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EP1805308A2 - Arn à domaines multiples comprenant au moins un aptamère pour délivrer arn à double brin dans un organisme parasite - Google Patents

Arn à domaines multiples comprenant au moins un aptamère pour délivrer arn à double brin dans un organisme parasite

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Publication number
EP1805308A2
EP1805308A2 EP05801497A EP05801497A EP1805308A2 EP 1805308 A2 EP1805308 A2 EP 1805308A2 EP 05801497 A EP05801497 A EP 05801497A EP 05801497 A EP05801497 A EP 05801497A EP 1805308 A2 EP1805308 A2 EP 1805308A2
Authority
EP
European Patent Office
Prior art keywords
plant
rna molecule
multidomain
pest
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05801497A
Other languages
German (de)
English (en)
Inventor
Thierry André Olivier Eddy BOGAERT
Pascale Feldmann
Geert Plaetinck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Devgen NV
Original Assignee
Devgen NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Devgen NV filed Critical Devgen NV
Priority to EP05801497A priority Critical patent/EP1805308A2/fr
Publication of EP1805308A2 publication Critical patent/EP1805308A2/fr
Withdrawn legal-status Critical Current

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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/16—Aptamers
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
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    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to RNAi and its use in gene silencing. Furthermore, the present invention relates to methods and constructs for delivering double stranded RNA (dsRNA) to pest organisms.
  • dsRNA double stranded RNA
  • RNAi double-stranded RNA inhibition
  • Double-stranded RNA inhibition is based on the introduction of RNA into a living cell to inhibit gene expression of a target gene in that cell.
  • the RNA has a region with double-stranded structure.
  • Double-stranded RNA (dsRNA) has the capability to render genes nonfunctional in a sequence-specific manner. When introduced into cells, dsRNA can activate mechanisms that target the degradation of cognate cytoplasmic mRNAs and thus can effectively silence full gene expression at the posttranscriptional level.
  • RNAi has been observed in many cell types from divergent eukaryotes, including protozoa, fungi, plants, invertebrates, and mammals. Once inside the cells, long dsRNA molecules are cleaved into double-stranded small interfering RNAs (siRNAs) that are 21-25 base pairs in length by an enzyme with RNase Ill-like activity (Dicer). Cleavage into siRNAs is an early step in the RNAi silencing mechanism. Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types.
  • siRNAs double-stranded small interfering RNAs
  • this technology may be used for instance with the aim of modifying or improving plant resistance towards pathogens and pests.
  • the latter technique may involve the uptake of the dsRNA by pest organisms when feeding on the plants.
  • dsRNA may be distributed to cells from the gut of the feeding organism in the same manner as nutrients. It is also conceivable that dsRNA residing in "infected” cells could undergo successive rounds of cellular exit and re-entry into adjacent "uninfected" cells.
  • dsRNA delivery of dsRNA to pest organisms by feeding has limits. Difficulties related to the delivery of dsRNA to feeding target organisms are numerous and may for instance involve the need to use very high amounts of dsRNA in order to be effective. Also, dsRNA may easily break down in the plants or during delivery to the target organism. Furthermore, in order to be effective, the dsRNA molecules should efficiently be taken up by the pest and delivered to the correct targeting site in the pest organisms. Since the advent of double-stranded RNA inhibition there has been recognized a need for specialized constructs designed for site-directed delivery of double-stranded RNA in a pest organism.
  • the present invention aims to provide improved methods and constructs useful in the delivery of double-stranded RNA in pest organisms, including nematodes, insects and fungi.
  • An object of the present invention is thus to provide dsRNA constructs with improved properties to be effectively taken up in the cells or tissues of the pest species.
  • the present invention provides new cellular delivery molecules for facilitating the delivery of a double-stranded ribonucleic acid molecule to a pest organism, as well as various uses of these molecules.
  • the present invention relates to a multidomain RNA molecule consisting of a (ribo)nucleotide sequence comprising at least one aptamer, and
  • double-stranded RNA comprising annealed complementary strands, one of which comprises a nucleotide sequence which is complementary to at least part of a pest target nucleotide sequence.
  • Double-stranded RNA molecules with homology to specific genes have been shown to cause silencing of said specific target genes through RNA interference.
  • RNA aptamer sequence(s) linked to dsRNA molecules capable of triggering gene silencing of specific target genes.
  • An aptamer sequence linked to a dsRNA molecule is herein also referred to as a chimeric aptamer-dsRNA molecule.
  • Such chimeric aptamer-dsRNA molecules can be made in vitro through chemical synthesis or by in vitro transcription from a DNA template or can be expressed in vivo from transgenic DNA or RNA.
  • Such chimeric aptamer-dsRNA molecules are more effective for silencing purposes than the conventionally known constructs because the aptamer part may:
  • gut cell proteins such as toxin-receptors, proteins inducing Bacillus thuringiensis (Bt) resistance in nematodes and insects (e.g. Cry proteins, glycosyltransferase proteins, Drosophila-EGGHEAD and BRAINIAC), etc) and as such improve the internalization of the dsRNA into the cells lining the pest gut or alimentary tract,
  • enterocyte surface molecules such as N-acetyl-D- galactosamine (GalNac) and mannose, invertebrate specific glycolipids, etc
  • RNA molecules of the present invention provide a powerful tool for various agronomic and research applications requiring the delivery of dsRNA into a target pest organism.
  • the present invention further provides various methods of using a multidomain RNA molecule described herein, including methods of facilitating delivery of a double-stranded ribonucleic acid molecule to a pest organism and methods for down-regulating expression of a target gene in a pest species.
  • the present invention relates to a method for delivering a double-stranded RNA molecule to a pest species, comprising: expressing in a plant cell or plant at least one multidomain RNA molecule according to the present invention, and feeding said plant cell or plant to said pest species.
  • the present invention relates to a method for delivering a double- stranded RNA molecule to a pest species, comprising: expressing in a plant cell or plant at least two multidomain RNA molecules according to the present invention, and wherein each multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single- stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide seq uence of the target gene, and feeding said plant cell or plant to said pest species.
  • the invention relates to a method for down-regulating expression of a target gene in a pest species comprising: expressing in a plant cell or plant at least one multidomain RNA molecule according to the present invention, and
  • the invention provides a method for down-regulating expression of a target gene in a pest species, comprising:
  • each multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single- stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, and
  • the present invention also relates to the use of a mult ⁇ domain RNA molecule according to the present invention for down-regulating expression of a target gene in a pest species.
  • the present invention further relates to a nucleic acid encoding a multidomain RNA molecule of the invention and to a vector comprising said n ucleic acid.
  • the present invention provides a host cell which comprises a nucleic acid encoding a multidomain RNA molecule of the invention or a vector comprising said nucleic acid.
  • the present invention relates to a composition or kit comp rising at least one multidomain RNA molecule as defined herein, and optionally further comprising at least one excipient, e.g. suitable for keeping the composition in a stabile condition.
  • the invention relates to methods for producing a transgenic plant that is resistant to a pest species, comprising:
  • the invention also relates to the use of a multidomain RNA molecule according to the present invention for producing a transgenic plant, in particular for producing a transgenic plant that is resistant to a pest species. More specifically the multidomain RfMA molecules of the invention can be used to generate pest resistant crops by making transgenic plants that express such chimeric aptamer-dsRNA molecules, directed against pest target gene transcripts.
  • the present invention further relates in another aspect to a transgenic plant resistant to a pest species, an essential derived variety thereof, plant part, plant cell or protoplast thereof obtainable by any of the methods according to the present invention.
  • the invention also provides a transgenic plant, an essentially derived variety thereof, plant part, plant cell or protoplast thereof which comprises a nucleic acid encoding a multidomain RNA molecule as defined herein, wherein said nucleic acid is heterologous to the genome of said transgenic plant, or an essentially derived variety thereof, plant part, plant cell or plant protoplast thereof.
  • the invention also provides a transgenic plant which comprises a vector comprising a nucleic acid encoding a multidomain RNA molecule according to the invention.
  • the invention further relates to a plant, an essentially derived variety thereof, plant part, plant cell or protoplast thereof that has been transformed with a nucleic acid encoding a multidomain RNA molecule as defined herein.
  • the invention in another aspect, relates to a plant, an essentially derived variety thereof, plant part, plant cell or protoplast thereof which expresses a (ribo)nucleotide sequence comprising at least one aptamer, and - at least one nucleotide sequence of interest forming double-stranded RNA, said double-stranded RNA comprising annealed complementary strands, one of which comprises a nucleotide sequence which is complementary to at least part of a pest target nucleotide sequence.
  • a plant an essentially derived variety thereof, plant part, plant cell or protoplast thereof which expresses a (ribo)nucleotide sequence comprising at least one aptamer, and - at least one nucleotide sequence of interest forming double-stranded RNA, said double-stranded RNA comprising annealed complementary strands, one of which comprises a nucleotide sequence which is complementary to at least part of a pest target nucleotide sequence.
  • the present invention relates to progeny or parts or derivatives of plants obtained from a plant or an essentially derived variety thereof accordi ng to the present invention.
  • Figure 1 is a schematic representation of DNA templates to generate multidomain RNA molecules according to the present invention.
  • 1 & 2 refer to DNA templates to generate aptamer-dsRNA complexes: 1 refers to DNA templates for generating a single transcript containing dsRNA by intramolecular base-pairing, while 2 refers to DNA templates for generating separate sense and antisense transcripts that can anneal to create dsRNA after transcription; P refers to a promoter (eukaryotic promoter, prokaryotic promoter, promoter for in vitro transcription i.e.
  • a promoter eukaryotic promoter, prokaryotic promoter, promoter for in vitro transcription i.e.
  • I, Il and III refer to regions that can contain synthetic aptamer sequences
  • S refers to a fragment of the target gene in sense orientation and AS to a fragment of the target gene in antisense orientation
  • a to F refer to single aptamer-dsRNA complexes, wherein A is a transcript with aptamer moiety at the 5' end, B is a transcript with aptamer moiety at the 3' end, C is a transcript with aptamer moiety within the loop structure of the dsRNA hairpin, D, E and F are transcripts containing different aptamers
  • G to I refer to aptamer-dsRNA complexes derived from separate sense and antisense transcripts, wherein G is a complex with an aptamer at the 5' end of one of the transcripts, H is a complex with an aptamer at the 3' end of one of the transcripts and I is an example of an aptamer-dsRNA complex containing different aptamer sequences.
  • Figure 2 represents DNA templates to generate different multidomain RNA molecules according to the present invention comprising nhx-2 - sup-35 aptamer-dsRNA fusions and controls.
  • P refers to a promoter (eukaryotic promoter, prokaryotic promoter, promoter for in vitro transcription i.e.
  • the white boxes refer to regions that contain a synthetic aptamer sequence against nhx-2 5 th extracellular loop; S-sup-35 refers to a fragment of sup-35 in sense orientation while AS-sup-35 refers to a fragment of sup-35 in antisense orientation; A refers to a transcript with aptamer moiety at the 5' end; B refers to a transcript with aptamer moiety at the 3' end; and C refers to a transcript with no aptamer moiety.
  • Figure 3 illustrates the survival of pha-1 mutant, at the restrictive temperature, upon sup- 35 RNAi.
  • Figure 4 illustrates the nucleic acid sequence of the Nhx-2 gene (protein sequence: NP_495614, nucleotide sequence: NM_063213 (bold and underlined: the fifth extracellular loop of the nhx-2 protein) (SEQ ID NO 1).
  • Figure 5 illustrates the sup-35 DNA sequence coding for the dsRNA sup-35 fragment (SEQ ID NO 2).
  • Figures 6 to 9 illustrate several examples of target sequences causing interference with a tubulin gene of specific pest organisms (SEQ ID NOs 3 to 6).
  • FIG. 10 illustrates a specific embodiment of the invention. Detailed description of the invention
  • the present invention relates to a multidomain RNA molecule consisting of a (ribo)nucleotide sequence comprising - at least one aptamer, and at least one nucleotide sequence of interest wherein the nucleotide sequence of interest is capable of forming double-stranded RNA, said double-stranded RNA comprising annealed complementary strands, one of which comprises a nucleotide sequence which corresponds (or is complementary) to a pest target nucleotide sequence.
  • an aptamer may be covalently bound to a double-stranded (ribo)nucleic acid molecule to form a multidomain RNA molecule.
  • ribo double-stranded
  • Such multidomain RNA molecule greatly facilitates uptake efficiency and allows for the efficient in vivo delivery of dsRNA into cells or tissues of pest organisms and thereby enhancing the potency of the dsRNA and the speed to kill the pest species.
  • RNA interference While the present invention is primarily directed to the delivery of a double-stranded ribonucleic acid molecule into a pest organism for the purposes of RNA interference, the multidomain RNA molecules described herein may also be used to facilitate the delivery of other non-coding RNAs, such as small temporal RNAs, small nuclear RNAs, small nucleolar RNAs or microRNAs, which may be used in applications other than RNA interference.
  • non-coding RNAs such as small temporal RNAs, small nuclear RNAs, small nucleolar RNAs or microRNAs, which may be used in applications other than RNA interference.
  • A. Multidomain RNA molecule consisting of a nucleotide sequence comprising at least one aptamer, and at least one nucleotide sequence of interest (capable of) forming double-stranded
  • RNA said double-stranded RNA comprising annealed complementary strands, one of which comprises a nucleotide sequence which is complementary to at least part of a pest target nucleotide sequence.
  • At least one in the context of the present invention means at least two, at least three, at least four, at least five, at least six, etc. and up to at least 10 or at least 15.
  • aptamer or “aptamer sequence”, or “aptamer domain” are used herein as synonym and are well known to a person of skill in the art. These terms refer to synthetic nucleic acid ligands capable of specifically binding a wide variety of target molecules, such as proteins or metabolites. As used herein aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. In a preferred embodiment, the aptamer specifically binds to a structure in the plant tissue or to a structure in the pest species.
  • aptamer (sequence) linked to a dsRNA molecule is herein also referred to as a "chimeric aptamer-dsRNA molecule", or as an “aptamer-dsRNA construct", or as “an aptamer-dsRNA complex", or as an “aptamer-dsRNA fusion”.
  • Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Analogous to Antibody-Antigen interactions aptamers bind to their corresponding antigens with high affinity and selectivity. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds and even entire organisms. Aptamers have specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
  • a typical aptamer is 10-15 kDa in s ⁇ z.e (30- 45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., will typically not bind other proteins from the same gene family).
  • a series of structural studies have shown that aptamers are capable of using the same types of binding interactions (hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion, etc.) that drive affinity and specificity in antibody- antigen complexes.
  • Aptamers are generally produced through an in vitro evolutionary process called "systematic evolution of ligands by exponential enrichment" (SELEX, DS Wilson and JW Szostak Annu. Rev. Biochem . 1999, 68:611-647).
  • the method is an iterative process based on selection and amplification of the anticipated tight binding aptamer.
  • the start library for selection of aptamers contains single stranded DNA oligonucleotides with a central region of randomized sequences (up to 10 15 different sequences) which are flanked by constant regions for subsequent transcription, reverse transcription and DNA amplification.
