WO2006070227A2 - Procede de regulation de l'expression genetique dans des champignons - Google Patents

Procede de regulation de l'expression genetique dans des champignons Download PDF

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WO2006070227A2
WO2006070227A2 PCT/IB2005/003495 IB2005003495W WO2006070227A2 WO 2006070227 A2 WO2006070227 A2 WO 2006070227A2 IB 2005003495 W IB2005003495 W IB 2005003495W WO 2006070227 A2 WO2006070227 A2 WO 2006070227A2
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spp
plant
fungal
cell
double
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PCT/IB2005/003495
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WO2006070227A3 (fr
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Marc Van De Craen
Phuay-Yee Goh
Marc Georges Logghe
Yee-Ling Khu
Katherine Mortier
Thierry Andre Olivier Eddy Bogaert
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Devgen Nv
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Priority claimed from GB0422094A external-priority patent/GB0422094D0/en
Application filed by Devgen Nv filed Critical Devgen Nv
Priority to US11/396,926 priority Critical patent/US20060247197A1/en
Publication of WO2006070227A2 publication Critical patent/WO2006070227A2/fr
Publication of WO2006070227A3 publication Critical patent/WO2006070227A3/fr
Priority to US12/778,278 priority patent/US8865968B2/en
Priority to US14/488,493 priority patent/US20150133523A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—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 fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the invention relates to methods for controlling fungal growth on cells or organisms, methods for preventing fungal infestation of cells or organism and methods for down- regulating gene expression in fungi using double-stranded RNA.
  • the invention also relates to transgenic plants resistant to fungal infestation.
  • RNA interference or "RNAi” is a process of sequence-specific down-regulation of gene expression (also referred to as “gene silencing” or “RNA-mediated gene silencing") initiated by double-stranded RNA (dsRNA) that is complementary in sequence to a region of the target gene to be down-regulated (Fire, A. Trends Genet. Vol. 15, 358-363, 1999; Sharp, P.A. Genes Dev. Vol. 15, 485-490, 2001).
  • dsRNA double-stranded RNA
  • RNAi RNA interference
  • RNAi comprises contacting the organism with a double-stranded RNA fragment (generally either as two annealed complementary single-strands of RNA or as a hairpin construct) having a sequence that corresponds to at least part of a gene to be down-regulated (the
  • RNAi can be performed by feeding the nematode with the RNAi fragment or with a bacterial strain that either contains the RNAi fragment or that upon ingestion by the nematode is capable of expressing the RNAi fragment.
  • RNAi by feeding reference may be made to International application WO 00/01846 by the present applicant, to 1998 East Coast Worm Meeting abstract 180 - Timmons and Fire (see www.elegans.swmed.edu/wli/[ecwm98p180]/), and again to WO 99/32619.
  • RNAi has also been proposed as a means of protecting plants against plant parasitic nematodes, i.e. by expressing in the plant (e.g. in the entire plant, or in a part, tissue or cell of a plant) one or more nucleotide sequences that form a dsRNA fragment that corresponds to a target gene in the plant parasitic nematode that is essential for its growth, reproduction and/or survival.
  • RNAi-mediated gene silencing in mammalian cellg using dsRNA fragments of 21 nucleotides in length (also termed small interfering RNAs or siRNAs). These short siRNAs demonstrate effective and specific gene silencing, whilst avoiding the interferon-mediated non-specific reduction in gene expression which has been observed with the use of dsRNAs greater than 30bp in length in mammalian cells (Stark G. R. et al., Ann Rev Biochem. 1998, 67: 227-264; Manche, L et al., MoI Cell Biol., 1992, 12: 5238-5248).
  • RNAi has been proposed as an alternative to the use of antisense technology for specific down-regulation or gene silencing of target genes in mammalian cells.
  • RNAi has been generally known in the art in plants, nematodes and mammalian cells for some years, to date little is known about the use of RNAi to down-regulate gene expression in fungi.
  • RNA- mediated gene silencing in the ascomycete fungus Magnaporthe oryzae (formerly Magnaporthe grisea; anamorph Pyricularia oryzae Cav. and Pyricularia grisae), the causal agent of rice blast disease, by a mechanism having molecular features consistent with RNAi.
  • Gene silencing was achieved by expression of dsRNA inside cells of the fungus: fungal protoplasts were transformed in the laboratory using DNA constructs capable of expressing the double-stranded RNA, such that the double-stranded RNA is transcribed within cells of the fungus.
  • RNAi in the filamentous fungus Neurospora crassa. Gene silencing was achieved by transforming fungal cells with a transgene capable of expressing the double-stranded RNA, allowing the double-stranded RNA to be transcribed within cells of the fungus. Liu et al., (2002) Genetics. 160: 463-470 describe RNA interference in the human pathogenic fungus Cryptococcus neoformans.
  • RNAi was achieved by transforming fungal cells in culture with a DNA construct encoding the double-stranded RNA, such that the double-stranded RNA was transcribed in situ in the fungal cells.
  • RNAi techniques requiring transformation of fungal cells with a DNA construct that directs production of dsRNA within the fungal cells are useful for experimental studies within the laboratory but are clearly not suitable for many potential practical applications of RNAi, for example applications which require dsRNA to be introduced into many fungal cells on a large scale or in the field, for example, to protect plants against plant pathogenic fungi or large scale treatment of substrates to protect against fungal infestation, or for pharmaceutical or veterinary use in the treatment or prevention of fungal infestation in humans or animals.
  • gene expression can be specifically down-regulated in fungi by contacting intact fungal cells (i.e. with an intact cell wall) with double-stranded RNA outside the cell (i.e. external to the cell wall), wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to (i.e. is complementary to at least part of) a target nucleotide sequence of a target gene of a fungus to be down-regulated.
  • RNAi in fungi avoids the need for complicated transformation procedures in order to introduce a transgene capable of directing expression of double-stranded RNA within cells of the fungus. Accordingly, there is no need for the fungus itself to be genetically manipulated and in particular no need to transform the fungal cells using non-natural procedures in order to introduce a DNA construct directing expression of dsRNA within the fungal cells.
  • the technique is simple and of great practical utility and opens up a whole range of different applications of RNAi in fungi that simply would not be practical using the prior art techniques.
  • the methods of the invention can find practical application in any area of technology where it is desirable to inhibit viability, growth, development or reproduction of the fungus, or to decrease pathogenicity or infectivity of the fungus.
  • the methods of the invention further find practical application where it is desirable to specifically down-regulate expression of one or more target genes in a fungus. Particularly useful practical applications include, but are not - A -
  • the invention relates to a method for controlling fungal growth in or on a cell or an organism or for preventing fungal infestation of a cell or an organism susceptible to fungal infection, comprising contacting fungal cells with a double- stranded RNA from outside the fungal cell(s), wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of a fungal target gene, whereby the double-stranded RNA is taken up into the fungal cells and thereby controls growth or prevents infestation.
  • the invention relates to a method for down- regulating expression of a target gene in a fungus, comprising contacting fungal cell(s) with a double-stranded RNA from outside the fungal cell(s), wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of the nucleotide sequence of the fungal target gene to be down-regulated, whereby the double-stranded RNA is taken up into the fungal cells and thereby down-regulates expression of the fungal target gene.
  • the methods of the invention rely on uptake into fungal cells of double-stranded RNA present outside of the fungus (i.e. external to the cell wall) and does not require expression of double-stranded RNA within cells of the fungus.
  • the present invention also encompasses methods as described above wherein the fungal cell(s) is contacted with a composition comprising the double-stranded RNA.
  • Said double-stranded RNA may be expressed by a prokaryotic (for instance but not limited to a bacterial) or eukaryotic (for instance but not limited to a yeast) host cell or host organism.
  • a prokaryotic for instance but not limited to a bacterial
  • eukaryotic for instance but not limited to a yeast
  • the methods of the invention rely on a GMO approach wherein the double stranded RNA is expressed by a cell or an organism infested with or susceptible to infestation by fungi.
  • said cell is a plant cell or said organism is a plant.
