotide sequence, DNA recombinant, cell or composition for the treatment of plant infections by pests. SUMMARY OF THE INVENTION The present invention relates to stabilized concatemer and / or RNA constructs capable of forming dsRNA, optionally comprising a sequence capable of protecting the dsRNA against RNA processing in a host cell. The invention also relates to methods for the production of these constructs and methods for using these constructs. The constructs according to the present invention are particularly useful in the control of pests affecting plants.