  • the start library is amplified by PCR and transcribed to an RNA start pool by T7 transcription.
  • Target specific RNA is selected from the pool by allowing the pool to interact with the target molecule, only tight binding RNA molecules with high affinity are removed from the reaction cycle, the tight binding RNA molecules are reverse transcribed to cDNA and amplified to double stranded DNA by PCR. These enriched binding sequences are transcribed back to RNA which is the source for the next selection and amplification cycle. Such selection cycles are usually repeated 5-12 times in order to obtain only sequences with highest binding affinities against the target molecule.
  • the invention provides constructs comprising aptamers that target the dsRNA to a high affinity binding site in the pest species. These can be localized on gut epithelial cells of feeding pests, on other cells in the body of the feeding pest or even on interacting cell surfaces of for instance fungi that feed on plant tissue.
  • the present invention relates to the use of multidomain RNA molecules consisting of at least one aptamer that enhances endocytosis in a pest gut cell or an aptamer that enhances binding to the gut or fungal cell surface, and at least one target sequence that will down-regulate a gene of the pest species.
  • a multidomain RNA molecule is constructed consisting of: at least one subunit or aptamer domain (sequence) that is capable of binding a protein or sugar moiety that is endocytosed into a pest cell, e.g. an enterocyte, or that is capable of binding to the endocytosis receptor of such pest cell , e.g.
  • the aptamer is produced by standard techniques (SELEX).
  • the multidomain RNA is obtained from the transcription of a synthetic gene that is generated in vitro, using standard molecular biology techniques.
  • the expression pattern of the transgene is either ubiquitous or localized to a region of the pest feeding site.
  • the choice of promoter for the expression of the transgene depends on the feeding behavior of the pest species targeted.
  • the transgenic RNA is expressed preferably in root tissues when the target species feeds exclusively on the root.
  • the pest ingests the multidomain RMA molecule.
  • the aptamer domain (sequence) of the molecule leads to the fast targeting of the molecule to for instance the gut cell membrane.
  • RNAi RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi ⁇ RNAi .
  • a plant or a surface or substance susceptible to pest infestation may be sprayed with a composition (or the like) comprising the multidomain RNA molecules, thereby protecting the plant or the surface or substance against infestation from multip Ie pests.
  • the present invention relates to a multidomain RNA molecule as described above - wherein at least one aptamer binds to a protein or sugar moiety that is endocytosed or transcytosed by an enterocyte of a pest species, or
  • At least one aptamer binds to a protein or sugar moiety that is endocytosed into a cell of a pest species, or
  • At least one aptamer binds to an endocytosis receptor molecule e.g. an enterocyte endocytosis receptor molecule or to a transcytosis receptor molecule, or
  • aptamers binding to carbohydrates, such as mannose may promote passage into the haemolymph, thereby creating a novel and efficive approach for delivering dsRNA to pest species by feed ing with multidomain RNA molecules according to the invention.
  • the aptamer is capable of targeting the multidomain RNA molecule and thus the dsRNA to which it is fused to a targeting site in a living pest organism.
  • targeting site refers to a specific cell or tissue in a living pest organism, as defined herein, to which the multidomain RNA molecule according to the present invention is targeted.
  • Endocytosis is defined herein as the cellular uptake of macromolecules and particulate substances by localized regions of the plasma membrane that surround the substance and pinch off to form an intracellular vesicle.
  • Receptor-mediated endocytosis is an essential process in all eukaryotes, including invertebrates such as insects or nematodes, and is required for general cellular functions, including uptake of nutrients (e.g., low- density lipoprotein [LDL] or transferrin) and recycling of membranes and membrane proteins.
  • nutrients e.g., low- density lipoprotein [LDL] or transferrin
  • Transcytosis is defined herein as the process by which a molecule may enter through one side of a cell and then migrate across the cell to exit on the other side. Transcytosis refers to the transport of substances across an epithelium by uptake into and release from coated vesicles. Also “receptor-mediated transcytosis” is an essential process in eukaryotes.
  • the term "enterocyte” means terminally differentiated cells comprising the majority of the external surface of the intestinal epithelium.
  • the enterocyte is lining the gut or intestine or alimentary tract of a pest species.
  • the multidomain RNA molecule thus may comprise an aptamer which allows endocytosis into the gut cell of a pest organism, e.g. an enterocyte.
  • the aptamer allows (or promotes or enables) transcytosis from the lumen of the gut to the coelumic fluid or haemolymph of the pest organism.
  • the multidomain RNA molecule may comprise an aptamer which allows endocytosis into a tissue cell of the pest organism, such as for instance, but not limited to, a muscle cell, a gonade cell, a nerve cell.
  • an aptamer allows (or promotes or enables) transcytosis from an endothelial eel I lining an organ to the lumen of said organ of the pest organism.
  • the multidomain RNA molecule comprises at least two aptamers, for instance one aptamer which allows (or promotes or enables) transcytosis from the gut cell of a pest organism to the coelumic fluid or haemolymph of the pest organism, and another aptamer which allows (or promotes or enables) endocytosis into a tissue cell of the pest organism.
  • the multidomain RNA molecule comprises at least one aptamer which is recognized by a receptor on the gut cell of a pest organism, for instance an endocytosis receptor molecule or a transcytosis receptor molecule and which recognition triggers the endocytosis or transcytosis of the complex between the aptamer and the dsRNA.
  • a receptor on the gut cell of a pest organism for instance an endocytosis receptor molecule or a transcytosis receptor molecule and which recognition triggers the endocytosis or transcytosis of the complex between the aptamer and the dsRNA.
  • the invention relates to a multidomain RNA molecule as described above wherein at least one aptamer binds specifically and with high affinity to specific gut cell proteins such as but not limited to toxin-receptors, proteins inducing Bacillus thuringiensis (Bt) resistance in nematodes and insects (such as but not limited to Cry proteins, glycosyltransferase proteins, Drosophila-EGGHEAD and BRAINIAC, etc).
  • gut cell proteins such as but not limited to toxin-receptors, proteins inducing Bacillus thuringiensis (Bt) resistance in nematodes and insects (such as but not limited to Cry proteins, glycosyltransferase proteins, Drosophila-EGGHEAD and BRAINIAC, etc).
  • the invention relates to a multidomain RNA molecule as described above wherein at least one aptamer binds specifically and with high affinity to enterocyte surface molecules such as carbohydrates, for instance but not limited to N-acetyl-D-galactosamine (GalNac) and mannose, or invertebrate specific glycolipids, etc.
  • enterocyte surface molecules such as carbohydrates, for instance but not limited to N-acetyl-D-galactosamine (GalNac) and mannose, or invertebrate specific glycolipids, etc.
  • the invention provides constructs comprising aptamers that protect dsRNAfrom degradation in the plant.
  • a plant is transformed with a transgene consisting of (1) a dsRNA sequence targeting an mRNA in the pest and (2) an aptamer designed to bind specifically with high affinity to an endogenous plant or secreted pest protein.
  • the invention thus relates to a multidomain RNA molecule as described above wherein at least one aptamer binds specifically and with high affinity to an endogenous plant protein.
  • the aptamer is designed to target the aptamer-dsRNA complex to a protein involved in the trafficking of molecules to the chloroplast, the mitochondria, plastids or other cell organelles.
  • the aptamer-dsRNA molecule is likely protected against processing and/or degradation by cytosolic plant Dicer. As such, more and longer dsRNA fragments will remain in the pla nt tissues.
  • the invention also relates to a multidomain
  • RNA molecule as described above wherein at least one aptamer binds specifically and with high affinity to a secreted pest protein.
  • the aptamer binds to an enzyme or protein that is secreted by a fungal species, said enzyme or protein involved in any of the processes used by said fungal species for uptake of nutrients upon feeding.
  • the multidomain RNA molecule may simultaneously be taken up by the fungus through binding of its aptamer moiety to said secreted enzyme or protein (or to a part thereof, which is re-uptaken).
  • a plant may also be transformed with a transgene consisting of (1 ) a sequence targeting an mRNA in the pest and (2) an aptamer designed to bind with high affinity and to specifically inhibit an enzyme, such as a nuclease which is present in the plant or which is secreted in the pest gut.
  • an enzyme such as a nuclease which is present in the plant or which is secreted in the pest gut.
  • the concentration of unprocessed dsRNA in the gut of the pest is increased.
  • the amount of dsRNA available for endocytosis is increased, thus improving the RNAi effect.
  • the invention thus provides constructs comprising aptamers that bind to and/or inhibit a plant or pest enzyme involved in the processing and/or degradation of the dsRNA.
  • the invention th us also relates to a multidomain RNA molecule as described above wherein at least one aptamer binds and/or inhibits a plant enzyme involved in processing and/or degradation of dsRNA.
  • the invention thus also relates to a multidomain RNA molecule as described above wherein at least one aptamer binds andvOr inhibits a pest enzyme involved in processing and/or degradation of dsRNA.
  • the invention encompasses several types of aptamers, such as but not limited to aptamers: - that are capable to bind to a protein or sugar that is endocytosed or transcytosed by an enterocyte of a pest species, that are capable to bind to a protein or sugar that is endocytosed into a cell of a pest species,
  • enterocyte endocytosis receptor molecule capable to bind to an enterocyte endocytosis receptor molecule or an enterocyte transcytosis receptor molecule, that bind specifically and with high affinity to particular proteins present at the enterocyte surface such as, but not limited to toxin-receptors, proteins inducing Bacillus thuringiensis (Bt) resistance in nematodes and insects,
  • enterocye surface such as but not limited to N-acetyl-D-galactosamine (GalNac) and mannose, or invertebrate specific glycolipids, etc.
  • GalNac N-acetyl-D-galactosamine
  • mannose or invertebrate specific glycolipids, etc.
  • bind specifically and with high affinity to an endogenous plant protein that bind specifically and with high affinity to a secreted pest protein, that bind and/or inhibit a plant enzyme involved in processing and/or degradation of dsRNA, or that bind and/or inhibit a pest enzyme involved in processing and/or degradation of dsRNA.
  • the multidomain RNA molecule of the invention comprises at least two, preferably at least three, more preferably at least four aptamers chosen from the group of aptamers as defined above.
  • Double stranded DNA and Target genes Double stranded DNA and Target genes
  • a "target gene” as used herein means a gene that needs to be silenced in the target (pest) species.
  • the target gene may be selected from the genome of any species as described herein According to a preferred embodiment, the target sequence is chosen from the genome of an organism, which organism is different from the organism in which the dsRNA capable of causing interference is expressed. This means that the dsRNA is expressed in one (host) cell or organism and is subsequently transferred to (or taken up by) another cell or organism comprising the target gene.
  • the dsRNA is expressed in the plant or a plant cell and the target gene is chosen from the genome of a bacterium, a virus or an invertebrate, more particularly from a plant pest species such as a nematode, fungus or an insect.
  • the expression "dsRNA” relates to double stranded RNA capable of causing RNA interference.
  • the dsRNA is expressed in a bacterial or fungal cell and the bacterial or fungal cell is taken up or eaten by the pest species.
  • the dsRNA is isolated from, 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 pest species.
  • Particular suitable target genes are genes that are involved in an essential biological pathway of the target pest species, meaning that the target gene is an essential gene to the target pest species and that gene silencing of the target gene has an adverse effect on the viability the growth, development, feeding, movement, and/or reproduction of the target pest species.
  • Suitable target genes are genes associated with infection, propagation or pathogenesis of the pest species in the host.
  • the sequence of the target RNA in the pest organism comprises seq uences of genes which are essential for development, neural function, reproduction or digestion of the pest organism.
  • the multidomain RNA molecule consist of a s&quence comprising at least one nucleotide sequence of interest which is (capable of) forming double-stranded RNA.
  • any suitable double-stranded RNA fragment capable of directing RNAi or RNA-mediated gene silencing of a target gene can be used.
  • a "double-stranded ribonucleic acid molecule (dsRNA)" refers to any RNA molecule, fragment or segment containing two strands forming an RNA duplex, notwithstanding the presence of single stranded overhangs of unpaired nucleotides.
  • the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence of th e target gene to be down-regulated.
  • the other strand of the double-stranded RNA is complementary to this target nucleotide sequence.
  • the "dsRNA” or “double-stranded RNA”, whenever said expression relates to RNA that is capable of causing interference, may be formed from two or more separate polynucleotide strands which together form a double stranded, folded or assembled structure which includes at least one double-stranded portion effective in gene silencing by RNAi.
  • said dsRNA may be formed form two separate (sense and antisense) RNA strands that are annealed together.
  • the sense and antisense strands of the dsRNA originate form distinct multidomain RNA molecules.
  • the RNA molecules may, when folded or assembled, include both double-stranded and single-stranded regions.
  • one of the strands originates from a multidomain RNA molecule according to the present invention, while the other strand is part of another RNA molecule, expressed in the same or in another cell, and which RNA molecule may or may not comprise other domains or sequences which protect it from degradation or which direct it to specific locations.
  • the dsRNA may comprise other sequences that are not complementary to a target gene or sequence but that have other functions.
  • the dsRNA may be formed from a single RNA polynucleotide molecule which includes regions of self-complementarity, such that when folded it is capable of forming a structure including one or more double-stranded portions (also referred as "dsRNA fragment") effective in gene silencing by RNAi.
  • the dsRNA may have a foldback stem-loop or hairpin structure wherein the two annealed strands of the dsRNA are covalently linked.
  • the sense and antisense strands of the dsRNA are formed from different regions of a single chimeric RNAi molecule that is partially self- complementary. The organization of sense and antisense portions making up the double stranded RNA is variable.
  • RNAs having this structure are convenient if the dsRNA is to be synthesized by expression in vivo, for example in a host cell or organism as discussed below, or by in vitro transcription.
  • the precise nature and sequence of the "loop" linking the two RNA strands is generally not material to the invention, except that it should not impair the ability of the double-stranded part of the molecule to mediate RNJAi.
  • the features of "hairpin" or “stem-loop" RNAs for use in RNAi are generally known in the art (references: WO 99/53050) According to specific embodiments of the invention, however, the loop linking the two RNA strands may comprise an aptamer sequence.
  • a double-stranded ribonucleic acid molecule may further include single stranded RNA molecules forming functional stem-loop structures, such as small temporal RNAs, short hairpin RNAs and microRNAs, thereby forming the structural equivalent of an RNA duplex with single strand overhangs.
  • the multidomain RNA molecules of the present invention may be isolated, purified, native or recombinant, and may be modified by the addition, deletion, substitution and/or alteration of one or more nucleotides, including non-natural Iy occurring nucleotides, including those added at 5" and/or 3' ends to increase nuclease resistance.
  • the methods of the invention are to be used for controlling growth or infestation of a pest organism in a host, it is preferred that the dsRNA is not harmful for organisms other than the target organism(s), and consequently that the double-stranded RNA does not share any significant homology with any host gene, or at least with any essential gene of the host.