  • the present invention thus also relates to a method for producing a plant resistant to a plant pathogenic fungus, comprising:
  • nucleotide sequence comprising a sense strand comprising the nucleotide sequence of (i) and an antisense strand comprising the complement of said nucleotide sequence of (i), wherein the transcript encoded by said nucleotide sequence is capable of forming a double-stranded RNA, (b) regenerating a plant from the transformed plant cell;
  • the double- stranded RNA is expressed from a recombinant construct, which construct comprises at least one regulatory sequence operably linked to said nucleotide sequence which is complementary to at least part of said nucleotide sequence of said fungal target gene to be down-regulated.
  • the fungal cell(s) can be any fungal cell, meaning any cell present within or derived from an organism belonging to the Kingdom Fungi.
  • the methods of the invention are applicable to all fungi and fungal cells that are susceptible to gene silencing by RNA interference and that are capable of internalising double-stranded RNA from their immediate environment.
  • the fungus may be a mould, or more particularly a filamentous fungus. In other embodiments of.the invention, the fungus may be a yeast.
  • the fungus may be an ascomycetes fungus, i.e. a fungus belonging to the Phylum Ascomycota.
  • the fungal cell is chosen from the group consisting of:
  • a fungal cell of, or a cell derived from a plant pathogenic fungus such as but not limited to Acremoniella spp., Alternaria spp. (e.g. Alternaria brassicola or Alternaria solani), Ascochyta spp. (e.g. Ascochyta pisi), Botrytis spp. (e.g. Botrytis cinerea or Botryotinia fuckeliana), Cladosporium spp., Cercospora spp. (e.g. Cercospora kikuchii or Cercospora zaea-maydis), Cladosporium spp. (e.g.
  • Cladosporium fulvum Colletotrichum spp. (e.g. Colletothchum lindemuthianum), Curvularia spp., Diplodia spp. (e.g. Diplodia maydis), Erysiphe spp. (e.g. Erysiphe graminis f.sp. graminis, Erysiphe graminis f.sp. hordei or Erysiphe pisi), Erwinia armylovora, Fusarium spp. (e.g. Fusarium nivale, Fusarium sporotrichioides, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum,
  • Fusarium spp. e.g. Fusarium nivale, Fusarium sporotrichioides, Fusarium oxysporum, Fusarium gramine
  • Gaeumanomyces spp. e.g. Gaeumanomyces graminis f.sp. tritici
  • Gibberella spp. e.g. Gibberella zeae
  • Nectria spp. e.g. Nectria heamatococca
  • Peronospora spp. e.g. Peronospora manshurica or Peronospora tabacina
  • Phoma spp. e.g. Phoma betae
  • Phakopsora spp. e.g. Phakopsora pachyrhizi
  • Phymatotrichum spp. e.g. Phymatotrichum omnivorum
  • Phytophthora spp. e.g.
  • Phytophthora cinnamomi Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora citrophthora, Phytophthora megasperma f.sp. soiae or Phytophthora infestans
  • Plasmopara spp. e.g. Plasmopara viticola
  • Podosphaera spp. e.g. Podosphaera leucotricha
  • Puccinia spp. e.g. Puccinia sorghi, Puccinia striiformis, Puccinia graminis f.sp.
  • Puccinia asparagi Puccinia recondita or Puccinia arachidis
  • Pythium spp. e.g. Pythium aphanidermatum
  • Pyrenophora spp. e.g. Pyrenophora tritici-repentens or Pyrenophora teres
  • Pyricularia spp. fe.g. Pyricularia oryzae
  • Pythium spp. e.g. Pythium ultimum
  • Rhincosporium secalis Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis), Rhizopus spp.
  • Uncinula necator Ustilago maydis (e.g. corn smut), Venturia spp. (e.g. Venturia inaequalis or Venturia pirina) or Verticillium spp. (e.g. Verticillium dahliae or Verticillium albo-atrum);
  • Venturia spp. e.g. Venturia inaequalis or Venturia pirina
  • Verticillium spp. e.g. Verticillium dahliae or Verticillium albo-atrum
  • a fungal cell of, or a cell derived from a fungus capable of infesting humans such as, but not limited to, 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 or Aspergillus terreus); Blastomyces dermatitidis; Paracoccidioides brasiliensis; Coccidioides immitis; Cryptococcus neoformans; Histoplasma capsulatum Var.
  • Candida spp. particularly Candida albicans
  • Dermatophytes including Epidermophyton spp., Trichophyton spp, and Microsporum spp.
  • Aspergillus spp. particularly Aspergillus flavus
  • fungi that attack foodstuffs, seeds, wood, paint, plastic, clothing etc.
  • fungi are the moulds, including but not limited to Stachybotrys spp., Aspergillus spp., Alternaria spp., Cladosporium spp., Penicillium spp. or Phanerochaete chrysosporium.
  • Preferred plant pathogenic fungi according to the invention are Cercospora spp. (e.g.
  • Cercospora kikuchii or Cercospora zaea-maydis causing e.g. black and yellow sigatoka in banana
  • Colletotrichum spp. e.g. Colletotrichum lindemuthianum
  • Curvularia spp. causing seedling blight
  • Diplodia spp. e.g. Diplodia maydis
  • Fusarium spp. e.g.
  • Phakopsora spp. e.g. Phakopsora pachyrhizi
  • Phakopsora spp. e.g. Phakopsora pachyrhizi
  • Puccinia sorghi Puccinia striiformis (yellow rust), Puccinia graminis f.sp. tritici, Puccinia asparagi, Puccinia recondita or Puccinia arachidis) causing e.g. common rust in corn; Rhizoctonia spp. fe. g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis) causing e.g. sheath blight in rice or early blight in potato,- Rhizopus spp. (e.g. Rhizopus chinensid) causing seedling blight; Trichoderma spp. (e.g.
  • Trichoderma virde causing seedling blight; or Verticillium spp. (e.g. Verticillium dahliae or Verticillium albo-atrum) causing e.g. verticillium wilt in cotton.
  • Verticillium spp. e.g. Verticillium dahliae or Verticillium albo-atrum
  • Particularly preferred plant pathogenic fungi according to the invention are Magnaporthe oryzae causing e.g. rice blast; Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis) causing e.g. sheath blight in rice; Curvularia spp., Rhizopus spp. (e.g.
  • Rhizopus chinensid Trichoderma spp. (e.g. Trichoderma virde) causing seedling blight in rice; Phakopsora spp. causing e.g. soybean rust; Phytophthora spp. (e.g. Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora citrophthora, Phytophthora megasperma f.sp. soiae or Phytophthora infestans) causing e.g. late blight in tomato and potato; Cercospora spp. (e.g.g.
  • Cercospora kikuchii or Cercospora zaea-maydis or Mycosphaerella spp. causing e.g. black and yellow sigatoka in banana; or Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum) causing e.g. Panama disease in banana.
  • a particularly preferred plant pathogenic fungus is Magnaporthe oryzae causing rice blast.
  • the fungal cell may be an intact fungal cell, meaning that the fungal cell has a cell wall.
  • the fungal cell is contacted with double-stranded RNA by contacting the intact fungal cell with the double-stranded RNA.
  • the cell wall of the fungal cell need not be removed prior to contact with the double-stranded RNA.
  • the method of the invention comprises contacting the fungal cell with at least one double-stranded RNA, wherein the dsRNA is added or contacted outside of the fungal cell and external to the cell wall of the fungal cell, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence of a target fungal gene to be down- regulated.
  • the dsRNA is taken up by the fungal cell(s) through the cell wall.
  • the term "fungal cell” encompasses fungal cells of all types and at all stages of development, including specialised reproductive cells such as sexual and asexual spores.
  • the fungal cell encompasses the fungus as such and also other life forms of the fungus, such as haustoria, conidia, mycelium, penetration peg, spore, zoospores etc.
  • these fungi have two names: the teleomorph name describes the fungus when reproducing sexually; the anamorph name refers to the fungus when reproducing asexually.
  • the holomorph name refers to the "whole fungus", encompassing both reproduction methods.
  • the fungal cell which is contacted with the dsRNA is 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.
  • the present invention relates to any gene of interest in the fungus (which may be referred to herein as the "target gene") that can be down-regulated.
  • down-regulation of gene expression and “inhibition of gene expression” are used interchangeably and refer to a measurable or observable reduction in gene expression or a complete abolition of detectable gene expression, at the level of protein product and/or mRNA product from the target gene. Down-regulation or inhibition of gene expression is "specific" when down-regulation or inhibition of the target gene occurs without manifest effects on other genes of the fungal cell.
  • RNA solution hybridization nuclease protection
  • Northern hybridization reverse transcription
  • gene expression monitoring with a microarray
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell analysis
  • the "target gene” may be essentially any gene that it is desirable to be inhibited because it interferes with growth or pathogenicity or infectivity of the fungus.
  • the method of the invention is to be used to prevent fungal growth and/or infestation then it is preferred to select a target gene which is essential for viability, growth, development or reproduction of the fungus, or any gene that is involved with pathogenicity or infectivity of the fungus, such that specific inhibition of the target gene leads to a lethal phenotype or decreases or stops fungal infestation.
  • the target gene is such that when its expression is down-regulated or inhibited using the method of the invention, the fungal cell is killed, or the reproduction or growth of the fungal cell is stopped or retarded.
  • This type of target genes is considered to be essential for the viability of the fungus cell(s) and is referred to as essential genes. Therefore, the present invention encompasses a method as described herein, wherein the target gene is an essential gene.
  • Particular essential target genes suitable for the methods of the present invention are genes involved in essential cellular functions that maintain cell viability, cell growth and development, and reproduction. Examples of still other suitable target genes involved in different cellular processes are described in Tables 1 and 2. According to a further non-limiting embodiment, the target gene is such that when it is down-regulated using the method of the invention, the infestation or infection by the fungus, the damage caused by the fungus, and/or the ability of the fungus to infest or infect host organisms and/or cause such damage, is reduced.
  • the terms "infest” and “infect” or “infestation” and “infection” are generally used interchangeably throughout. This type of target genes is considered to be involved in the pathogenicity or infectivity of the fungus.
  • the present invention extends to methods as described herein, wherein the target gene is involved in the pathogenicity or infectivity of the fungus, preferably the fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia.
  • the advantage of choosing the latter type of target gene is that the fungus is blocked to infect further plants or plant parts and to form further generations.
  • a further advantage of using a target gene involved in pathogenicity or infectivity is that the dsRNA can be taken up by the fungus when it is growing inside the plant, so that the spores formed are unable to infect further plant parts.
  • target genes are conserved genes or fungus-specific genes.
  • the invention thus relates to RNAi-mediated down-regulation or inhibition of one or more of the Magnaporthe grisea target genes listed above in Table 1 , and also to down- regulation of the homologous/orthologous target genes in other fungal species as listed in Table 2. Therefore, the present invention extends to methods as described herein wherein the fungal target gene is involved in any of the cellular functions as defined in Table 1 (rightmost column).
  • a non-limiting example is for instance a method as described herein wherein the fungal target gene is a gene involved in the function of a ribosome, proteasome, spliceosome, APC complex; or a gene involved in nuclear transport, translation initiation, transcription (e.g. transcription activation), intracellular membrane traffic, DNA replication, mitotic spindle formation, vesicle transport or the cytoskeleton.
  • the fungal target gene is a gene involved in the function of a ribosome, proteasome, spliceosome, APC complex
  • a gene involved in nuclear transport, translation initiation, transcription (e.g. transcription activation), intracellular membrane traffic, DNA replication, mitotic spindle formation, vesicle transport or the cytoskeleton e.g. transcription activation
  • any suitable double-stranded RNA fragment capable of directing RNAi or
  • RNA-mediated gene silencing or inhibition of a fungal target gene may be used in the methods of the invention.
  • dsRNA is used to inhibit growth or to interfere with the pathogenicity or infectivity of the fungus.
  • the invention thus relates to isolated 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 fungus.
  • the target gene may be any of the target genes described herein, or a part thereof that exerts the same function.
  • an isolated 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 fungus.
  • the target gene may be any of the target genes described herein, or a part thereof that exerts the same function.
  • an isolated double-stranded may be any of the target genes described herein, or a part thereof that exerts the same function.
  • RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene, wherein said fungal target gene is essential for the viability, growth, development or reproduction of the fungus, preferably said fungal target gene is involved in any of the cellular functions as defined in Table 1; or wherein said fungal target gene is involved in the pathogenicity or infectivity of the fungus, preferably said fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia, said nucleotide sequence being capable of inhibiting expression of the target gene.
  • said target gene comprises a sequence which is at least 75%, 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% or 99% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 3, 42, 99, 100, 39, 60, 111 , 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1 , 41 , 184, 185, 97, 98, 37, 59, 124, 9, 45, 188, 189, 106, 13, 47, 109, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 186, 187, 103, 104, 105, 29, 55, 118, 17, 49, 108, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 27, 54, 122, 21, 51, 120, 15, 48 and 110, or the complement thereof, or
  • the growth inhibition can be quantified as being greater than about 5%, 10%, more preferably about 20%, 25%, 33%, 50%, 60%, 75%, 80%, most preferably about 90%, 95%, or about 99% as compared to a target cell that has been treated by control dsRNA.
  • an isolated double- stranded RNA wherein at least one of said annealed complementary strands comprises the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 99, 100, 111 , 112, 113, 114, 115, 116, 101, 102, 97, 98, 124, 106, 109, 126, 119, 127, 103, 104, 105, 118, 108, 121 , 125, 123, 107, 122, 120 and 110, or a double- stranded fragment of at least 17, preferably at least 18, 19, 20 or 21 , more preferably at least 22, 23 or 24 basepairs in length thereof.
  • said isolated double-stranded RNA comprises the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 192, 201, 202, 193, 190, 191 , 196, 199, 200, 194, 195, 198 and 197, or a double stranded fragment of 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof .
  • the double-stranded RNA does not share any significant homology with any host gene, or at least not 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 region. If genomic sequence data is available for the host organism one may cross-check sequence identity with the double-stranded RNA using standard bioinformatics tools.
  • the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence of the target gene to be down-regulated.
  • the other strand of the double-stranded RNA is able to base- pair with the first strand.
  • target region or “target nucleotide sequence” of the target fungal gene may be any suitable region or nucleotide sequence of the gene.
  • the target region should comprise at least 17, at least 18 or at least 19 consecutive nucleotides of the target gene, more preferably at least 20 or at least 21 nucleotide and still more preferably at least 22, 23 or 24 nucleotides of the target gene.
  • the double-stranded RNA will share 100% sequence identity with the target region of the fungal target gene.
  • 100% sequence identity over the whole length of the double stranded region 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 terms "corresponding to” or “complementary to” are used herein interchangeable, and when these terms are used to refer to sequence correspondence between the double-stranded RNA and the target region of the target gene, they are to be interpreted accordingly, i.e. as not absolutely requiring 100% sequence identity.
  • the % sequence identity between the double-stranded RNA and the target region will generally be at least 80% or 85% identical, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99%.
  • RNA equivalent substantially means that in the DNA sequence(s), the base “T” may be replaced by the corresponding base “U” normally present in ribonucleic acids.
  • the dsRNA contains a sequence which corresponds to the target region of the target gene 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 dsRNA may contain one or more substitute bases in order to optimise performance in RNAi. Substitution of even a single nucleotide may have an effect on activity of the dsRNA in RNAi. It will be apparent to the skilled reader how to vary each of the bases of the dsRNA in turn and test the activity of the resulting siRNAs (e.g. in a suitable in vitro test system) in order to optimise the performance of a given dsRNA.
  • 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.
  • RNAs short interfering RNAs
  • the minimum length of dsRNA preferably is at least about 80-100 bp in order to be efficiently taken up by certain pest organisms.
  • invertebrates such as the free living nematode C. elegans or the plant parasitic nematode Meloidogyne incognita, these longer fragments are more effective in gene silencing, possibly due to a more efficient uptake of these long dsRNA by the invertebrate.