  • the double-stranded RNA shows less than 30%, more preferably less that 20%, more preferably less than 10%, and even more preferably less than 5% nucleic acid sequence identity with any gene of the host cell. % sequence identity should be calculated across the full length of the double-stranded RNA sequence capable of causing RNA interference. If genomic sequence data is available for the host organism then it is simple to cross-check sequence identity with the double-stranded RNA using standard bioinformatics tools .
  • the double-stranded RNA sequence capable of causing RNA interference does not have 20 contiguous nucleotides in common with a sequence of an organism other than the target organism.
  • the target organism is a plant pathogen, such as a plant parasitic nematode or an insect
  • the double-stranded RNlA does not have 20 contiguous nucleotides in common with a nucleotide sequence from a plant or a mammal (a human in particular).
  • the "target region” of the target pest gene may be any suitable region of the gene.
  • the target region should comprise at least 17 or 18 consecutive nucleotides of the target gene, more preferably at least 19, 20 or 21 nucleotide and still more preferably at least 22, 23, 24 or 25 nucleotides of the target gene.
  • complementarity as used herein relates to DNA-DNA and RNJA-RNA complementarity as well as to DMA-RNA complementarity.
  • RNA equivalent means that in a DNA sequence(s), the base “T” may be replaced by the corresponding base "U” normally present in ribonucleic acids.
  • nucleotide sequence which is complementary to means a sequence that is complementary to at least part of a nucleotide sequence of a target gene.
  • complementary when used in the context of the present invention for a dsRNA, means having substantial sequence identity to one of the strands of the target gene.
  • the complementary sequence will generally comprise a nucleotide sequence having more than about 75% sequence identity to the corresponding sequence of the target gene, however, a higher homology might produce a more efficient inhibition of expression of the target gene.
  • sequence identity is about 80%, 85%, 90%, 95%, and even more preferably more than about 99%.
  • the expression “more than about” has the same meaning as "at least”.
  • the double-stranded RNA will share 100% sequence identity with the target region of the target pest gene.
  • 100% sequence identity is not essential for functional RNA inhibition.
  • RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for RNA inhibition.
  • the term "corresponding to”, when used to refer to sequence correspondence between the double- stranded RNA and the target region of the target gene, is therefore to be interpreted accordingly as not absolutely requiring 100% sequence identity.
  • the dsRNA must contain a sequence which corresponds to the target region of the target gene (i.e. wherein one strand of the dsRNA is complementary to at least part of a target (e.g. pest) nucleotide sequence) it is not absolutely essential for the whole of the dsRNA to correspond to the sequence of the target region.
  • the dsRNA may contain short non-target regions flanking the target-specific sequence, provided that such sequences do not affect performance of the dsRNA in RNA inhibition to a material extent.
  • the multidomain RNA molecule of the invention comprises multiple dsRNA fragments, each fragment comprising annealed complementary strands, one of which is complementary to a least part of a target nucleotide sequence to be silenced.
  • Multiple dsRNA fragments as used in the present invention are also generally referred to as "concatemers”.
  • the present invention provides a multidomain RNA molecule consisting of a nucleotide sequence comprising:
  • multiple dsRNA fragments each comprising annealed complementary strands, one of which comprises a nucleotide sequence which is complementary.
  • multiple in the context of the present invention means 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.
  • Non-limiting examples of suitable concatemers for use in the present invention include concatemer cloverleaf, concatemer dumbbell, concatemer hairpin, concatemer stem dsRNA.
  • said multidomain RNA molecule comprises multiple dsRNA fragments that are complementary to different (e.g. distinct) sequences in one target gene. In another embodiment, said multidomain RNA molecule comprises multiple dsRNA fragments that are complementary to different (e.g. distinct) target genes. In yet another embodiment, said multidomain RNA molecule comprises at least one repeat of one dsRNA fragment. As used herein "one repeat" means two copies of the same dsRNA fragment. In yet another embodiment, said multidomain RNA molecule comprises at least two or three copies, preferably st least four, five or six copies, more preferably at least seven, eight, nine ten or more copies of one (e.g. the same) dsRNA fragment.
  • said multiple dsRNA fragments are repeats of a single dsRNA fragment.
  • the dsRNA fragments are not separated by non-hybridizing RNA fragments.
  • the dsRNA fragments are separated by a linker or spacer sequence.
  • the linker or spacer sequence is double stranded and the strands are complementary, thus also forming a double stranded region.
  • the li nker sequence may comprise a short random nucleotide sequence that is not complementary to target sequences.
  • the dsRNA fragments are not separated by a linker, a spacer or a lock sequence as described further.
  • the present invention encompasses multidomain RNA molecules comprising at least one aptamer and one dsRNA fragment comprised of annealed complementary strands, one of which has a nucleotide sequence which is complementary to a least part of a target nucleotide sequence of a pest target gene, and which comprises one or more additional dsRNA fragments, each comprised of annealed complementary strands, wherein at least one complementary strands of each dsRNA fragment, comprises each independently a nucleotide sequence which is complementary to
  • multidomain RNA molecule also encompasses multidomain RNA molecules comprising copies of one or more ds RNA fragments and further comprising other dsRNA fragments, that are different from the repeated or copied dsRNA fragments. Therefore, the invention also relates to multidomain RNA molecule comprising in addition to at least one aptamer, one or more repeats of dsRNA fragments and further comprising at least one dsRNA fragment which is distinct from the repeated fragment(s).
  • the length of each of the dsRNA fragments may be at least 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp or more, for example about 30 bp, about 40bp, about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110 bp or about 120 bp.
  • Preferred dsRNA fragments in a concatemer comprising multidomain molecule have a length between 17 and 300 bp, preferably between 21 and 250 bp, preferably between 40 and 150 bp, more preferably between 50 and 120 bp or any number in between.
  • the terms "another target gene” or "a further target gene” are use interchangeably and mean for instance a second, a third or a fourth, etc. target gene.
  • the dsRNA fragments target at least one target gene that is essential for viability, growth, development or reproduction of the pest and target at least one gene involved in pathogenicity or infectivity.
  • the dsRNA fragments target multiple genes of the same category, for example, the dsRNA fragments target at least two essential genes or at least two genes involved in pathogenicity or at least two genes involved in any of the cellular functions.
  • the dsRNA fragments target at least two target genes, which target genes are involved in a different cellular function.
  • the dsRNA fragments target two or more genes involved in protein synthesis (e.g. ribosome subunits), protein degradation (e.g. proteasome subunits), formation of microtubule cytoskeleton (e.g. beta-tubulin gene), and the like.
  • the dsRNA fragments in the multidomain RNA molecule may be combined as follows: a) when multiple dsRNA fragments targeting a single target gene are combined, they may be combined in the original order (i.e. the order in which the regions appear in the target gene) in the multidomain RNA molecule, b) alternatively, the original order of the fragments may be ignored so that they are scrambled and combined randomly or deliberately in any order into the multidomain RNA molecule, c) alternatively, one single fragment may be repeated several times, for example from 1 to 10 times, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, in the multidomain RNA molecule, or d) the dsRNA fragments (targeting a single or different target genes) may be com bined in the sense or antisense orientation.
  • the present invention thus encompasses a multidomain RNA molecule comprising at least one aptamer and multiple dsRNA fragments targeting different target genes which origi nates from a single target (or pest) species, or wherein said different target genes originates from distinct target (or pest) species, for instance pest species belonging to the same (in one embodiment) or to different (in other embodiments) genera, families, orders or even phyla.
  • the multidomain RNA molecules comprising such multiple dsRNA fragments and targeting multiple target genes, are characterized by stacking multiple RNAi capacity, resulting in synergetic effects, and capable of triggering multiple RNAi effects in the target cell or target organism. Protection against RNA processing
  • the dsRNA may further contain DNA bases, non-natural bases or non-natural backbone linkages or modifications of the sugar-phosphate backbone, for example to enhance stability during storage or enhance resistance to degradation by nucleases.
  • the double-stranded RNA fragment capable of causing interference will itself preferably be greater than 17 bp in length, preferably 19bp in length, more preferably greater than 20bp, more preferably greater than 21 bp, or greater than 22bp, or greater than 23bp, or greater than 24bp, or greater than 25bp in length.
  • moieties capable of protecting the double stranded RNA portion (causing RNA interference) against RNA. processing.
  • Chimeric RNA ⁇ constructs according to the present invention may thus comprise different dsRNA core types, optionally comprising linker types, optionally comprising different lock types designed to protect the dsRNA core against RNA processing in the host cell expressing the dsRNA construct.
  • a sequence capable of protecting the dsRNA against RNA processing is also referred to as a "lock".
  • a sequence capable of protecting the dsRNA against RNA processing is also referred to as a "lock”.
  • core refers to the dsRNA portion, which core may comprise at least one dsRNA fragment or which may comprise multiple dsRNA fragments.
  • dsRNA core refers to the core of the dsRNA molecule. Different dsRNA core types are for example a single stem comprising one dsRNA, a single stern comprising multiple dsRNA fragments (concatemer) which dsRNA fragments are each independently complementary to one target gene or to different target sequences of one target gene or are complementary to different target genes.
  • lock refers to a sequence capable of protecting the dsRNA or a portion thereof against RNA processing.
  • Different lock types include a GC rich clamp, a short loop of about 4 or of about 5 base pairs, a mismatch lock, or a protein binding RNA structure such as an IRES, a 5' region of a virus, an Iron responsive element or other RNA motifs that are recognized by proteins.
  • RNA molecules of the present invention may be combined withi n the multidomain RNA molecules of the present invention, for example embedding th e dsRNA in viroids or in natural unprocessed RNA structures (such as mi RNA, tRNA, ribosomal RNA, components of the spliceosome or other non-coding RNA's transcribed from RNA polymerase I, Il or III promoters).
  • Embeding the dsRNA in a viroid-like dsRNA structure is described and illustrated for instance in Navarro and Flores (2000 EMBO Journal 19(11) p 2662.
  • the dsRNA may be incorporated within the viroid as such, or in trie viroid mutated to avoid internal cleavage (for example by ribozymes) or to avoid translation. Mutations can be based on information from Dais et al. (1991 , NAR 19(8), p 1893). These type of constructs may be transported to the chloroplasts, where it can receive extra protection against dsRNA processing.
  • the dsRNA can be compartmentalized in an intermediate host cell, before it is transferred to the target host cell.
  • the multidomain RNA molecule may be compartmentalized in a pla nt cell, for example, it may be located in the chloroplast, mitochondrion or plastid, before it is transferred to the plant pest species, for example the plant pest nematode or insect. Compartmentalization may occur in a variety of ways, such as for example via the use of viroid structures, or via the use of signal sequences, for example chloroplast, mitochondrial or plastid signal sequences. These organelles are from prokaryotic origin and may offer a protective environment away from the plant RNA processing machinery.
  • Linker refers to a molecule enabling linking of a lock to a dsRNA core.
  • Different linker types are conditionally self-cleaving RNA moieties such as linkers that are cleaved at low pH or at high pH or that are cleaved in hydrophobic conditions, or are one of an intron, or a non-complementary RNA sequence.
  • the chimeric RNAi molecule may comprise an interstem base pairing moiety or can be in the form of a triple RNA.
  • the multiple dsRNA fragments of the multidomain are conditionally self-cleaving RNA moieties such as linkers that are cleaved at low pH or at high pH or that are cleaved in hydrophobic conditions, or are one of an intron, or a non-complementary RNA sequence.
  • the chimeric RNAi molecule may comprise an interstem base pairing moiety or can be in the form of a triple RNA.
  • RNA molecule are connected without linker.
  • a linker is present between the at least one aptamer and the dsRNA in the multidomain RNA molecule.
  • the multiple dsRNA fragments of the multidomain RNA molecule are connected by one or more linker.
  • the linkers may be used to disconnect smaller dsRNA region s in the pest organism.
  • the linker sequence may promote division of a long dsRNA into smaller dsRNA under particular circumstances, resulting i n the release of separate dsRNA fragments under these circumstances and leading to more efficient gene silencing by these smaller dsRNA fragments.
  • suitable conditionally self-cleaving linkers are RNA sequences that are self-cleaving at suitable pH conditions. Suitable examples of such RNA sequences are described b>y Borda et al. (Nucleic Acids Res. 2003 May 15, 31(10):2595-600), which document is incorporated herein by reference.
  • the linkers are self cleaving in the endosomes. This may be advantageous when the multidomain RNA molecule of tri e invention is taken up by the pest via endocytosis or transcytosis, and are therefore compartmentalized in the endosomes of the pest.
  • the endosomes may have a low pH environment, leading to the cleavage of the linker.
  • Linkers that are self-cleaving i n hydrophobic conditions are particularly useful in multidomain RNA molecules of trie present invention when used to be transferred from one cell to another via the transit in a cell wall, for example when crossing the cell wall of a pest.
  • Particular plant pest organisms of interest for application of this technique are plant parasitic fungi or plant parasitic viruses or bacteria.
  • An intra may be used as a linker.
  • An "intron” as used herein may be any non-coding RNIA sequence of a messenger RlMA.
  • Particular suitable intron sequences for the multidomain RNA molecules of the present invention are (1) U-rich (35-45%); (2) have an average length of 100 bp (varying between about 50 and about 500 bp) which base pairs may be randomly chosen or may be based on known intron sequences; (3) strat at the 5' end with -AT:GT- or -CG:GT- and/or (4) have at their 3' end -AG/GC- or AG:AA.
  • a linker sequence may be present between the dsRMA fragments or not. Preferably, no linker sequence is present between the dsRMA fragments.
  • the linker may for instance comprise a short random nucleotide sequence that is not complementary to target sequences but that is the result of the cloning.
  • a by itself non-complementary RNA sequence ranging from about 1 base pair to about 10000 base pairs, for instance of at least 10, 20, 30, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 1500, 2000, 3000, 10000 base pairs, or any number in-between, may also be used as a linker.
  • target gene(s) to be targeted by a multidomain RNA molecule depends on the choice of target gene which is to be silenced in the target organism or organisms in order to achieve the desired effect of pest control.
  • target gene(s) was (were) chosen from one or more of the following categories of genes:
  • essential genes encompass genes that are vital for one or more target organisms and result in a lethal or severe (e.g. movement, feeding, paralysis, drinking, fertility) phenotype when silenced .
  • pathogenicity genes are genes that are involved in the pathogenicity or infectivity of the pest.
  • pest specific genes encompass genes that have no substantial homologous counterpart in non-pest organisms as can be determined by bioinformatics homology searches, for example by BLAST searches. The choice of a pest specific target gene results in a species-specific RNAi effect, with no effect or no substantial (adverse) effect in non-target organisms.
  • “conserved pathway” genes encompass genes that are involved in the same biological pathway or cellular process, or encompass genes that have the same functionality in different species resulting in a specific and potent RNA.i effect and more efficient pest control.
  • the multidomain RNA molecules target genes inducing improved delivery / uptake / endocytosis in the pest, such as for example chitin synthase genes, genes encoding peritrophic membrane proteins, excreted
  • RNAses proteins involved in the secretion of Rnase in the gut
  • tight junction genes proteins involved in the acidification of the gut (especially for lepidoteren insect, such as ion channels) and any proteins involved in the maintenance and/or regeneration of gut epithelium.