  • RNA duplexes consisting of either 27-mer blunt or short hairpin (sh) RNAs with 29 bp stems and 2-nt 3' overhangs are more potent inducers of RNA interference than conventional 21-mer siRNAs.
  • molecules based upon the targets identified above and being either 27-mer blunt or short hairpin (sh) RNA's with 29-bp stems and 2-nt 3'overhangs are also included within the scope of the invention.
  • the double-stranded RNA fragment (or region) will itself preferably be at least 17 bp in length, preferably 18 or 19bp in length, more preferably at least 20bp, more preferably at least 21 bp, or at least 22 bp, or at least 23 bp, or at least 24 bp, 25 bp, 26 bp or at least 27 bp in length.
  • the expressions "double-stranded RNA fragment" or “double-stranded RNA region” refer to a small entity of the double-stranded RNA corresponding with (part of) the target gene.
  • the double stranded RNA is preferably between about 17-1500 bp, even more preferably between about 80 - 1000 bp and most preferably between about 17-27 bp or between about 80-250 bp; such as double stranded RNA regions of about 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 27 bp, 50 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp or 1500 bp.
  • the upper limit on the length of the double-stranded RNA may be dependent on i) the requirement for the dsRNA to be taken up by the fungal cell and ii) the requirement for the dsRNA to be processed within the cell into fragments that direct RNAi.
  • the chosen length may also be influenced by the method of synthesis of the RNA and the mode of delivery of the RNA to the cell.
  • the double-stranded RNA to be used in the methods of the invention will be less than 10,000 bp in length, more preferably 1000 bp or less more preferably 500 bp or less, more preferably 300 bp or less, more preferably 100 bp or less.
  • the optimum length of the dsRNA for effective inhibition may be determined by experiment.
  • the double-stranded RNA may be fully or partially double-stranded.
  • Partially double- stranded RNAs may include short single-stranded overhangs at' one or both ends of the double-stranded portion, provided that the RNA is still capable of being taken up by fungai cells and directing RNAi.
  • the methods of the invention can encompass simultaneous or sequential provision of two or more different double-stranded RNAs or RNA constructs to the same fungal cell, so as to achieve down-regulation or inhibition of multiple target genes or to achieve a more potent inhibition of a single target gene.
  • multiple targets are hit by the provision of one double-stranded RNA that hits multiple target sequences, and a single target is more efficiently inhibited by the presence of more than one copy of the double stranded RNA fragment corresponding to the target gene.
  • the double-stranded RNA construct comprises multiple dsRNA regions, at least one strand of each dsRNA region comprising a nucleotide sequence that is complementary to at least part of a target nucleotide sequence of a fungal target gene.
  • the dsRNA regions in the RNA construct may be complementary to the same or to different target genes and/or the dsRNA regions may be complementary to targets from the same or from different fungus species.
  • the double stranded RNA region comprises multiple copies of the nucleotide sequence that is complementary to the target gene.
  • the dsRNA hits a further target sequence of the same target gene.
  • the term "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.
  • a further target gene or "at least one other target gene” mean for instance a second, a third or a fourth, etc. target gene.
  • DsRNA that hits more than one of the above-mentioned targets, or a combination of different dsRNA against different of the above mentioned targets may be developed or used in the methods of the present invention.
  • the invention relates to an isolated double stranded RNA construct comprising at least two copies of the RNA equivalent of at least one of the nucleotide sequences represented by any of SEQ ID NOs 99, 100, 111, 112, 113, 114, 115, 116, 101 ,
  • said double-stranded RNA comprises the RNA equivalent of the nucleotide sequence as represented in SEQ ID NO 117, or a double stranded fragment of at least 17, preferably at least 18, 19, 20 or 21 , more preferably at least 22, 23 or 24 basepairs in length thereof.
  • the present invention extends to methods as described herein, wherein the dsRNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a fungal target gene, and which comprises at least one additional dsRNA region, at least one strand of which comprises a nucleotide sequence which is complementary to at least part of the nucleotide sequence of at least one other fungal target gene.
  • Such further target gene may be any of the target genes herein described.
  • the dsRNA hits at least one target gene that is essential for viability, growth, development or reproduction of the fungus and hits at least one gene involved in pathogenicity or infectivity as described hereinabove.
  • the dsRNA hits multiple genes of the same category, for example, the dsRNA hits at least 2 essential genes or at least 2 genes involved in pathogenicity or at least two genes involved in any of the cellular functions as described in Table 1.
  • the dsRNA hits 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) such as the genes shown in Figure 3.
  • the present invention extends to methods as described herein, wherein said fungal target gene comprises a sequence which is at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 3, 42, 99, 100, 39, 60, 111 , 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1 , 41 , 184, 185, 97, 98, 37, 59, 124, 9, 45, 188, 189, 106, 13, 47, 109, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 186, 187, 103, 104, 105, 29, 55, 118, 17, 49, 108, 25, 53, 121 , 19, 50, 125, 31, 56, 123, 11 , 46, 107, 27, 54, 122, 21 , 51 , 51
  • the dsRNA regions (or fragments) in the double stranded RNA may be combined as follows: a) when multiple dsRNA regions targeting a single target gene are combined, they may be combined in the original order (ie the order in which the regions appear in the target gene) in the RNA construct, 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 double stranded RNA construct, 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 ds RNA construct, or , d) the dsRNA regions (targeting a single or different target genes) may be combined in the sense or antisense orientation.
  • the target gene(s) to be combined may be chosen from one or more of the following categories of genes: e) "essential" genes or "pathogenicity genes" as described above encompass genes that are vital for one or more target fungi and result in a lethal or severe (e.g. feeding, reproduction, growth) phenotype when silenced.
  • the choice of a strong lethal target gene results in a potent RNAi effect.
  • multiple dsRNA regions targeting the same or different (very effective) lethal genes can be combined to further increase the potency, efficacy or speed of the RNAi effect in fungal control.
  • "weak” genes encompass target genes with a particularly interesting function in one of the cellular pathways described herein, but which result in a weak phenotypic effect when silenced independently.
  • multiple dsRNA regions targeting a single or different weak gene(s) may be combined to obtain a stronger RNAi effect.
  • "fungus specific” genes encompass genes that have no substantial homologous counterpart in non-fungus organisms as can be determined by bioinformatics homology searches, for example by BLAST searches. The choice of a fungal specific target gene results in a species specific RNAi effect, with no effect or no substantial (adverse) effect in non-target organisms.
  • h) conserved genes encompass genes that are conserved (at the amino acid level) between the target organism and non-target organism(s). To reduce possible effects on non-target species, such effective but conserved genes are analysed and target sequences from the variable regions of these conserved genes are chosen to be targeted by the dsRNA regions in the RNA construct. Here, conservation is assessed at the level of the nucleic acid sequence. Such variable regions thus encompass the least conserved sections, at the level of the nucleic acid sequence, of the conserved target gene(s).
  • RNA constructs according to the present invention target multiple genes from different biological pathways, resulting in a broad cellular RNAi effect and more efficient fungal control.
  • all double stranded RNA regions comprise at least one strand that is complementary to at least part or a portion of the nucleotide sequence of any of the target genes herein described.
  • the other double stranded RNA regions may comprise at least one strand that is complementary to a portion of any other fungal target gene (including known target genes).
  • an isolated double stranded RNA construct further comprising at least one additional sequence and optionally a linker.
  • the additional sequence is chosen from the group comprising (i) a sequence facilitating large-scale production of the dsRNA construct; (ii) a sequence effecting an increase or decrease in the stability of the dsRNA; (iii) a sequence allowing the binding of proteins or other molecules to facilitate uptake of the RNA construct by a fungal cell(s); (iv) a sequence which is an aptamer that binds to a receptor or to a molecule on the surface or in the cytoplasm of a fungal cell(s) to facilitate uptake, endocytosis and/or transcytosis by the fungal cell(s); or (v) additional sequences to catalyze processing of dsRNA regions.
  • the linker is a conditionally self-cleaving RNA sequence, preferably a pH sensitive linker or a hydrophobic sensitive linker. In one embodiment, the linker is an intron.
  • the multiple dsRNA regions of the double-stranded RNA construct are connected by one or more linkers.
  • the linker is present at a site in the RNA construct, separating the dsRNA regions from another region of interest. Different linker types for the dsRNA constructs are provided by the present invention.
  • the multiple dsRNA regions of the double-stranded RNA construct are connected without linkers.
  • the linkers may be used to disconnect smaller dsRNA regions in the pest organism.
  • the linker sequence may promote division of a long dsRNA into smaller dsRNA regions under particular circumstances, resulting in the release of separate dsRNA regions under these circumstances and leading to more efficient gene silencing by these smaller dsRNA regions.
  • RNA sequences that are self- cleaving at high pH conditions. Suitable examples of such RNA sequences are described by
  • a linker is located at a site in the RNA construct, separating the dsRNA regions from another, e.g. the additional, sequence of interest, which preferably provides some additional function to the RNA construct.
  • the dsRNA constructs of the present invention are provided with an aptamer to facilitate uptake of the dsRNA by the fungus.
  • the aptamer is designed to bind a substance which is taken up by the fungus. Such substances may be from a fungal or plant origin.
  • an aptamer is an aptamer that binds to a transmembrane protein, for example a transmembrane protein of a fungus.
  • the aptamer may bind a (plant) metabolite or nutrient which. is taken up by the fungus.
  • the linkers are self-cleaving in the endosomes.
  • the constructs of the present invention may be taken up by the fungus via endocytosis or transcytosis, and are therefore compartmentalized in the endosomes of the fungus species.
  • the endosomes may have a low pH environment, leading to cleavage of the linker.
  • linkers that are self-cleaving in hydrophobic conditions are particularly useful in dsRNA constructs of the 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 fungus pest organism.
  • An intron may also be used as a linker.
  • An "intron” as used herein may be any non- coding RNA sequence of a messenger RNA.
  • Particular suitable intron sequences for the constructs 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) start at the 5' end with -AG:GT- or - CG:GT- and/or (4) have at their 3' end -AG:GC- or -AG:AA.
  • a non-complementary RNA sequence ranging from about 1 base pair to about
  • 10,000 base pairs may also be used as a linker.
  • RNAs added externally to a fungal cell are taken up into the cell by the natural mechanisms by which fungal cells take up material from their immediate environment, such as for example pathways of endocytosis.
  • Double-stranded RNAs taken up into the cell are then processed within the cell into short double-stranded RNAs, called small interfering RNAs (siRNAs), by the action of an endogenous endonuclease.
  • the resulting siRNAs then mediate RNAi via formation of a multi-component RNase complex termed the RISC or RNA interfering silencing complex.
  • the double- stranded RNA added to the exterior of the cell wall may be any dsRNA or dsRNA construct that can be taken up into the cell and then processed within the cell into siRNAs, which then mediate RNAi, or the RNA added to the exterior of the cell could itself be an siRNA that can be taken up into the cell and thereby direct RNAi.
  • siRNAs are generally short double-stranded RNAs having a length in the range of from 19 to 25 base pairs, or from 20 to 24 base pairs. In preferred embodiments siRNAs having 19, 20, 21 , 22, 23, 24 or 25 base pairs, and in particular 21 or 22 base pairs, corresponding to the target gene to be down-regulated may be used.
  • siRNAs may include single-stranded overhangs at one or both ends, flanking the double-stranded portion.
  • the siRNA may contain 3' overhanging nucleotides, preferably two 3' overhanging thymidines (dTdT) or uridines (UU). 3 1 TT or UU overhangs may be included in the siRNA if the sequence of the target gene immediately upstream of the sequence included in do ⁇ ble-stranded part of the dsRNA is AA. This allows the TT or UU overhang in the siRNA to hybridise to the target gene.
  • dTdT 3' overhanging thymidines
  • UU uridines
  • siRNAs which are RNA/DNA chimeras are also contemplated. These chimeras include, for example, the siRNAs comprising a double- stranded RNA with 3' overhangs of DNA bases (e.g.
  • dTdT double-stranded RNAs which are polynucleotides in which one or more of the RNA bases or ribonucleotides, or even all of the ribonucleotides on an entire strand, are replaced with DNA bases or deoxynucleotides.
  • the dsRNA may be formed from two separate (sense and antisense) RNA strands that are annealed together by (non-covalent) basepairing.
  • 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 stands of the dsRNA are formed from different regions of single polynucleotide molecule that is partially self- complementary.
  • RNAs having this structure are convenient if the dsRNA is to be synthesised 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 RNAi.
  • RNAs for use in RNAi are generally known in the art (see for example WO 99/53050, in the name of CSIRO, the contents of which are incorporated herein by reference).
  • the loop structure may comprise linker sequences or additional sequences as described above.
  • the double-stranded RNA or construct may be prepared in a manner known per se.
  • double-stranded RNAs may be synthesised in vitro using chemical or enzymatic RNA synthesis techniques well known in the art. In one approach the two separate RNA strands may be synthesised separately and then annealed to form double- strands.
  • double-stranded RNAs or constructs may be synthesised by intracellular expression in a host cell or organism from a suitable expression vector. This approach is discussed in further detail below.
  • the amount of double-stranded RNA with which the fungal cell is contacted is such that specific down-regulation of the one or more target genes is achieved.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell. However, in certain embodiments higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded RNA may yield more effective inhibition. For any given fungal gene target the optimum amount of dsRNA for effective inhibition may be determined by routine experimentation.
  • the fungal cell can be contacted with the double-stranded RNA in any suitable manner, permitting direct uptake of the double-stranded RNA by the fungus.
  • the fungal cell can be contacted with the double-stranded RNA in pure or substantially pure form, for example an aqueous solution containing the dsRNA.
  • the fungus may be simply "soaked" with an aqueous solution comprising the double-stranded RNA.
  • the fungal cell can be contacted with the double-stranded RNA by spraying the fungal cell with a liquid composition comprising the double-stranded RNA.
  • the double-stranded RNA may be linked to a food component of the fungi, such as a food component for a mammalian pathogenic fungus, in order to increase uptake of the dsRNA by the fungus.
  • the fungal cell may be contacted with a composition containing the double-stranded RNA.
  • the composition may, in addition to the dsRNA, contain further excipients, diluents or carriers. Preferred features of such compositions are discussed in more detail below.
  • the double-stranded RNA may also be incorporated in the medium in which the fungus grows or in or on a material or substrate that is infested by the fungus or impregnated in a substrate or material susceptible to infestation by fungus.
  • target nucleotide sequences of the fungal target genes herein disclosed are particularly important to design the dsRNA constructs according to the present invention.
  • target nucleotide sequences are preferably at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides in length.
  • Non-limiting examples of preferred target nucleotide sequences are given in the examples.
  • the present invention encompasses isolated nucleotide sequences consisting of at least one sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 39, 60, 111 , 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1 , 41 , 184,
  • the present invention provides an isolated nucleotide sequence encoding a double stranded RNA or double stranded RNA construct as described herein.
  • the present invention provides fungal target genes, which comprise a sequence as herein represented by SEQ ID NO 192, 117, 201,
  • the present invention relates to an isolated nucleic acid sequence consisting of a sequence represented by any of SEQ ID NOs 3, 99, 100, 192, 39, 111, 112, 113, 114, 115, 116, 117, 201 , 202, 5, 101, 102, 193, 184, 97, 98, 190, 191, 37, 124, 9, 188, 106, 196, 13, 109, 199, 200, 33, 126, 23, 119, 35, 127, 7, 186, 103, 104, 105, 194, 195, 29, 118, 17, 108, 198, 25, 121 , 19, 125, 31 , 123, 11, 107, 197, 27, 122, 21 , 120, 15 and 110, or a fragment of at least 17 preferably at least 18, 19, 20 or 21 , more preferably at least 22, 23 or 24 nucleotides thereof.