  • septate junction genes genes encoding proteins involved in the acidification of the gut (especially for lepidoteren insect, such as ion channels) and any proteins involved in the maintenance and/or regeneration of gut epithelium.
  • the multidomain RNA molecules and methods of the present invention are particularly useful to target multiple sequences simultaneously. These multiple sequences may originate from one target gene. Alternatively, the multiple target sequences may originate from multiple target genes. These multiple target genes may originate from one and the same pest species. Alternatively, these multiple target genes may originate from different pest species from the same or different order. Therefore, one multidomain RNA molecule of the present invention, for example in the form of a concatemer cloverleaf, a concatemer stem, or a concatemer hairpin, may simultaneously target multiple sequences originating from the same and /or multiple target genes of the same and or different pest species, such as from nematodes, insects, bacteria and/or fungi.
  • the multidomain RNA molecule targets multiple target genes originating from multiple species.
  • a multidomain RNA molecule may target multiple genes from multiple plant pest organisms, and by expressing the multidomain RNA molecule in the plant, the plant acquires resistance against multiple plant pests simultaneously.
  • a plant or a surface or substance susceptible to pest infestation may be sprayed with a composition (or the like) comprising the multidomain RNA molecule, thereby protecting the plant or the surface or substance against infestation from multiple pests.
  • the plant acquires resistance against nematodes and insects, or against nematodes, insects and/or fungi.
  • the multidomain RNA molecule allows the plant to acquire resistance against multiple nematodes of a different genus, family, order or class, and/or against insects of a different genus, family or order, and/or against fungi of a different genus, family or order.
  • the multidomain RNA molecule targets multiple target genes originating from different species from the same order.
  • one multidomain RNA molecule which targets genes of different bacterial, viral, fungal, insect or nematode species may be used as an effective and broad spectrum bacteria, virus, fungus, insect killer or broad spectrum nematode killer.
  • Combination of dsRNA fragments with at least one aptamer into one multidomain RNA molecule according to the present invention, wherein said dsRNA fragments are targeting multiple target sequences from different pest species is favorable to enlarge the pest species spectrum of the RNAi effect of the dsRNA molecules.
  • the multidomain RNA molecule targets multiple target genes originating from the same organism, for example from the same pest species.
  • a construct offers the advantage that several weak target genes from the same organism can be silenced together to efficiently control the pest organism, while silencing one or more of the weak target genes separately is not effective to control the pest.
  • several strong target genes from the same organism can be silenced simultaneously, in order to further improve the efficacy of the pest control, or to avoid thes occurrence of resistance of the pest organisms by mutation.
  • target organism or “target species” or “pest organism” or “pest species” are used herein as synonym and refer to any organism or species which needs to be killed or paralyzed.
  • Suitable target species are chosen from the group comprising virus, bacteria , yeast, fungi, insects, mites, protozoa, metazoa (comprising nematodes), algae, plants , animals (including mammals).
  • Most suitable for the methods of the present invention are target species which are pest organisms.
  • target species or “pest species” refers to plant pest organisms such as nematodes, insects, fungi, bacteria and viruses and preferably refers to any organism classified in the taxonomical group of the Insecta, or of the Nematoda, or of the Fungi.
  • Nematodes as used herein comprises species of the order Nematoda. Many species of nematodes are parasitic and cause health problems to humans and animals (for example species of the orders Ascaradida, Oxyurida, Strongylida, Stronglyloides and Trichocephalida), as well as to plants and fungi (for example species of the orders Aphelenchida, Tylenchida ad others).
  • nematodes refers to plant parasitic nematodes and nematodes living in the soil.
  • Plant parasitic nematodes include, but are not limited to, ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorus spp.; semiparasites such as Tylenchulus spp.; migratory endoparasites such as Pratylenchus spp., Radopholus spp., and Scutellonerna.
  • ectoparasites such as Xiphinema spp., Longidorus spp., and Trichodorus spp.
  • semiparasites such as Tylenchulus spp.
  • migratory endoparasites such as Pratylenchus spp., Radopholus spp., and Scutellonerna.
  • the nematodes are plant parasitic nematodes, in particular root parasitic soil nematodes such as, for example, those of the genera Heterodera and Globodera (cyst-forming nematodes) an d Meloidogyne (root knot nematodes).
  • RNA constructs of the present invention are particularly suitable to control harmful species of the genus Meloidogyne, such as for example, Meloidogyne incognita, and of the genus Heterodera, such as for example, Heterodera glycines (soybean cyst nematode) and also of the genus Globodera, such, as for example, Globodera rostochiensis (potato cyst nematode,) and also representatives of migrating endoparasites, such as, for example, Pratylenchus penetrans or Radopholus similes and representatives of ectoparasites, such as, for example, Trichodorus spp. and Xiphinema spp.
  • the use of the RNA constructs according to the invention is in no way restricted to these genera, but also extends in the same manner to other nematodes.
  • insects as used herein comprises all insect species.
  • said insect species comprises species of the order Lepidoptera.
  • the insects are insects that damage plants, comprising amongst others Leptidopteran insect pests, such as Heliothis spp., Helicoverpa spp., Spodoptera spp., Ost ⁇ nia 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 nubilaffs, Spodoptera frugiper
  • Leptidopteran insect pests such as
  • lnsecta examples include, but are not limited to, members of the orders Coleoptera (Anobium, Ceutorhynchus, Rhynchophorus, Cospopolites, Lissorhopterus spp., Lissorhopterus oryzophilus, Meligethes, Echinocnemus squamos, Hypothenemus, Hylesinus, Acalymma, Lema, Psylliodes, Leptinotarsa, Gonocephalum, Agriotes, Dermolepida, Heteronychus, Phaedon, Tribolium, Sitophilus spp., Sitophilus zeamais, D/abrotica spp.
  • Diabrotica virgifera virgifera Daibrotica undecimpunctata howardi, Diabrotica barberi
  • Oulema oryzae Chaetocnema pulicaria, Epilachna varivestis, Cerotoma trifurcata, Leptinotarsa decemlineata, Anthonomus spp., Anthonomus grandis, or Anthrenus spp.
  • Lepidoptera e.g.
  • Ephestia Mamestra, Earias, Pectinophora, Ostrinia, Trichoplusia, Pieris, Laphygma, Agrotis, Amathes, Wiseana, Tryporyza, Diatraea, Sporganothis, Cydia, Archips, Plutella, Chilo, Heliothis, Helicoverpa (especially Helicoverpa armigera), Spodoptera or Tineola spp.), Diptera (e.g.
  • Musca Aedes, Anophe/es, Culex, Glossina, Simulium, Stomoxys, Haematobia, Tabanus, Hydrotaea, Lucilia, Chrysomia, Callitroga, Dermatobia, Gasterophilus, Hypoderma, Hylemyia, Atherigona, Chlorops, Phytomyza, Ceratitis, Liriomyza, and Melophagus spp.), Phthiraptera, Hemiptera (e.g. Laodelphax striatellus, Sogatella furcifera, Rhopalosiphum maidis, Macrosiphum euphorbiae, Aphis spp.
  • Hemiptera e.g. Laodelphax striatellus, Sogatella furcifera, Rhopalosiphum maidis, Macrosiphum euphorbiae, Aphis spp.
  • Triatoma Triatoma, Rhodnius, Psylla, Myzus spp., Myzus persica ⁇ , Megoura, Phylloxera, Adelyes, Nilaparvata spp., Nilaparvata lugens, Nephrotettix spp., Nephotettix virescens, or Cimex spp.), Orthoptera (e.g. Locusta, Gryl/us, Schistocerca or Acheta spp.), Dictyoptera (e.g. Blattella, Periplaneta or Blatta spp.), Hymenoptera (e.g.
  • lsoptera e.g. Odontotermes and Reticulitermes spp.
  • Siphonaptera e.g. Ctenocephalides or Pulex spp.
  • Thysanura e.g. Lepisma spp.
  • Dermaptera e.g. Forficula spp.
  • Psocoptera e.g. Pe ⁇ psocus spp.
  • Thysanoptera e.g. Thrips tabaci.
  • Fungi as used herein comprises all species of the order Fungi.
  • the target gene originates from a plant parasitic fungus such as Magnaporthe oryzae (rice blast, formerly Magnaporthe grisae; anamorph Pyricularia oryzae Cav.
  • FRhizoctonia spp. particularly Rhizoctonia solani and Rhizoctonia oryzae; Gibberella fujikuroi; Sclerotinium spp.; Helminthospo ⁇ um sigmoideum; Pythium spp.; Altemaria spp., particularly Alternara solani; Fusarium spp., particularly Fusarum solani and Fusarium germinearum; Acremoniella spp.; Leptosphaeria salvinii; Puccinia spp., particularly Puccinia recondita and Puccinia striiformis; Septoriar nodorum; Pyrenophora teres; Rhincosporium secalis; Erysiphe spp., particularly Erysiphe* graminis; Cladosporium spp.; Pyrenophora spp.; Tilletia spp.; Phyt
  • Bacteria that damage plants and that can be controlled with the constructs and methods of the present invention are for example Agrobacterium ssp.; Arachnia ssp.; Clavibacter ssp.; Corynebacterium ssp.; Erwinia ssp.; Fusobacterium ssp.; HaMa ssp.; Pseudomonas ssp.; Spiroplasma ssp.; Streptomyces ssp.; Xanthomonas ssp.; XyIeIIa ssp. and Xylophilus ssp.
  • Viruses that damage plants and that can be controlled with the constructs and methods of the present invention are for example African cassava mosaic virus; Alfalfa mosaic virus; American plum line pattern virus; Andean potato latent virus; Andean potato mottle virus; Apple chlorotic leaf spot virus; Apple mosaic virus; Apple stem grooving virus;
  • the pest organism can be any species.
  • the pest organism is any insect or nematode of economic importance, such as, for example, organisms that cause disease, a household pest, or an agricultural pest, or that are associated with plants diseases, e.g. in corn, potatoes, soybeans, sugarbeets, turf, trees, orchards and vineyards, garden vegetables, etc..
  • the pest organism can be at any stage of development, however, it is preferred that when the organism is an insect, it is in a larval or adult developmental stage when the dsRNA is delivered.
  • the invention further relates to a nucleic acid encoding any of the multidomain RNA molecules as herein described.
  • the invention relates to a nucleic acid as described above comprising an in frame signal sequence for directing the encoded multidomain RNA molecule to a specific localization within the plant cell such as the cytoskeleton or a plant organelle, such as a chloroplast, a plastide, a mitochondrion.
  • the nucleic acid or the vector comprising the nucleic acid comprises a promoter which allows expression of the RNA molecule in a plant organel, such as a chloroplast, a plastide, a mitochondrion.
  • expression constructs also referred herein as recombinant DNA constructs, to facilitate introd uction into a host cell and for instance a plant cell and/or facilitate expression and/or facilitate maintenance of the nucleotide sequence encoding the multidomain RNA molecules according to the invention.
  • the expression constructs may be inserted into a plasm ⁇ d or a vector, which may be commercially available.
  • the expression construct is an expression vector, suitable for transformation into host organisms such as yeast, bacteria, fungi or plants or plant cells and suitable for maintenance and expression of a multid omain RNA molecule according to the present invention in a transformed host cesll.
  • An "expression vector” is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host.
  • Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors.
  • the invention thus also relate to a vector comprising any of the nucleic acids described above. Said vector may further comprise regulatory sequences for controlling expression of the nucleic acid in said host cell.
  • the expression construct is a plant expression vector, suitable for transformation into plants and suitable for maintenance and expression of a multidomain RNA molecule according to the present invention in a transformed plant cell.
  • the invention thus also relates to a vector comprising any of the nucleic acids described above.
  • Said vector may further comprise regulatory sequences for controlling expression of the nucleic acid in a plant cell.
  • regulatory sequences and “control sequence” used herein are to be taken in a broad context and refer to regulatory nucleic acid sequences capable of driving and/or regulating expression of the sequences to which they are ligated and/or operably linked.
  • the control sequences differ depending upon the intended host organism and upon the nature of the sequence to be expressed.
  • control sequences For expression of a protein in prokaryotes, the control sequences generally include a promoter, a ribosomal binding site, and a terminator. In eukaryotes, control sequences generally include promoters, terminators and, in some instances, enhancers, and/or 5' and 3' untranslated sequences.
  • the term 'control sequence' is intended to include, at a minimum, all components necessary for expression, and may also include additional advantageous components.
  • the control sequence is operable in a plant; preferably the control sequence is a sequence derived from a plant sequence.
  • control sequence encompasses a promoter or a sequence capable of activating or enhancing expression of a nucleic acid molecule in a cell, tissue or organ.
  • Promoters useful for the expression of dsRNA are a promoter from an RNA Poll, an RNA PoIII, an RNA PoIIII, T7 RNA polymerase or SP6 RNA polymerase. . These promoters are typically used for in vitro-production of dsRNA, which dsRNA is then included in an antipesticidal agent, for example in an anti-pesticidal liquid, spray or powder.
  • promoters suitable for the constructs and methods according to the present invention are constitutive plant promoters, such as the CaMV35S promoter, doubled CaMV35S promoter, GOS2 promoter, Figwort mosaic viruse (FMV) 34S promoter, rubisco promoter, actin promoter or ubiquitin promoter.
  • constitutive plant promoters such as the CaMV35S promoter, doubled CaMV35S promoter, GOS2 promoter, Figwort mosaic viruse (FMV) 34S promoter, rubisco promoter, actin promoter or ubiquitin promoter.
  • the plants preferably express the dsRNA in plant parts easily accessible to the plant pest.
  • Preferred tissues to express the dsRNA in are the roots, leafs, stems, rhizomes, shoots, tubers, anthers, petioles, seeds, flowers, fruits. Therefore, tissue-preferred promoters may be used, such as a root specific promoter or a leaf specific promoter. Suitable examples of a root preferred promoter are PsMTA (Fordam-Skelton, A.P., et al.,1997 Plant Molecular Biology 34: 659-668), Class III Chitinase promoter, etc....
  • leaf- and stem-specific or photosynthetic tissue-specific promoters that are also photoactivated are promoters of two chlorophyll binding proteins (cab1 and cab2) from sugar beet (Stahl D.J., et al., 2004 BMC Biotechnology 2004 4:31), ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS (Nomura M. et al., 2000 Plant MoI. Biol. 44: 99-106), A (gapA) and B (gapB) subunits of chloroplast glyceraldehyde-3-phosphate dehydrogenase (Conley T.R. et al. 1994 MoI. Cell Biol. 19: 2525-33; Kwon H. B. et al. 1994 Plant Physiol. 105: 357-67), promoter of the cab1 and cab2 from sugar beet (Stahl D.J., et al., 2004 BMC
  • Solanum tuberosum gene encoding the leaf and stem specific (ST-LS1) protein (Zaidi M.A. et al., 2005 Transgenic Res. 14:289-98), stem-regulated, defense-inducibl e genes, such as JAS promoters (patent publication no. 20050034192/US-A1 ), flower-specific promoters such as chalcone synthase promoter (Faktor O. et al. 1996 Plant MoI. Biol. 32: 849) and fruit-specific promoters such as that of RJ39 from strawberry (WO 98 31812).