  • Sequence homologues can be of two types:(i) where homologues exist in different species they are known as orthologues. e.g. the ⁇ -globin genes in mouse and human are orthologues.(ii) paralogues are homologous genes in within a single species, e.g. the ⁇ - and ⁇ - globin genes in mouse are paralogues
  • Preferred homologues are genes comprising a sequence which is at least about 85% or 87.5%, still more preferably about 90%, still more preferably at least about 95% and most preferably at least about 99% identical to a sequence selected from the group of sequences represented by SEQ ID NOs 3, 42, 99, 100, 39, 60, 111 , 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1, 41 , 184, 185, 97, 98, 37, 59, 124, 9, 45, 188, 189, 106, 13, 47, 109, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 186, 187, 103, 104, 105, 29, 55, 118, 17, 49, 108, 25, 53, 121 , 19, 50, 125, 31 , 56, 123, 11, 46, 107, 27, 54, 122, 21, 51 , 120, 15, 48 and 110, or the complement thereof' Method
  • Further preferred homologues are genes comprising at least one single nucleotide polymorphism (SNP) compared to a gene comprising a sequence as represented by any of SEQ ID NOs 3, 42, 99, 100, 39, 60, 111, 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1, 41 , 184, 185, 97, 98, 37, 59, 124, 9, 45, 188, 189, 106, 13, 47, 109, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 186, 187, 103, 104, 105, 29, 55, 118, 17, 49, 108, 25, 53, 121 , 19, 50, 125, 31 , 56, 123, 11 , 46, 107, 27, 54, 122, 21, 51 , 120, 15, 48 or 110.
  • SNP single nucleotide polymorphism
  • the invention encompasses target genes which are fungal orthologues of a gene comprising any of SEQ ID Nos 206 to 458.
  • Preferred orthologues are represented by any of SEQ ID NOs 206 to 353. More preferred orthologues are represented by any of SEQ ID NOs 206 to 337.
  • the dsRNA may be expressed by (e.g. transcribed within) a host cell or host organism, the host cell or organism being an organism susceptible or vulnerable to infestation with a fungus.
  • RNAi-mediated gene silencing of one or more target genes in the fungus may be used as a mechanism to control growth of the fungus in or on the host organism and/or to prevent or reduce fungal infestation of the host organism.
  • expression of the double-stranded RNA within cells of the host organism may confer resistance to a particular fungus or to a class of fungi.
  • expression of the double-stranded RNA within cells of the host organism may confer resistance to more than one fungus or more than one class of fungi.
  • the host organism is a plant and the fungus is a plant pathogenic fungus.
  • the fungal cell is contacted with the double-stranded RNA by expressing the double-stranded RNA in a plant or plant cell that is infested with or susceptible to infestation with the plant pathogenic fungus.
  • plant encompasses any plant material that it is desired to treat to prevent or reduce fungal growth and/or fungal infestation. This includes, inter alia, whole plants, seedlings, propagation or reproductive material such as seeds, cuttings, grafts, explants, etc. and also plant cell and tissue cultures.
  • the plant material should express, or have the capability to express, dsRNA corresponding to one or more target genes of the fungus.
  • the invention provides a plant, preferably a transgenic plant, or propagation or reproductive material for a (transgenic) plant, or a plant cell culture expressing or capable of expressing at least one double-stranded RNA, wherein the double- stranded RNA comprises 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 fungus, such that the double-stranded RNA is taken up by a fungal cell upon plant- fungus interaction, said double stranded RNA being capable of inhibiting the target gene or down-regulating expression of the target gene by RNA interference.
  • the target gene may be any of the target genes herein described, for instance a target gene that is essential for the viability, growth, development or reproduction of the fungus, preferably said fungal target gene is involved in any of the cellular functions as defined in Table 1 ; or for instance a fungal target gene that is involved in the pathogenicity or infectivity of the fungus, preferably said fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia.
  • the fungal cell can be any fungal cell, but is preferably a fungal cell of a plant pathogenic fungus.
  • Preferred plant pathogenic fungi include, but are not limited to, those listed above.
  • a plant to be used in the methods of the invention, or a transgenic plant according to the invention encompasses any plant, but is preferably a plant that is susceptible to infestation by a plant pathogenic fungus, including but not limited to the following plants: rice, corn, soybean, cotton, potato, banana or tomato, cereals including wheat, oats, barley, rye, vine, apple, pear, sorghum, millet, beans, groundnuts, rapeseed, sunflower, sugarcane. Most preferably the plant is rice, corn, soybean, cotton, potato, banana or tomato.
  • the present invention extends to methods as described herein wherein the plant is wheat, sorghum, millet, beans, groundnuts, rapeseed, sunflower, sugarcane, rice, corn, soybean, cotton, potato, banana or tomato.
  • the plant is rice, corn, soybean, cotton, potato, banana or tomato.
  • the present invention extends to methods as .described herein, wherein the plant is rice and the target gene is a gene from a fungus selected from the group consisting of: Magnaporthe spp. fe.g. Magnaporthe oryzae or Magnaporthe grisae), Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis),
  • Fusarium spp. e.g. Fusarium roseum
  • Acremoniella spp. e.g. Acremoniella atra
  • Pythium spp. e.g. Pythium arrhenomanes, P. myriotylum, or P. dissotocum
  • C ⁇ rvularia spp. e.g. Curvularia oryzae, Curvularia lunatas
  • T ⁇ choderma spp. e.g. Trichoderma virde
  • Rhizopus spp. e.g.
  • Rhizopus chinensis in another embodiment the present invention extends to methods as described herein, wherein the plant is corn and the target gene is a gene from a fungus selected from the group consisting of: Colletotrichum spp. fe.g. Colletotrichum lindemuthianum), Gibberella spp., Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum), Diplodia spp. fe.g. Diplodia maydis) or Puccina spp. fe.g. Puccinia sorgh, Puccinia ⁇ triiformis (causing yellow rust),
  • a fungus selected from the group consisting of: Colletotrichum spp. f
  • the present invention extends to methods as described herein, wherein the plant is potato and the target gene is a gene from a fungus selected from the group consisting of Phytophthora spp. fe.g.
  • Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis or a fungal species that causes wilt, rot or scurf; in another embodiment the present invention extends to methods as described herein, wherein the plant is banana and the target gene is a gene from a fungus selected from the group consisting of Mycosphaerella spp., Cercospora spp. (e.g. Cercospora kikuchii or Cercospora zaea-maydis) or Fusarium spp. (e.g.
  • the present invention extends to methods as described herein, wherein the plant is tomato and the target gene is a gene from a fungus selected from the group consisting of Phytophthora spp. (e.g.
  • Phytophthora cinnamom Phytophthora cactorum, PhytophtHora phaseoli, Phytophthora parasitica, Phytophthora- citrophthora, Phytophthora megasperma f.sp. soiae or Phytophthora infestans) or a fungal species that causes foliar disease, wilt or fruit rot.
  • the plant is rice and the fungus is Magnaporthe oryzae causing e.g. rice blast.
  • the plant is rice and the fungus is Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis) causing e.g. sheath blight.
  • the plant is rice and the fungus is Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis), Fusarium spp. (e.g. Fusarium roseum), Acremoniella spp. ( e.g.
  • Acremoniella atra Acremoniella atra
  • Pythium spp. e.g. Pythium arrhenomanes, P. myriotylum, or P. dissotocum
  • Curvularia spp. e.g. Curvularia oryzae, Curvularia lunatas
  • Trichoderma spp. e.g. Trichoderma virde
  • Rhizopus spp. e.g. Rhizopus chinensis
  • seedling blight in another specific embodiment the plant is soybean and the fungus is Phakopsora spp. (e.g. Phaopsora pachyrhizi) causing e.g.
  • soybean rust in another specific embodiment the plant is potato and the fungus is Phytophthora spp. (e.g. Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora citrophthora, Phytophthora megasperma f.sp. soiae or Phytophthora infestans) causing e.g. late blight. ; in another specific embodiment the plant is banana and the fungus is Cercospora spp. (e.g.
  • the plant is banana and the fungus is Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum) causing e.g Panama disease; in another specific embodiment the plant is tomato and the fungus is Phytophthora spp. (e.g.
  • Phytophthora cinnamomi Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora citrophthora, Phytophthora megasperma f.sp. soiae or Phytophthora infestans) causing e.g. late blight;
  • the plant in yet another specific embodiment the plant is corn and the fungus is Colletotrichum spp. (e.g. Colletot ⁇ chum lindemuthianum) causing e.g. anthracnose.