  • Suitable promoters are pathogen-induced promoters, such as nematode induced plant promoters, or feeding-site specific promoters, examples of which are Wun-1 (Hansen et al. 1 996. Physiol. MoI. Plant Pathol. 48: 161-170); Lea- 14, Lemmi 9 (Van der Eycken W et al. Plant J. 1996 9(1 ): 45-54; Escobar C et al. MoI Plant Microbe Interact. 1999, 12(5):440-9), pin-2 (Keil et al. 1989. EMBO J. 8:1323- 1330) and TobRB7 (Opperman et al. 1994. Science, 263: 221-223).
  • Wun-1 Haansen et al. 1 996. Physiol. MoI. Plant Pathol. 48: 161-170
  • Lea- 14, Lemmi 9 Van der Eycken W et al. Plant J. 1996 9(1 ): 45-
  • the vectors comprise a constitutive promoter.
  • the vectors comprise an inducible promotes r.
  • the vectors comprise a tissue-specific promoter, for instance a root-specific promoter in case of alleviating pest infestations where the pest predominantly feeds on the roots of the plant, or for instance a leaf-specific promoter in case of alleviating pest infestations where the pest predominantly feeds on the leafs of the plant. Promoters which initiate transcription only in certain tissues or cells are herein referred to as "tissue-specific" or "ce ll- specific" promoters, respectively.
  • the present invention relates to a vector according to the invention wherein the promoter is selected from the group comprising tissue specific promoters such as any selected from the group comprising root specific promoters of genes encoding PsMTA Class III Chitinase, photosynthetic tissue- specific promoters such as promoters of cab1 and cab2, rbcS, gapA, gapB and ST- LS1 proteins, JAS promoters, chalcone synthase promoter and the promoter of RJ39 from strawberry.
  • tissue specific promoters such as any selected from the group comprising root specific promoters of genes encoding PsMTA Class III Chitinase, photosynthetic tissue- specific promoters such as promoters of cab1 and cab2, rbcS, gapA, gapB and ST- LS1 proteins, JAS promoters, chalcone synthase promoter and the promoter of RJ39 from strawberry.
  • transcription termination sequences may also be incorporated in the expression construct.
  • transcription termination sequence encompasses a control sequence at the end of a transcriptional unit, which signals 3' processing and poly- adenylation of a primary transcript and termination of transcription. Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in the expression construct.
  • the expression constructs of the invention may further include an origin of replication which is required for maintenance and/or replication in a specific cell type.
  • an origin of replication which is required for maintenance and/or replication in a specific cell type.
  • an expression construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule).
  • Preferred origins of replication include, but are not limited to the f1-ori and colE1 ori.
  • the expression construct may optionally comprise a selectable marker gene.
  • selectable marker gene includes any gene, which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with an expression construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance or visual markers. Examples of selectable marker genes include genes encoding neomycin phosphotransferase (nptll), hygromycin phosphotransferase (hpt) or Basta.
  • suitable selectable marker genes include resistance genes against ampicillin (Ampr), tetracydine (Tcr), kanamycin (Kanr), phosphinothricin, and chloramphenicol (CAT).
  • Other suitable marker genes provide a metabolic trait, for example manA.
  • Visual marker genes may also be used and include for example beta- glucuronidase (GUS), luciferase and Green Fluorescent Protein (GFP).
  • the multidomain RNA molecule is expressed in plant cell organelles to protect dsRNA from processing.
  • the invention relates to a nucleic acid as described above comprising an in frame signal sequence for directing the encoded multidomain RNA molecule to a specific localization within the plant cell such as the cytoskeleton or a plant organelle, such as a chloroplast, a plastide, a mitochondrion for instance signaling the multidomain RNA molecule towards an intracellular compartment of the host cell in another mechanism to protect dsRNA from processing.
  • the nucleic acid or the vector comprising the nucleic acid comprises a promoter which allows expression of the RNA molecule in a plant organelle, such as a chloroplast, a plastide, a mitochondrion (see above).
  • the multidomain RNA molecule can be compartmentalized in an intermediate host cell or organism before it is transferred to the target host cell, e.g. a pest cell.
  • the multidomain RNA molecule may be compartmentalized in a plant cell, for instance it may be located in the chloroplast, mitochondrion or plastid, before it is transferred to the plant pest species, for example a plant pest nematode or insect. Compartmentalization may occur in a variety of ways, such as for example via the use of viroid structures, or via the use of signal sequences as described above, for example chloroplast, mitochondrial or plastid signal sequences. These organelles are form prokaryotic origin and may offer a protective environment away from the plant RMA processing machinery.
  • a major advantage of compartmentalization of the multidomain RNA molecule thus includes that the molecules are protected from nuclear/cytoplasmic processing (dicing) of dsRNA.
  • compartmentalization provides for an accumulation of dsRMA sequences.
  • sense and antisense RNA fragments corresponding to a selected gene of a particular pest species are cloned behind differe nt promoters driving expression (i) separate plant tissues or (ii) within the same cell but in separate cellular compartments. These promoters are tissue or organel specific and allow strong simultaneous expression in different cellular compartments or in adjacent tissues.
  • the sense and antisense strands may be targeted to different plant tissue or cell types.
  • the sense strand may be expressed in the nerve ce Hs while the antisense is expressed in the palisade tissue. This may be achieved by using different promoters to drive the expression of the sense and the antisense strands. Tine advantage of this technique is that the sense and antisense strands never come together in the plant cell, and therefore no degradation or auto-silencing or RNA interference can occur within the plant by Dicer.
  • the strands are set free and mixed, allowing the annealing of the dsRNA in the gut lumen and then base pairing between the sense and antisense strands may occur to form long dsRMA. Subsequently, this dsRNA may be taken up efficiently and leads to the desired RKlAi response, leading to degradation of the target mRNA in the pest and death of the pest.
  • This approach can be accomplished by feeding the pest with two bacterial strains " for instance provided in a composition, one strain producing the sense, the other produci ng the antisense strand.
  • RNA delivery molecule consisting of different modules.
  • a delivery molecule may consist for example of a polypeptide sequence comprising (i) at least one RNA-binding domain, (ii) at least one targeting polypeptide able to bind to a cellular endocytosis and/or transcytosis receptor molecule and (iii) optionally at least one peptide linker and/or at least one purification tag.
  • RNA delivery-promoting molecule can be used to further facilitate the uptake and the correct delivery of dsRNA to a suitable target site in a plant-feeding pest organism for the purpose of RNA interference.
  • RNA delivery module RNA delivery molecule
  • RNA delivery vehicle RNA delivery vehicle
  • the RNA delivery molecule consisting of different modules, comprises: at least one RNA binding module, at least one targeting module able to be endocytosed and/or transcytosed or able to bind to a cellular endocytosis and/or transcytosis receptor molecule, optionally at least one linker for linking the dsRNA binding module to the targeting module, 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 may bind double-stranded RNA generically or specifically, single- stranded RNA generically or specifically.
  • the RNA binding molecule may bind dsRNA and/or ssRNA structure-specifically.
  • RNA binding proteins include but are not limited to coliphage HK022 NUN protein, Bacillus subtilis LicT protein, or bacteriophage MS2 coat protein or essential parts, or homologues thereof.
  • a second module of the RNA delivery molecule comprises a targeting module.
  • targeting module and “targeting protein” are used herein as synonyms and both refer to a protein, or an essential part, or a homologue thereof capable of targeting the RNA delivery molecule to a targeting site in a living pest organism.
  • the targeting module preferably comprises a protein which is capable of being endocytosed and/or transcytosed in a cell of the pest organism, or a protein able to bind an endocytosis and/or transcytosis receptor molecule present on a cell or a tissue of the pest organism, or any combinations thereof.
  • the present invention relates to a host cell or organism comprising a nucleic acid or a vector as defined herein.
  • host cells which may be used in accordance with the present invention include a bacterial, yeast, fungal, or plant cell.
  • Host cells may be prokaryotic cells such as E. coli and A. tumefaciens, or may be eukaryotio cells such as yeast, or plant cells. It is preferred that host cells are monocotyledonous or dicotyledonous plant cells.
  • the present invention also encompasses a cell, e.g. a host cell, comprising any of the multidomain RNA molecules, dsRNA, nucleic acid or a vector as defined 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 , yeast cells or plant cells).
  • prokaryotic cells such as, but not limited to, gram- positive and gram-negative bacterial cells
  • eukaryotic cells such as, but not limited to , yeast cells or plant cells.
  • said cell is a bacterial cell or a plant cell.
  • the present invention also encompasses a transgenic plant, reproductive or propagation material for a transgenic plant comprising such a plant cell.
  • the vector or nucleic acid molecule according to the invention may either be integrated into the genome of the host cell or it may be maintained in some form extra - chromosomally.
  • the present invention relates to methods for delivering dsRNA to a pest species, to methods for down-regulating the expression of a target gene in a pest species and to methods for producing transgenic plants resistant to pest species.
  • Methods according to the present invention include feeding the multidomain RNA molecule(s) to the organism to deliver the dsRNA to the organism tissues. It is envisaged that the methods of the invention will have use in controlling plant diseases caused by feeding organisms. Methods of pest control of organisms, and of protecting plants against organisms are provided.
  • the "dsRNA” or “double stranded RNA”, whenever said expression relates to RNA that is capable of causing interference, may be formed form two separate (sense and antisense) RNA strands that are annealed together.
  • the sense and antisense strands of the dsRNA originate form distinct RN,A molecules, wherein at least one of the RNA molecules is a multidomain RNA molecule as described herein.
  • the multidomain RNA molecule may have a foldback stern- loop or hairpin structure wherein the two annealed strands of the dsRNA are covalently linked.
  • the sense and antisense strands of the dsRNA are formesd from different regions of a single multidomain RNA molecule that is partially self- complementary.
  • the origin of sense and antisense strands making up the double- stranded RNA is variable.
  • Non-limiting examples of expression strategies for expressing multidomain RNA molecules are represented in Figures 1 and 2.
  • the multidomain RNA molecules may be taken up by the pest organism in several ways.
  • the invention relates to a method for delivering a double-stranded RNA molecule to a pest species, comprising:
  • the invention relates to a method for delivering a double-stranded RNA molecule to a pest species, comprising feeding the pest species with at least one multidomain RNA molecule according to the present invention whereby said multidomain RNA molecule is taken up into the gut of the pest species, and whereby the multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulates expression of the target gene in a pest cell.
  • feeding may refer to feeding of the pest species with a host cell or organism, e.g. plant cell, bacteria, fungi, yeast, etc., or a mixture thereof comprising, expressing, sprayed or coated with at least one multidomain RNA molecule of the invention.
  • a host cell or organism e.g. plant cell, bacteria, fungi, yeast, etc., or a mixture thereof comprising, expressing, sprayed or coated with at least one multidomain RNA molecule of the invention.
  • a mixture of bacteria or plant(s) cell(s) may be fed to a pest species, whereby the mixture comprises at least one bacterium or plant (cell) that comprises or expresses a first multidomain RNA molecule comprising a sense or antisense RNA strand and at least another bacterium or plant (cell) that comprises or expresses another multidomain RNA molecule, said other RNA molecule being capable of forming double stranded RNA with a portion of the sense or anti-sense RNA of said first multidomain RNA molecule (for the purpose of causing RNA interference in a pest species).
  • the invention relates to a method for delivering a double-stranded RNA molecule to a pest species:
  • RNA molecule - expressing in a plant cell or plant at least one multidomain RNA molecule according to the present invention and at least one other RNA molecule comprising single-stranded RNA
  • said multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of said other RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene
  • RNA molecule refers to an RNA molecule which can be or can not be another multidomain RNA molecule, and which RNA molecule may or may not comprise domains or sequences which protect it from degradation or which direct it to specific locations.
  • the "other RNA molecule” may thus refer to another multidomain RNA molecule as defined herein, or to an RNA molecule which does not comprise an aptamer sequence.
  • the invention in another embodiment, relates to a method for delivering a double-stranded RNA molecule to a pest species comprising feeding the pest species with at least one multidomain RNA molecule according to the present invention and with at least one other RNA molecule comprising single-stranded RNA, wherein said multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of said other RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and whereby the multidomain RNA molecule is taken up into the gut of the pest species, and whereby the multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulates expression of the target gene in a pest cell.
  • the invention relates to a method for delivering a double- stranded RNA molecule to a pest species comprising: - expressing in a plant cell or plant at least two multidomain RNA molecules according to the present invention wherein each multidomain RNA molecule comprises at least one aptamer sequence and wherein each multidomain RNA molecule further comprises single-stranded RNA, forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and feeding said plant cell or plant to said pest species.
  • the invention provides a method for delivering a double-stranded RNA molecule to a pest species, comprising feeding the pest species with at least two multidomain RNA molecules according to the present invention, wherein each multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and whereby the multidomain RNA molecules are taken up into the gut of the pest species, and whereby the multidomain RNA molecules are transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulate expression of the target gene in a pest cell.
  • the present invention also relates to method for down-regulating expression of a target gene in a pest species.
  • the invention relates to a method for down- regulating expression of a target gene in a pest species, comprising: expressing in a plant cell or plant at least one multidomain RNA molecule according to the invention, and - feeding said plant cell or plant to said pest species.
  • Another method comprises feeding the pest species with at least one multidomain RNA molecule according to the invention, wherein the RNA molecule comprises at least one aptamer sequence and further comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, whereby the multidomain RNA molecule is taken up into the gut of the pest species, and whereby the multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulates expression of the target gene in a pest cell.
  • a method for down-regulating expression of a target gene in a pest species comprisi ng: expressing in a plant cell or plant at least one multidomain RNA molecule according to the invention and at least one other RNA molecule comprising single- stranded RNA, wherein said multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of said other RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, and
  • a method for down-regulating expression of a target gene in a pest species comprises feeding the pest species with at least one multidomain RNA molecule according to the present invention and with at least one other RNA molecule comprising single-stranded RNA, wherein said multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of said other RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, and whereby the multidomain RNA molecule is taken up into the gut of the pest species, and whereby the multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulates expression of the target gem e in a pest cell.
  • a method for down-regulating expression of a target gene in a pest species comprising:
  • each multidomain RNA molecule comprises at least one aptamer sequence
  • each multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated
  • a method for down-reg ulating expression of a target gene in a pest species comprises feeding the pest species with at least two multidomain RNA molecules according to the present invention, wherein each multidomain RNA molecule comprises at least one aptamer sequence, and wherein each multidomain RNA molecule further comprises single-stranded RNA,
  • double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA co mprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, and whereby the multidomain RNA molecules are taken up into the gut of the pest species, and whereby the multidomain RNA molecules are transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulate expression of the target gene in a pest cell.
  • the invention also relates to a method for producing a transgenic plant that is resistant to a pest species, comprising expressing in a plant cell at least one multidomain RNA molecule according to the invention and regenerating a plant from said plant cell.