  • the plant is corn and the fungus is Diplodia spp. Ce.g.
  • Diplodia maydis Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum) or Gibberella spp. causing e.g. ear, kernel and stalk rots.
  • the plant is corn and the fungus is Puccinia spp. (e.g. Puccinia sorghi, Puccinia striiformis, Puccinia graminis f.sp.
  • the plant in another specific embodiment the plant is cotton and the fungus is Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum) causing e.g. fusarium wilt.
  • the plant in another specific embodiment the plant is cotton and the fungus is Verticillium spp. (e.g.
  • Verticillium dahliae or Verticillium albo-atrum causing e.g. verticillium wilt
  • the plant in another specific embodiment the plant is potato and the fungus is Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis) causing e.g. early blight.
  • the plant in another specific embodiment the plant is potato and the fungus is a fungal species causing e.g. wilts, rots or scurf; in another specific embodiment the plant is tomato and the fungus is a fungal species causing e.g. foliar disease, wilts or fruit rots.
  • Transgenic plants according to the invention extend to all plant species specifically described above being resistant to the respective fungus species as specifically described above.
  • Preferred transgenic plants are plants (or reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell) wherein said fungal target gene comprises a sequence which is at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 3, 42, 99, 100, 39, 60, 111 , 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1 , 41 , 184, 185, 97, 98, 37, 59, 124, 9, 45, 188, 189, 106, 13, 47, 109, 33, 57, 126, 23, 52, 119, 35, 58, 127,
  • the plant may be provided in a form wherein it is actively expressing (transcribing) the double-stranded RNA in one or more cells, cell types or tissues.
  • the plant may be "capable of expressing", meaning that it is transformed with a transgene which encodes the desired dsRNA but that the transgene is not active in the plant when (and in the form in which) the plant is supplied.
  • a recombinant DNA construct comprising the nucleotide sequence encoding the dsRNA or dsRNA construct according to the present invention operably linked to at least one regulatory sequence.
  • the regulatory sequence is selected from the group comprising constitutive promoters or tissue specific promoters as described in the invention.
  • the target gene may be any target gene herein described.
  • the regulatory element is a regulatory element that is active in a plant cell. More preferably, the regulatory element is originating from a plant.
  • regulatory sequence is to be taken in a broad context and refer to a regulatory nucleic acid capable of effecting expression of the sequences to which it is operably linked.
  • promoters and nucleic acids or synthetic fusion molecules or derivatives thereof which activate or enhance expression of a nucleic acid so called activators or enhancers.
  • operably linked refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
  • the transgene nucleotide sequence encoding the double- stranded RNA could be placed under the control of an inducible or growth or developmental stage-specific promoter which permits transcription of the dsRNA to be turned on, by the addition of the inducer for an inducible promoter or when the particular stage of growth or development is reached.
  • the transgene encoding the double-stranded RNA is placed under the control of a strong constitutive promoter such as any selected from the group comprising the CaMV35S promoter, doubled CaMV35S promoter, ubiquitin promoter, actin promoter, rubisco promoter, GOS2 promoter, Figwort mosaic viruse (FMV) 34S promoter..
  • the transgene encoding the double-stranded RNA is placed under the control of a tissue specific promoter 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 promoter of RJ39 from strawberry
  • a tissue specific promoter 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 promoter of RJ39 from strawberry
  • a tissue specific promoter such as any selected from the group comprising root specific promoters of
  • the plants could preferably express the dsRNA in a plant part that is first accessed or damaged by the plant pest.
  • preferred tissues to express the dsRNA are the roots, leaves and stem. Therefore, in the methods of the present invention, a plant tissue-preferred promoter may be used, such as a root specific promoter, a leaf specific promoter or a stem-specific promoter. Suitable examples of a root specific promoter are PsMTA (Fordam-Skelton, A.P., et al., 1997 Plant Molecular Biology 34: 659- 668.) and the Class III Chitinase promoter.
  • 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.
  • promoters useful for the expression of dsRNA include, but are not limited to, promoters from an RNA Poll, an RNA PoIII, an RNA PoIIII 1 T7 RNA polymerase or SP6 RNA polymerase. These promoters are typically used for in v/fro-production of dsRNA, which dsRNA is then included in an antifungal agent, for example in an anti-fungal liquid, spray or powder. Therefore, the present invention also encompasses a method for generating any of the double-stranded RNA or RNA constructs of the invention. This method comprises the steps of a.
  • the present invention also encompasses a cell comprising any of the nucleotide sequences or recombinant DNA constructs described herein.
  • the invention further encompasses prokaryotic cells (such as, but not limited to, gram-positive and gram- negative bacterial cells) and eukaryotic cells (such as, but not limited to, 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.
  • transcription termination sequences 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 recombinant 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) in a cell.
  • Preferred origins of replication include, but are not limited to, fl-ori and colE1 ori.
  • the recombinant 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 selectable markers include resistance genes against ampicillin (Ampr), tetracycline (Tcr), kanamycin (Kanr), phosphinothricin, and chloramphenicol (CAT) gene.
  • 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). Plants that have been stably transformed with a transgene encoding the dsRNA may be supplied as seed, reproductive material, propagation material or cell culture material which does not actively express the dsRNA but has the capability to do so.
  • the present invention encompasses a plant (e.g. a rice plant), or a seed (e.g. a rice seed), or a cell (e.g. a bacterial or plant cell), comprising any of the nucleotide sequences encoding the dsRNA or dsRNA construct as described herein.
  • the present invention also encompasses a plant (e.g. a rice, barley, rye, wheat, miller, lovegrass or crabgrass plant), or a seed (e.g. a rice, barley, rye, wheat, miller, lovegrass or crabgrass seed), or a cell (e.g. a bacterial or plant cell), comprising any of the dsRNA or dsRNA constructs described herein.
  • these plants or seeds or cells comprise a recombinant construct wherein the nucleotide sequence encoding the dsRNA or dsRNA construct according to the present invention is operably linked to at least one regulatory element as described above.
  • the plant or seed or cell is rice, or a rice seed or a rice cell.
  • General techniques for expression of exogenous double-stranded RNA in plants for the purposes of RNAi are known in the art (see Baulcombe D, 2004, Nature. 431 (7006): 356- 63. RNA silencing in plants, the contents of which are incorporated herein by reference).
  • double-stranded RNA in plants for the purposes of down-regulating gene expression in plant pests such as nematodes or insects are also known in the art. Similar methods can be applied in an analogous manner in order to express double-stranded RNA in plants for the purposes of down-regulating expression of a target gene in a plant pathogenic fungus. In order to achieve this effect it is necessary only for the plant to express (transcribe) the double-stranded RNA in a part of the plant which will come into direct contact with the fungus, such that the double-stranded RNA can be taken up by the fungus.
  • dsRNA Depending on the nature of the fungus and its relationship with the host plant, expression of the dsRNA could occur within a cell or tissue of a plant within which the fungus is also present during its life cycle, or the RNA may be secreted into a space between cells, such as the apoplast, that is occupied by the fungus during its life cycle. Furthermore, the dsRNA may be located in the plant cell, for example in the cytosol, or in the plant cell organelles such as a chloroplast, mitochondrion, vacuole or endoplastic reticulum.
  • the dsRNA may be secreted by the plant cell and by the plant to the exterior of the plant.
  • the dsRNA may form a protective layer on the surface of the plant.
  • the invention relates to a composition for controlling fungal growth and/or preventing or reducing fungal infestation, comprising at least one double- stranded RNA, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene and optionally further comprising at least one suitable carrier, excipient or diluent.
  • the target gene may be any target gene described herein.
  • the fungal target gene is essential for the viability, growth, development or reproduction of the fungus, for instance the fungal target gene is involved in any of the cellular functions as presented in Table 1; or the fungal target gene is involved in the pathogenicity or infectivity of the fungus, for instance the fungal target gene is involved in the formation of germ tubes, conidia attachment, formation of appressoria, formation of the penetration peg or formation of conidia.