  • the invention also relates to a method for producing a transgenic plant that is resistant to a pest species, comprising
  • each multidomain RNA molecule comprises single-stranded RNA, (capable of) forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and regenerating a plant from said plant cell.
  • the invention provides a method for producing a transgenic plant that is resistant to a pest species, comprising expressing in a plant cell at least one multidomain RNA molecule according to the invention and at least one other RNA molecule comprising single-stranded RNA, wherein said multidomain RNA molecule comprises single-stranded RNA, capable of forming double-stranded RNA with the single-stranded RNA of said other RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and regenerating a plant from said plant cell.
  • the invention further relates to any of the methods described above wherein said at least one multidomain RNA molecule comprises one aptamer chosen from the group of aptamers as defined earlier.
  • the invention further relates to any of the methods described above, wherein at least one multidomain RNA molecule comprises two aptamers chosen from the group of aptamers as defined earlier.
  • the invention further relates to any of the methods described above wherein said at least one multidomain RNA molecule comprises three aptamers chosen from the group of aptamers as defined earlier.
  • the invention further relates to any of the methods described herein wherein said pest species is any pest species described herein, preferably the pest species is chosen from the group comprising yeast, fungi, insects and nematodes.
  • the present invention provides methods for the production of transgenic plants, plant cells or plant tissues comprising the introduction of a nucleic acid or vector according to the invention into the genome of said plant, plant cell or plant tissue.
  • the present invention also relates to a transgenic plant resistant to a pest species, an essential derived variety thereof, plant part, plant cell or protoplast thereof obtainable by any of the methods as described herein.
  • the invention relates to a transgenic plant, an essentially derived variety thereof, plant part, plant cell or protoplast thereof which comprises Q nucleic acid encoding a multidomain RNA molecule as defined herein, wherein said n ucleic acid is heterologous to the genome of said transgenic plant, or an essentially derived variety thereof, plant part, plant cell or plant protoplast thereof.
  • the invention provides a transgenic plant which comprises a vector as described herein.
  • 'transgenic plant' includes reference to a plant, which comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a vector.
  • Transgenic' is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of the heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • a plant an essentially derived variety thereof, plant part, plant cell or protoplast thereof that has been transformed with a nucleic acid encoding at least one multidomain RNA molecule as defined herein.
  • a plant an essentially derived variety thereof, plant part, plant cell or protoplast thereof that has been transformed with a nucleic acid encoding at least one multidomain RNA molecule as defined herein and that has been transformed with a nucleic acid encoding at least one other RNA molecule which comprises single-stranded RNA, whereby said multidomain RNA molecule comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of said other RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementaryto at least pa rt of a target nucleotide sequence of the target gene.
  • RNA molecule refers to an RNA molecule which can be or can not be another multidomain RNA molecule, and which RNA molecule may or may not comprise domains or sequences which protect it from degradation or which direct it to specific locations.
  • the "other RNA molecule” may thus refer to another multidomain RNA molecule as defined herein, or to an RNA molecule which does not comprise an aptamer sequence.
  • each multidomain RNA molecules comprise single-stranded RNA forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene.
  • the present invention also provides a plant, an essentially derived variety thereof, plant part, plant cell, or protoplast thereof which expresses at least one multidomain RMA molecule as defined herein, wherein the multidomain RNA molecule comprises at least one aptamer sequence and further comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementa ry to at least part of a target nucleotide sequence of the target gene to be down-regulated.
  • the invention also further relates to a plant, an essentially derived variety, plant part, pla nt cell or protoplast thereof which expresses at least one multidomain RNA molecule as defined herein, and which expresses at least one other RNA molecule comprising single- stranded RNA, wherein said multidomain RNA molecule comprises single-stranded RNA (capable of) forming double-stranded RNA with the single-stranded RNA of said oth er RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene.
  • the invention also relates to a plant, an essentially derived variety, plant part, plant cell or protoplast thereof which expresses at least two multidomain RNA molecules as defined herein, wherein each multidomain RNA molecule comprises single-stranded RNA, (capable of) forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene.
  • transformation refers to the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for the transfer.
  • Tine polynucleotide may be transiently or stably introduced into the host cell or organism and may be maintained non-integrated, for example, as a plasmid, or alternatively, may be integrated into the host genome. Transformation may be transient or stable.
  • the invention thus also relates to such a stably or transiently transformed transgenic plant, plant cell or plant tissue.
  • the invention further relates to any plant which comprises any of the subject vectors in accordance with the invention.
  • the invention also relates to any of the transgenic plants described herein comprising a nucleic acid encoding a multidomain RNA molecule as defined herein characterized in that said plant has increased resistance to a pest organism, for instance increased resistance of between 30% to 80% compared to control plants.
  • the invention also relates to the progeny of a plant or essentially derived variety thereof obtainable by a method of the present invention or as described herein which has been obtained in a generative or vegetative manner.
  • the present invention also includes parts or derivatives of obtainable by a method of the present invention or as described herein, such as but not limited to leaves, stems, roots, shoots, cuttings or explants and the like, protoplasts, somatic embryos, anthers, petioles, cells in culture, seeds, flowers, fruits and tubers.
  • the invention in another embodiment relates to a method for controlling a pest species comprising feeding said pest species with at least one multidomain RNA molecu le as defined herein wherein the multidomain RNA molecule comprises at least one aptamer, and at least one nucleotide sequence of interest forming double-stranded RNA, said double-stranded RNA comprising annealed complementary strands, o ne of which comprises a nucleotide sequence which is complementary to at least part of a pest target nucleotide sequence, whereby the multidomain RNA molecule is taken up into the gut of the pest species, and whereby the multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell; and whereby the double-stranded RNA causes RNAi interference with the target gene in a pest cell such that the pest species is killed or paralyzed.
  • the multidomain RNA molecule comprises at least one aptamer, and at least one nucleotide sequence of
  • the invention relates to a method for controlling a pest species comprising feeding said pest species with at least one multidomain RNA molecule as defined herein and at least one other RNA molecule comprising single-stranded RN> ⁇ .
  • said multidomain RNA molecule comprises single-stranded RNA (capable of) forming double-stranded RNA with the single-stranded RNA of another RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and whereby said multidomain RNA molecule is taken up into the gut of the pest species, and whereby said multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell; and whereby the double-stranded RNA causes RNAi interference with the target gene in a pest cell such that the pest species is killed or paralyzed.
  • the invention in another embodiment relates to a method for controlling a pest species comprising feeding said pest species with at least two multidomain RNA molecules as defined herein, and wherein each multidomain RNA molecule comprises at least one aptamer sequence, and wherein each multidomain RNA molecule further comprises single-stranded RNA forming double-stranded RNA with the single-stranded RNA of another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene, and whereby each multidomain RNA molecule is taken up into the gut of the pest species, and whereby each multidomain RNA molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell, and whereby the double-stranded RNA causes RNAi interference with the target gene in a pest cell such that the pest species is killed or paralyzed.
  • the invention also relates to a method for controlling a pest species comprising feeding the pest species with a transgenic plant which is resistant to said pest species, or any progeny or part thereof as defined herein.
  • a method for protecting a plant against a pest organism comprising expressing in said plant of a multidomain RNA molecule as defined herein.
  • the host organism is a plant and the pest species is a plant pathogenic pest.
  • the host organism is a plant and the pest species is a plant pathogenic pest.
  • plant as used herein encompasses any plant material such as inter alia a plant cell, plant tissue (including callus), plant part, whole plant, ancestors and progeny.
  • a plant part may be any part or organ of the plant and include for example a seed, fruit, stem, leaf, shoot, flower, anther, root or tuber.
  • the plant material should express, or have the capability to express, multidomain RNA molecules comprising dsRNA corresponding to one or more target genes of the pest species to be killed or paralyzed.
  • plant also encompasses suspension cultures, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, and microspores.
  • the plant as used herein refers to all plants including algae, ferns and trees.
  • the plant belongs to the superfamily of Viridiplantae, further preferably is a monocot or a dicot.
  • the plant is susceptible to infestation by a plant pathogenic and/or parasitic nematode, by a fungus or an insect.
  • Particular plants useful in the methods of the present invention are crop plants including for example monocots such as sugar cane and cereals (including wheat, oats, barley, sorghum, rye, millet, com, rice) and dicots such as potato, tomato, vine, apple, pear, banana, sunflower, soybean, canola, alfalfa, rapeseed and cotton.
  • monocots such as sugar cane and cereals (including wheat, oats, barley, sorghum, rye, millet, com, rice)
  • dicots such as potato, tomato, vine, apple, pear, banana, sunflower, soybean, canola, alfalfa, rapeseed and cotton.
  • Particular trees that can be used in the methods of the present invention are pine, eucalyptus and poplar.
  • administering a DNA to a cell may be achieved by a variety of means, each well known by the person skilled in the art. Examples of useful techniques are shot-gun, ballistics, electroporation, transfection and transformation.
  • RNAi general techniques for expression of exogenous double-stranded RNA in plants for the purposes of RNAi are known in the art (Baulcombe D, 2004, Nature. 431 (7006):356-63. RNA silencing in plants). RNA silencing in plants, the contents of which are incorporated herein by reference).
  • RNA molecules(s) are also known in the art. Similar methods can be applied in an analogous manner in order to express the multidomain RNA molecule(s) in plants for the purposes of down-regulating expression of a target gene in a plant pest species. In order to achieve this effect it is necessary only for the plant to express (transcribe) the multidomain RNA molecule(s) in a part of the plant which will come into direct contact with the pest species, such that the multidomain RNA molecule(s) can be taken up by the pest species.
  • the multidomain RNA molecule(s) could occur within a cell or tissue of a plant within which the pest species is also present during its life cycle, or the multidomain RNA molecule(s) may be secreted into a space between cells, such as the apoplast, that is occupied by the pest species during its life cycle.
  • the multidomain RNA molecule(s) may be located in the plant cell, for example in the cytosol, or in the plant cell organelles such as chloroplast, mitochondrion, vacuole or endoplasmatic reticulum.
  • the multidomain RNA molecule may be secreted by the plant cell and by the plant to the exterior of the plant. As such, the multidomain RNA molecule may form a protective layer on the surface of the plant.
  • the present invention thus relates to a method for the production of a transgenic cell or organism, comprising the step of administering a nucleic acid or a vector as described herein to said cell or organism.
  • said cell is a plant cell or said organism is a plant.
  • the invention further relates to any transgenic cell or transgenic organism obtainable by the above described method, preferably said transgenic cell or organism is plant cell or plant organism.
  • the methods of the present invention for the production of transgenic organism may further comprise the steps of cultivating the transgenic cell under conditions promoting growth and development. Where the transgenic organism is a plant, these methods may further comprise the steps of regenerating a plant from plant tissue, allowing growth to reach maturity and to reproduce. Alternatively, the transgenic plant tissue may take other forms or may form part of another plant, examples of which are chimera plsnts and grafts (for example a transformed rootstock grafted to an untransformed scion). Whenever used herein, "transfer" of the dsRNA from the plant to the pest species means that the dsRNA is produced in the plant cell and is being taken up, relocated or brought into contact with the pest organisms.
  • a plant parasitic nematode or an insect for example may take up the dsRNA produced in the plant by feeding from the plant cell cytoplasm.
  • a fungal cell which is contacted with the dsRNA may be a plant pathogenic fungal cell in a life stage outside a plant cell, for example in the form of a hypha, germ tube, appressorium, conidium (asexual spore), ascocarp, cleistothecium, or ascospore (sexual spore outside the plant).
  • the fungal cell which is contacted with the dsRNA is a plant pathogenic fungal cell in a life stage inside a plant cell, for example a pathogenic form such as a penetration peg, a hypha, a spore or a haustorium.
  • a pathogenic form such as a penetration peg, a hypha, a spore or a haustorium.
  • transfer of dsRNA means contacting with a composition comprising the dsRNA or the dsRNA construct.
  • the invention relates to the use of a multidomain RNA molecu Ie as described herein for various agronomic and research applications requiring the delivery of dsRNA into a target pest organism.
  • the invention relates to the use of a multidomain RNA molecule as described herein for delivering dsRNA to a pest species. It shall be understood from the present description that the present multidomain RNA molecule(s) are particularly suitable for improving the delivery of dsRNA to pest organisms which are feeding on a plant expressing dsRNA.
  • the multidomain RNA molecule(s) according to the present invention may also be very useful for improving the delivery of dsRNA to pest organisms by any other way, including but not limited to injection of dsRISIA, soaking the organisms in dsRNA solution or by feeding the pest organisms Escherichia coli bacteria that simultaneously express sense and antisense RNAs and that can acquire dsRNA.
  • the present multidomain RNA molecules are also very useful for down-regulating the expression of target genes in pest species.
  • the invention therefore also provides for the use of said multidomain RNA molecules as described herein for down-regulating the expression of target genes in pest species.
  • the present invention relates to the use of a multidomain RNA molecule according to the invention for producing a transgenic plant resistant to pest organisms.
  • the present invention relates to the use of a transgenic plant resistant to a pest organism as described herein for controlling pest population growth, and/or for reducing infestation by a pest species and/or for killing or paralyzing a pest organism, and/or for preventing or reducing the amount and the number of chemical (e.g. pesticide, fungicide, nematicide) applications, and/or for reducing the environmental impact of chemical applications pesticides and/or for reducing disease incidence in a crop and/or for improving crop yield.
  • chemical e.g. pesticide, fungicide, nematicide
  • the invention relates to the use of a plant, an essentially derived variety thereof, plant part, plant cell or protoplast thereof that has been transformed with a nucleic acid encoding at least one multidomain RNA molecule as herein described for improving resistance to a pest organism, and/or for controlling pest population growth, and/or for preventing or reducing infestation by a pest species and/or for killing or paralyzing a pest organism, and/or for reducing the amount and the number of chemical (e.g. pesticide, fungicide, nematicide) applications, and/or for reducing the environmental impact of chemical applications pesticides and/or for reducing disease incidence in a crop and/or for improving crop yield.
  • chemical e.g. pesticide, fungicide, nematicide
  • the invention relates to the use of a plant, an essentially derived variety thereof, plant part, plant cell or protoplast thereof that has been transformed with a nucleic acid encoding at least two multidomain RNA molecules as herein described for improving resistance to a pest organism, and/or for controlling pest population growth, and/or for preventing or reducing infestation by a pest species and/or for killing or paralyzing a pest organism, and/or for reducing the amount and the number of chemical
  • pesticide e.g. pesticide, fungicide, nematicide
  • fungicide fungicide
  • nematicide for reducing the environmental impact of chemical applications pesticides and/or for reducing disease incidence in a crop and/or for improving crop yield.
  • the invention relates to the use of a plant, an essentially derived variety, plant part, plant cell or protoplast thereof which expresses at least one multidomain RNA molecule as defined herein, wherein the multidomain RNA molecule comprises at least one aptamer sequence and further comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated for improving resistance to pest organism, and/or for controlling pest population growth, and/or for preventing or reducing infestation by a pest species and/or for killing or paralyzing a pest organism, and/or for reducing the amount and the number of chemical (e.g.
  • the invention relates to the use of a plant, an essentially derived variety, plant part, plant cell or protoplast thereof which expresses at least two multidomain RNA molecules as defined herein, wherein each multidomain RNA molecule comprises single-stranded RNA, forming double-stranded RNA with the single-stranded RNA of another RNA molecule or another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene for improving resistance to pest organism, and/or for controlling pest population growth, and/or for preventing or reducing infestation by a pest species and/or for killing or paralyzing a pest organism, and/or for reducing the amount and
  • the invention relates to the use of progeny or parts or derivatives of plants obtainable from any plant or essentially derived variety thereof as described herein for improving resistance to pest organism, and/or for controlling pest population growth, and/or for preventing or reducing infestation by a pest species and/or for killing or paralyzing a pest organism, and/or for reducing the amount and the number of chemical (e.g. pesticide, fungicide, nematicide) applications, and/or for reducing the environmental impact of chemical applications pesticides and/or for reducing disease incidence in a crop and/or for improving crop yield.
  • chemical e.g. pesticide, fungicide, nematicide
  • the method of the invention may also be used as a tool for experimental research, particularly in the field of functional genomics.
  • Targeted down- regulation of pest genes by RNAi can be used in in vitro or in vivo assays in order to study gene function.
  • Assays based on targeted down-regulation of specific pest genes, leading to a measurable phenotype may also form the basis of compound screens for novel pesticides.
  • compositions and kits In a further aspect the invention relates to a composition for reducing pest population growth and/or for killing or paralyzing a pest organism and/or for improving plant resistance to pest organisms, for improving resistance to pest organism, and/or for controlling pest population growth, and/or for preventing or reducing infestation by a pest species and/or for killing or paralyzing a pest organism, for reducing disease incidence in a crop and/or for improving crop yield, said composition comprising at least one multidomain RNA molecule as herein described.
  • the invention relates to a composition
  • a composition comprising at least one multidomain RNA molecule described herein and a physiological or agronomical acceptable carrier, excipient or diluent.
  • the invention also encompasses the use of said composition as a pesticide for a plant or for propagation or reproductive material of a plant.
  • the invention relates to a composition
  • a composition comprising at least one multidomain RNA molecule described herein, and a physiological or agronomical acceptable carrier, excipient or diluent.
  • the composition may contain further components which serve to stabilise the dsRNA and/or prevent degradation of the dsRNA during prolonged storage of the composition.
  • composition may still further contain components which enhance or promote uptake of the multdiomain RNA molecule by the pest organism.
  • components which enhance or promote uptake of the multdiomain RNA molecule by the pest organism may include, for example, chemical agents which generally promote the uptake of RNA into cells e.g. lipofectamin etc., and enzymes or chemical agents capable of digesting the fungal cell wall, e.g. a chitinase.
  • composition may be in any suitable physical form for application to the pest, to substrates, to cells (e.g. plant cells), or to organism infected by or susceptible to infection by a pest species.
  • cells e.g. plant cells
  • the invention provides a composition comprising at least one multidomain RNA molecule as herein described, wherein each multidomain RNA molecule comprises single-stranded RNA, (capable of) forming double-stranded RNA with the single-stranded RNA of another RNA molecule or another multidomain RNA molecule, said double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene.
  • the invention also relates to a method for producing a multidomain RNA molecule as described herein comprising: introducing into a host cell an isolated DNA molecule encoding any of the multidomain RNA molecules as described herein, a nucleic acid encoding any of the multidomain RNA molecules of the invention or a vector comprising said nucleic acid; - growing the host cell under conditions suitable for expression of the multidomain
  • RNA molecule and isolating the multidomain RNA molecule produced by the host cell.
  • the term "host cell” may any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant or animal cell.
  • the present invention also relates to a kit comprising at least one multidomain RNA molecule described herein.
  • the present invention also relates to a kit comprising at least two multidomain RNA molecule as described herein.
  • the present invention also relates to a kit comprising at least one nucleic acid encoding a multidomain RNA molecule as described herein.
  • the invention also relates to a kit comprising any vector described herein comprising a nucleic acid encoding any of the multidomain RNA molecules as described herein. It should be understood that these nucleic acid sequences according to the present invention may be comprised in one or in several separate vectors.
  • the invention relates to a kit comprising at least one composition as defined herein.
  • composition of the invention may be supplied as a "kit- of-parts" comprising the multidomain RNA molecule in one and a suitable diluent or carrier for the RNA in a further separate container.
  • the invention also relates to the supply of the multidomain RNA molecule alone without any further components.
  • the multidomain RNA molecule may be supplied in a concentrated form, such as a concentrated aqueous solution. It may even be supplied in frozen form or in freeze-dried or lyophilized form. The latter may be more stable for long term storage and may be de- frosted and/or reconstituted with a suitable diluent immediately prior to use.
  • the present invention further relates to the medical use of any of the multidomain RNA molecules, constructs, nucleotide sequences, recombinant DNA constructs or compositions thereof described herein.
  • the composition is a pharmaceutical or veterinary composition for treating or preventing pest infections of humans or animals, respectively.
  • compositions will comprise at least one multidomain RNA molecule according to the invention, wherein the multidomain RNA molecule comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene of a pest species to be down-regulated and at least one carrier, excipient suitable for pharmaceutical use or for veterinary use, respectively.
  • the composition may be a composition suitable for topical use, such as application on the skin of an animal or human, for example as liquid compositions to be applied to the skin as drops, or by brushing, or a spray, also creams, ointments, etc. for topical application and transdermal patches.
  • Target human pathogenic and animal pathogenic fungi include, but are not limited to the following:
  • Candida spp. particularly Candida albicans
  • Dermatophytes including Epidermophyton spp., Trichophyton spp., and Microsporum spp.
  • Aspergillus spp. particularly Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus group
  • Coccidioides immitis Crytococcus neoformans
  • Microsporum gypseum Trichophyton mentagrophytes; Apergillus spp.; Cryptococcus neoformans.
  • the composition may be a composition suitable for agronomical use, such as a spray, a coating, a powder and the like.
  • the present invention provides pesticidal compositions developed to be used in agriculture or horticulture. These pesticidal compositions may be prepared in a manner known per se.
  • the active compounds can be converted into the customary formulations, such as solutions, emulsions, wettable powders, water dispersible granules, suspensions, powders, dusting agents, foaming agents, pastes, soluble powders, granules, suspo-emulsion concentrates, microcapsules, fumigants, natural and synthetic materials impregnated with active compound and very fine capsules and polymeric substances.
  • the pesticidal compositions according to the present invention may comprise a synergist.
  • the multidomain RNA molecules, constructs, nucleotide sequences, or compositions thereof according to the invention, as such or in their formulations, can also be used in a mixture with known fungicides, bactericides, acaricides, nematicides or insecticides, to widen, for example, the activity spectrum or to prevent the development of resistance. In many cases, this results in synergistic effects, i.e. the activity of the mixture exceeds the activity of the individual components.
  • RNA molecules, constructs, nucleotide sequences or compositions thereof according to the invention can also be used in a mixture with other known active compounds, such as herbicides, fertilizers and/or growth regulators.
  • the present invention also relates to fibrous pesticide composition and its use as pesticide, wherein the fibrous composition comprises a non-woven fiber and an effective amount of at least one of the multidomain RNA molecules, nucleotide sequences, recombinant DNA constructs or compositions thereof described herein, covalently attached or stably adsorbed to the fiber.
  • the fiber is biodegradable and the adsorbed the multidomain RNA molecules or compositions thereof as described herei n, can be slowly released into a localized area of the environment to control pests in that area over a period of time.
  • the present invention also encompasses solid formulations of slow-release pesticidal composition comprising the molecules or constructs as described herein, and their use as pesticide.
  • the formulations release the multidomain RNA molecule as described herein (a) into the environment (soil, aqueous medium, plants) in a controlled and slow fashion (complete release within several days up to a few months).
  • the present invention also relates to surfactant-diatomaceous earth compositions for pesticidal use in the form of dry spreadable granules comprising at least one multidomain RNA molecule as described herein.
  • the granules comprises in addition to the diatomaceous earth, a surfactant composition designed to provide binding, rewetting and disintegration properties to the granules.
  • diatomaceous earth is meant a silica material characterized by a large surface area per unit volume.
  • Diatomaceous earth is a naturally occurring material and consists mainly of accumulated shells or frustules of intricately structured amorphous hydrous silica secreted by diatoms.
  • the present invention also provides solid, water-insoluble lipospheres and their use as pesticide, wherein said lipospheres are formed of a solid hydrophobic core having a layer of a phospholipid embedded on the surface of the core, containing at least one multidomain RNA molecule as described herein in the core, in the phospholipid, adhered to the phospholipid, or a combination thereof.
  • the pesticidal compound containing lipospheres have several advantages including stability, low cost of reagents, ease of manufacture, high dispersibility in an aqueous medium, a release rate for the entrapped compound that is controlled by the phospholipid coating and the carrier.
  • the invention further relates to pesticidal formulations in the form of microcapsules having a capsule wall made from a urea/dialdehyde precondensate and comprising at least one multidomain RNA molecule as described herein.
  • the composition may be a coating that can be applied to a substrate in order to protect said substrate from infestation by a pest species, such as a fungus and/or to prevent, arrest or reduce growth of the pest species on the substrate and thereby prevent damage caused by said pest species.
  • a pest species such as a fungus
  • the composition can be used to protect any substrate or material that is susceptible to infestation by or damage caused by a pest species, for example, foodstuffs and other perishable materials, and substrates such as wood.
  • Preferred target pest species for this embodiment is are fungus species, including, but are not limited to, the following: Stachybotrys spp.; Apergillus spp.; Alterna ⁇ a spp.; Cladosporium spp.
  • the composition will comprise at least one multidomain RNA molecule according to the invention, wherein the multidomain RNA molecule comprises an aptamer specifically binding to an interacting cell surface of said fungus species, and further comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene of the pest species to be down-regulated and optionally at least one carrier, excipient suitable for the intended use.
  • the nature of the excipients and the physical form of the composition may vary depending upon the nature of the substrate that it is desired to treat.
  • the composition may be a liquid that is brushed or sprayed onto or imprinted into the material or substrate to be treated, or a coating that is applied to the material or substrate to be treated.
  • the present invention further encompasses a method for treating and/or preventing fungal infestation on a substrate comprising applying an effective amount of any of the compositions described herein to said substrate.
  • the present invention also relates to methods for treating and/or preventing pest infestation on a substrate comprising applying an effective amount of a multidomain RNA molecule, nucleic acid, vector, or a composition thereof as described herein to said substrate.
  • the present invention also relates to methods for treating and/or preventing pest growth and/or pest infestation of a plant or propagative or reproductive material of a plant comprising applying an effective amount of a multidomain RNA molecule, nucleic acid, vector, or a composition thereof as described herein to a plant or to propagation or reproductive material of a plant.
  • the invention in another embodiment, relates to a method for controlling pest growth on a cell or an organism or for preventing pest infestation of a cell or an organism susceptible to infection to said pest species, comprising contacting said pest species with any of the multidomain RNA molecule, nucleic acid, vector, or a composition thereof described herein, whereby the multidomain RNA molecule is taken up by said pest species and thereby controls growth or prevents infestation.
  • Bacteria can be engineered to produce any of the dsRNA or dsRNA constructs of the invention. These bacteria can be eaten by the pest species. When taken up, the dsRNA can initiate an RNAi response, leading to the degradation of the target mRNA and weakening or killing of the feeding pest.
  • said multidomain RNA molecule is expressed by a prokaryotic, such as a bacterial, or eukaryotic, such as a yeast, host cell or host organism.
  • a prokaryotic such as a bacterial, or 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 the Leguminosea (for example Soy).
  • recombinant bacteria could be mixed with the seeds (ie coating) and used as soil improve rs.
  • dsRNA producing bacteria can be sprayed directly onto the crops, for instance Bacillus thuringiensis species.
  • Possible applications include intensive greenhouse cultures, for instance crops that are less interesting from a GMO point of view, as well as broader field crops such as soy.
  • This approach has several advantages, eg: since the problem of possible dicing by a plant host is not present, it allows the delivery of dsRNA into the gut lumen of the feeding pest; the use of bacteria as insecticides does not involve the generation of transgenic crops, especially for certain crops where transgenic variants are difficult to obtain; there is a broad and flexible application in that different crops can be simultaneously treated on the same field and/or different pests can be simultaneously targeted, for instance by combining different bacteria producing distinct dsRNAs.
  • the invention encompasses the GMO approaches and thus relates to a method as described above wherein said double- stranded RNA is expressed by said cell or organism infested with or susceptible to infestation by said pest species, for instance said cell is a plant cell or said organism is a plant.
  • the invention further relates to a method for increasing plant yield comprising introducing in a plant any of the multidomain RNA molecules, nucleic acid, vector or composition thereof in an expressible format.
  • RNA molecules for delivering dsRNA from a plant to a feeding organism are numerous.
  • a first major advantage of the multidomain RNA molecule according to the present invention is that it permits to deliver dsRNA more efficiently intracellular ⁇ in target organisms.
  • One of the advantages is that feeding organisms dsRNA which is efficiently endocytosed can lead to more efficient uptake of dsRNA (lower concentrations of dsRNA needed to reach an effect).
  • RNA molecules eaten by insects can enter gut cells, through endocytosis (binding of toll receptor) or transporter related mechanisms.
  • >80bp dsRNAs are more effective in feeding experiments than small 21- mers because their charges make them bind non-specifically to the enterocyte cell surface.
  • the multidomain RNA molecule could consist of a domain that binds a plant or insect protein or sugar moiety that is efficiently endocytosed in the insect gut for instance binding to transferrins, lectins, etc... in the gut.
  • the multidomain RNA molecule comprising the aptamer and the dsRNA is taken up by the target organism by feeding can enter the gut cells through endocytosis, e.g. by entering endocytic vesicles, and from there is capable of entering the cell cytoplasm. In the endosome the aptamer-dsRNA molecule may be degraded. A fraction of the dsRNA will then enter the cell in a manner similar to the delivery mechanism for antisense RNAs, as is well known in the literature.
  • the gut of an insect is a hostile environment, having low or high pH values and RNases which degrade RNA.
  • High pH such as found in lepidopteran guts degrades RNA chemically enhanced.
  • Transcytosis of the RNA molecules to the haemolymph enables to quickly remove the dsRNA from an RNase rich and/or pH hostile environment.
  • the multidomain RNA molecule according to the present invention comprises an aptamer that is capable of binding a protein that is transcytosed through the gut of a target organism to the haemolymph or coelomic fluid, or that binds to a transcytosis receptor
  • the chimeric multidomain RNA molecule which is taken up by the target organism by feeding can enter the gut and pass through the gut to the haemolymph or coelomic fluid.
  • Transcytosis of the multidomain RNA molecules to the haemolymph or coelomic fluid allows to direct the dsRNA to a broad range of target tissues including muscles, CNS, and other.
  • RNA molecules may even comprise small RNA fragments, e.g. 21 mers, and efficiently deliver these to the gut cell of a target pest organism. Feeding a target organism dsRNA that has been bound to an aptamer according to the present invention and that is efficiently delivered results in an improved uptake of the dsRNA. As a result thereof, lower amounts of dsRNA need to be used in order to obtain a suitable effect in the target organism.
  • Another advantage of the present multidomain RNA molecule is that when forming a multidomain molecule of aptamers and RNA capable of forming dsRNA for the purpose of causing interference, the aptamer moiety may effectively protect the dsRNA molecules from degradation in the plant and in the gut of the target organism, which is a very hostile environment.
  • the present multidomain RNA molecule may thus allow expression of long as well as short dsRNA fragments in plants, to be delivered by feeding to a target organism.
  • long (of e.g. 80 bp or more) dsRNA fragments are expressed in a plant for delivery by feeding to a target organism.
  • Expression of long dsRNA fragments involves several disadvantages. For instance, it makes it necessary to protect these long RNA fragments in the plant cytoplasm from Dicer activity in the endogenous plant.
  • RNA fragments Dicing of longer RNA fragments may result in diced fragments that may create dominant negative effects in plants through tittering away RISC which is needed for normal plant growth and physiology, or which may down regulate plant genes or chromatic or even the transgenic dsRNA expressing gene.
  • RISC ribonucleic acid
  • 21 bp may be effectively fed to target organisms and taken up in the gut enterocytes leading to target knockdown. As a consequence thereof, it may be not longer required to express long RNA fragments of 80 bp or longer in the plants, and shorter -and thus more specific and selective— target fragments can be expressed in plants.
  • RNA molecules comprising aptamers which protect the RNA, may obviate the need of additionally protecting these fragments from dicing activity in the endogenous plant.
  • Another advantage can arise if one uses an aptamer sequence that binds a cellular plant protein or structure specifically, permitting the dsRNA molecule to be accumulated in the plant in a selected compartment (e.g. nucleolus, nucleus, cytoplasm, trna or ribosome, phloem) for more efficient accumulation or delivery to the pest.
  • a selected compartment e.g. nucleolus, nucleus, cytoplasm, trna or ribosome, phloem
  • Example 1 Non-limiting examples of aptamer-dsRNA fusions that recognize a lethal gene in C. elepans
  • dsRNA molecules are linked to an aptamer, for instance and aptamer that recognizes nhx-2, an Na-H exchanger, encoded by the gene BO495.4, required for feeding of the animals.
  • Nhx-2 is localized to the epithelial cells of the ⁇ /vorm intestine.
  • Nhx-2 is a transmembrane protein, displaying several extracellular and intracellular loops, separated by 10-12 transmembrane domains.
  • Nhx-2 has been shown to be internalized by the cellular endocytosis machinery, an ideal target pathway to increase the uptake of dsRNA.
  • the aptamer is designed to bind specifically and with high affinity to an extracellular loop of nhx-2.
  • the aptamer is fused to dsRNA targeting the silencing of sup-35, a suppressor of the pha-1 temperature sensitive mutation.
  • dsRNA targeting the silencing of sup-35, a suppressor of the pha-1 temperature sensitive mutation.
  • the aptamer Upon feedingysoaking on the aptamer- dsRNA fusion, the aptamer targets the fusion to the intestinal cells, leading to an increased uptake of the dsRNA.
  • the increased RNAi effects induced by sup-35 dsRNA are quantified by counting the number of pha-1 animals reaching the L4 stage at the non- permissive temperature (see below for details).
  • Step 1 Selection and production of the "nhx-2-ap" aptamer.
  • an aptamer that recogn izes nhx-2 is selected.
  • the Nhx-2 complete sequence protein sequence: NP_495614, nucleotide sequence: NM_063213
  • Figure 4 SEQ ID NO 1
  • the fifth extracellular loop of the nhx-2 protein in bold and underlined on Figure 4 is chosen as the recognition target site for the aptamer, because it is the largest predicted extracellular loop in the protein.
  • a synthetic peptide, corresponding to the 5 th nhx-2 extracellular loop, is synthesized using standard methods, for example, provided by the company NeoMPS (NeoMPS SA - 7 rue de Boulogne - 67100 France - France).
  • NeoMPS NeoMPS SA - 7 rue de Boulogne - 67100 France - France
  • a control aptamer or scrambled aptamer, composed of identical nucleotides composition, but in random order is also generated.
  • the synthetic peptide is then used as a bait to identify an aptamer designed to specifically recognize and bind the 5 th extracellular loop of nhx-2.
  • Step 1 a highly selective and potent aptamer, against the 5 th extracellular loop of nhx-2 is identified.
  • Step 2 Constructions and in vitro production of a multidomain RNA molecule
  • a multidomain RNA molecule is constructed using standard molecular biology techniques.
  • the multidomain RNA molecule contains (1 ) the aptamer described in step 1 and (2) a dsRNA fragment targeting the silencing of sup-35.
  • RNAi-induced silencing of sup-35 was previously shown to suppress the lethality of the pha-1 temperature sensitive mutant.
  • the Sup-35 DIMA sequence, coding for the dsRNA sup-35 fragment is represented in Figure 5 (SEQ ID NO 2).
  • the DNA encoding for the aptamer is fused, in frame, at either the 5' position of the DNA encoding the sense RNA ( Figure 2A) or at the 3' position of the DNA encoding the antisense RNA ( Figure 2B).
  • the scrambled (i.e. randomly shaken) aptamer is used to generate a fusion to the dsRNA at either the 5' position of the DNA encoding the sense RNA or at the 3' position of the DNA encoding the antisense RNA.
  • single sup-35 specific dsRNA is used (Figure 2C). The five RNA samples are generated using standard in vitro transcription assay methods (see Figure 3).
  • RNA fusions comprise:
  • the Naked dsRNA comprises sup-35 (according to Figure 2-C)
  • Step 3 Improved RNAi effect induced by nhx-2 ap / sup-35 ds - in vivo testing in C. elepans
  • RNAi effect The efficacy of the four multidomain RNA molecules and the naked dsRNA is tested in in vivo assays.
  • C. elegans animals are soaked in media containing in vitro synthesized multidomain RNA molecules (see Step 2) in increasing concentrations (DRCs).
  • DRCs concentrations
  • the soaking RNAi method is used to knock down sup-35 (without aptamer fusion) by RNAi in a pha-1 mutant background.
  • the invention presented above, where the dsRNA is fused to an aptamer, will improve the effect achieved with sup-35 dsRNA only. For example, the RNAi effect will be achieved when using significantly lower dsRNA concentrations.
  • the materials required to perform the soaking RNAi method include: pha-1 (e2123) or wild-type N2 Bristol strains; M9 medium and IM9 + PEG; dsRNA of target gene (3.8 mg/ml) e.g. obtained by T7 RiboMAXTM Express RNAi System, Promega or obtained by Megascript RNAiTM kit, Ambion; lipofectamine (2mg/ml), Invitrogen; big drop 3 cm NGIVI plates
  • the soaking RNAi method comprises the following steps: :produce dsRNA using T7 " polymerase transcription of the template DNA following the provided protocol of RNA. dsRNA kit manufacturers (e.g. Promega, Ambion etc. ); grow pha-1 or N2 until L4 stage (15°C); remove L4 hermaphrodites with M9 and wash 3 times; transfer the worms to a fresh unseeded NGM plate and let dry for 15 min.
  • RNAi Effectiveness of RNAi can be examined by setting-up dose-response curve analysis to identify the dsRNA concentration to achieve a certain phenotype (e.g. pha-1 rescue, lethality) to a certain arbitrary level.
  • a serial 3-fold dilution series of dsRNA (starting at 3.8 ⁇ g/ ⁇ l) can be applied to identify the effective concentrations for the claimed constructs, in comparison to the regular dsRNA construct.
  • Example 2 Construction of multidomain RNA molecules that recognize lethal genes in pest species
  • the aptamer-dsRNA molecule is principally a fusion construct that contains a target RNA sequence and, an aptamer at the 3' or 5' end, with recognition sequence for an protein, such as for instance nhx-2 (NM_063213).
  • the gene sup-35 is used to exemplify the invention.
  • the target gene is beta-tubulin (Genbank accession for beta-tubulin in C. elegans NMJD66966), see Table 2.
  • the length of the tested target RNA sequence is 80-300 base pairs. Target gene from the target species are cloned accordingly.
  • beta-tubulin fragment in four different plant pest species Meloidogyne incognita, Caenorhabditis elegans, IMilaparvata lugens and Magnaporthe grisea.
  • RNAi fragment of target gene beta- tubulin sequence is provided in figures 6 to 9 (SEQ ID NOs 3 to 6), respectively.
  • Example 3 Non-limiting examples of aptamer-dsRNA fusions that recognize carbohydrate molecules (or sugar moieties)
  • dsRNA molecules are linked to an aptamer that recognizes carbohydrate molecules (or sugar moieties).
  • Carbohydrate molecules (or sugar moieties) are for instance localized to the epithelial cells of the worm intestine.
  • the aptamer is designed to bind specifically and with high affinity to the specific carbohydrate molecules (or sugar moieties), such GalNac or mannose.
  • the aptamer is fused to dsRNA targeting the silencing of, for instance sup-35, a suppressor of the pha-1 temperature sensitive mutation.
  • the aptamer Upon feeding/soaking on the aptamer-dsRNA fusion , the aptamer targets the multidomain RNA molecule to the glycoprotein on the intestinal cells, leading to an increased uptake of the dsRNA.
  • the increased RNAi effects induced by sup-35 dsRNA are quantified by counting the number of pha-1 animals reaching the L4 stage at the non- permissive temperature (see above for details).
  • Step 1 Selection and production of the aptamer.
  • a synthetic peptide, corresponding to the carbohydrate molecule (or sugar moiety) is synthesized using standard methods, for example, provided by the company NeoMPS (NeoMPS SA - 7 rue de Boulogne - 67100 France. For further steps and detail see Exaple 1
  • Example 4 In planta test for efficient pest control
  • the multidomain RNA molecules of the present invention are cloned behind the CaMV35S promoter, a root specific promoter or a feeding site specific promoter in a binary vector suitable for plant transformation.
  • the binary vectors are transferred to Agrobacterium rhizogenes by three-parental mating (e.g. by E. coli HB101 containing pRK2013 helper plasmid).
  • the binary vectors are transferred from Esherichia coli into Agrobacterium tumifaciens. Subsequently crops plants (such as tomato, soybean, cotton or Tobacco) are transformed with the constructs via agrobacterium-mediated transformation.
  • Agrobacterium without binary vector is used as a control.
  • Further controls are multidomain RNA molecules which do not comprise an aptamer as described in the present invention or a linker as described in the present invention.
  • Stability of the multidomain RNA molecules of the present invention in plant cells The stability of the expressed constructs are analyzed with quantitative real-time PCR to determine the quantity of the expressed construct of the invention present in the transgenic plant cell relative to the quantities present in control transgenic plants.
  • the method to monitor PCR in real-time is described previously and is based on Taqman probes or intercalating dyes (SYBR green).
  • the expressed multidomain RNA molecules are quantified relative towards a standard dilution series of the template.
  • the results are normalized by using the quantitative PCR data of a set of housekeeping genes from the same samples (Vandesompele et al., Genome Biology 2002, 3:research0034.1-0034.11).
  • Tomato e.g. Lycopersicum esculentum cv. Marmande
  • cotton ⁇ Gossypium hirsutum cotyledons are transformed with A. rhizogenes and transformed hairy roots are tested for nematode resistance.
  • the necessary number of independent transformed lines e.g. 15
  • replicates per line e.g. 10
  • Egg masses can be put to hatch and the fecundity of the parasite are investigated.
  • the offspring are used to test infectivity/viability of the second generation.
  • An analogous assay is performed whereby the hairy roots are transformed with the multidomain RNA molecule with a fungal target gene sequence and whereby the hairy roots are inoculated with a fungus, for example Magnaporte grisea.
  • Plant tissues (such as tomato tissue) are transformed with the constructs of the present invention and regenerated into whole plants.
  • Whole transgenic plants are inoculated with the pest species and the phenotype of the plant is monitored.

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Abstract

L'invention concerne des procédés et des produits de synthèse permettant d'administrer un ARN en double brin (RNAdb) à des organismes de parasites. Plus précisément, l'invention concerne une molécule d'ARN multidomaine comprenant une séquence nucléotidique renfermant : (i) au moins un aptamère et (ii) au moins une séquence nucléotidique étudiée formant un ARN en brin double, celui-ci comprenant des brins complémentaires recuits, un de ceux-ci présentant une séquence nucléotidique complémentaire d'au moins partiellement une séquence nucléotidique cible de parasites. L'invention concerne également des acides nucléiques codant les molécules d'ARN multidomaine et diverses utilisations des molécules d'ARN multidomaine en agriculture.
EP05801497A 2004-10-25 2005-10-25 Arn à domaines multiples comprenant au moins un aptamère pour délivrer arn à double brin dans un organisme parasite Withdrawn EP1805308A2 (fr)

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PCT/EP2005/011439 WO2006045590A2 (fr) 2004-10-25 2005-10-25 Molecules destinees a l'administration d'arn en brin double a des organismes de parasites

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US7612194B2 (en) 2001-07-24 2009-11-03 Monsanto Technology Llc Nucleic acid sequences from Diabrotica virgifera virgifera LeConte and uses thereof
US20050256071A1 (en) * 2003-07-15 2005-11-17 California Institute Of Technology Inhibitor nucleic acids
CA2693280C (fr) 2004-04-09 2017-09-12 Monsanto Technology Llc Compositions et procedes pour le controle des invasions d'insectes sur les plantes
CA2812343C (fr) 2005-09-16 2017-12-12 Monsanto Technology Llc Methodes de controle genetique de l'infestation de plantes par des parasites, et compositions a cet effet
EP2270181B1 (fr) 2005-09-16 2015-10-21 deVGen N.V. L'ARN à double brin pour la lutte contre les insectes
US20100324113A1 (en) * 2006-06-01 2010-12-23 Sullenger Bruce A Delivery Method
US8847013B2 (en) 2008-01-17 2014-09-30 Pioneer Hi Bred International Inc Compositions and methods for the suppression of target polynucleotides from lepidoptera
US8809625B2 (en) 2008-01-17 2014-08-19 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides from Lygus
US8367895B2 (en) 2008-01-17 2013-02-05 Pioneer Hi-Bred International, Inc. Compositions and methods for the suppression of target polynucleotides from the family aphididae
WO2011153418A2 (fr) 2010-06-04 2011-12-08 Broglie Karen E Compositions et procédés utilisables en vue de la lutte contre les punaises des bois
KR102386113B1 (ko) * 2013-12-20 2022-04-12 더 유니버서티 어브 퀸슬랜드 이중층 수산화물 입자 상으로 흡착된 식물-보호 이중-가닥 RNA를 포함하는 식물-보호 RNAi 조성물
EP3322295B1 (fr) 2015-06-19 2022-09-28 The University of Queensland Composition
CN112899301B (zh) * 2021-01-29 2023-04-11 中国热带农业科学院热带生物技术研究所 一种木薯普通花叶病毒诱导的基因沉默系统及其应用

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