  • the invention relates to a composition as described above, wherein the fungal target gene comprises a sequence which is at least 75%, preferably at least 80%, 85%, 90%, more preferably at least 95%, 98% or 99% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 3, 42, 99, 100, 39, 60, 111, 112, 113, 114, 115, 116, 117, 5, 43, 101 , 102, 1 , 41 , 184, 185, 97, 98, 37, 59, 124, 9, 45, 188, 189, 106, 13, 47, 109, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 186, 187, 103, 104, 105, 29, 55, 118, 17, 49, 108, 25, 53, 121 , 19, 50, 125, 31 , 56, 123, 11, 46, 107, 27, 54, 122, 21 , 51 , 120
  • the present invention further relates to a composition
  • a composition comprising at least one double-stranded RNA, at least one double-stranded RNA construct, at least one nucleotide sequence and/or at least one recombinant DNA construct as descried herein, optionally further comprising at least one suitable 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 dsRNA by the fungal cell.
  • components which enhance or promote uptake of the dsRNA by the fungal cell 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 fungal cells, to substrates, to cells (e.g. plant cells), or to organism infected by or susceptible to infection by fungi.
  • composition of the invention may be supplied as a "kit-of- parts" comprising the double-stranded RNA in one container and a suitable diluent or carrier for the RNA in a separate container.
  • the invention also relates to supply of the double- stranded RNA alone without any further components.
  • the dsRNA 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 lyophilised 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 double-stranded RNAs, double-stranded RNA constructs, nucleotide sequences, recombinant DNA constructs or compositions described herein.
  • the composition is a pharmaceutical or veterinary composition for treating or preventing fungal disease or infections of humans or animals, respectively.
  • Such compositions will comprise at least one double-stranded RNA or RNA construct, or nucleotide sequence or recombinant DNA construct encoding the double- stranded RNA or RNA construct, wherein the double-stranded RNA comprises annealed complementary strands, one of which has a nucleotide sequence which corresponds to a target nucleotide sequence of a fungal target gene that causes the disease or infection, and at least one carrier, excipient or diluent suitable for pharmaceutical use.
  • 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 composition to be applied to the skin as drops, gel, aerosol, or by brushing, or a spray, cream, ointment, etc. for topical application or as transdermal patches.
  • the fungal dsRNA is produced by bacteria (e.g. lactobacillus) which can be included in food and which functions as an oral vaccine against the fungal infection.
  • bacteria e.g. lactobacillus
  • target human pathogenic and animal pathogenic fungi include, but are not limited to the following:
  • Candida spp. particularly Candida albicans
  • Dermatophytes including
  • Aspergillus terreus group Blastomyces dermatitidis; Paracoccidioides brasiliensis;
  • Fonsecaea spp. Penicillium
  • Zygomycetes group of fungi particularly Absidia corymbifera, Rhizomucor pusillus', and Rhizopus arrhizus. ;
  • Candida spp. In animals: Candida spp.; Microsporum spp., particularly Microsporum canis, Microsporum gypseum; Trichophyton mentagrophytes; Aspergillus spp.; or Cryptococcus neoformans.
  • the composition may be a coating that can be applied to a substrate in order to protect the substrate from infestation by a fungus and/or to prevent, arrest or reduce fungal growth on the substrate and thereby prevent damage caused by the fungus.
  • the composition can be used to protect any substrate or material that is susceptible to infestation by or damage caused by a fungus, for example foodstuffs and other perishable materials, and substrates such as wood.
  • Preferred target fungal species for this embodiment include, but are not limited to, the following: Stachybotrys spp.,
  • the nature of the excipients and the physical form of the composition may vary depending upon the nature of the substrate that 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 invention further encompasses a method for treating and/or preventing a fungal disease or condition, comprising administering to a subject in need of such treatment and/or prevention, any of the compositions as herein described, said composition comprising at least one double-stranded RNA or double stranded RNA construct comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a nucleotide sequence of a fungal target gene that causes the fungal disease or condition.
  • compositions are used as a fungicide for a plant or for propagation or reproductive material of a plant, such as on seeds.
  • the composition can be used as a fungicide by spraying or applying it on plant tissue or spraying or mixing it on the soil before or after emergence of the plantlets.
  • the present invention provides a method for treating and/or preventing fungal growth and/or fungal infestation of a plant or propagation or reproductive material of a plant, comprising applying an effective amount of any of the compositions herein ' described to a plant or to propagation or reproductive material of a plant.
  • the invention relates to the use of any double-stranded RNA or RNA construct, or nucleotide sequence or recombinant DNA construct encoding the double-stranded RNA or RNA construct described herein, or to any of the compositions comprising the same, used for controlling fungal growth; for preventing fungal infestation of plants susceptible to fungal infection; or for treating fungal infection of plants.
  • Specific plants to be treated for fungal infections caused by specific fungal species are as described earlier and are encompassed by the said use.
  • the invention further relates to a kit comprising at least one double stranded RNA, or double stranded RNA construct, or nucleotide sequence, or recombinant DNA construct, or cell, or composition as described earlier for treating fungal infection in plants.
  • the present invention extends to a method for increasing plant yield comprising introducing in a plant any of the nucleotide sequences or recombinant DNA constructs as herein described in an expressible format. Plants encompassed by this method are as described earlier. Preferably, said plant is rice.
  • 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 fungal genes by RNAi can be used in in vitro or in vivo assays in order to study gene function, in an analogous approach to that which has been described in the art for the nematode worm C.elegans and also Drosophila melanogaster. Assays based on targeted down-regulation of specific fungal genes, leading to a measurable phenotype may also form the basis of compound screens for novel anti-fungal agents. Description of figures and tables
  • FIG. 1 Effect of dsRNA on the mycelium growth of the Magnaporthe grisea. Data are shown for the dsRNA of the beta-tubulin target gene (MG00604.4 (nt 1151-1344, see table 3) (a), MG00884.4 (nt 845 - 1044, see table 3) (b), MG07031.4 (nt 251 - 500, see table 3) (c), and MG04484.4 (nt 211 - 409, see table 3) (d).
  • Each assay consists of 4 replicates, in a 96- well format and is compared to an assay with control dsRNA such as that from GST.
  • Each dsRNA is added to 1250 fungal spores at 0.5 mg/ml, and absorbance readings are taken at 0, 24 and 30 hours after addition of dsRNA.
  • the percent inhibition by dsRNA of beta-tubulin is significant at P ⁇ 0.05 at 24 and 30 hours.
  • the dotted line indicates background absorbance at O. D. 595 nm.
  • Figure 3 List of target genes including the coding sequences. The start and stop codons are at the beginning and at the end of the underlined sequence.
  • Figure 4 List of hairpin sequences. The sequence in bold represents the SI intron (SEQ ID NO 204).
  • Table 1 Examples of suitable fungal target genes.
  • the DNA and protein sequences given in the table originate from the rice blast fungus Magnaporthe grisea (also known as Magnaporthe oryzae). Identifiers correspond to the gene identifiers of the M. grisea genome project.
  • the homologous gene of the budding yeast Saccharomyces cerevisiae is also given, identifiers correspond to gene identifiers of the Saccharomyces Genome Database (SGDTM), together with gene function assigned on the basis of yeast data.
  • SGDTM Saccharomyces Genome Database
  • Table 2 Examples of suitable fungal target genes.
  • the DNA and protein sequences given in the table correspond with fungal orthologues of the rice blast fungus Magnaporthe grisea genes of Table 1. Identifiers correspond to accession number and version number.
  • Table 3 Overview of cloning details of cDNA's of Magnaporthe grisea target genes PCR conditions were as follows: A: Expand High Fidelity PCR system (Roche) 5' 94°C, 30 cycles (1' 94 0 C, 1' 58°C, 1' 72°C) 10' 72°C; B: Expand High Fidelity PCR system (Roche) 5' 94°C, 30 cycles (45" 94°C, 45" 58 0 C, 45" 72°C), 10' 72 0 C; C: Expand High Fidelity PCR system (Roche) 5' 94 0 C, 30 cycles (45" 94 0 C, 45" 58 0 C, 45" 72°C), 10' 72°C; D:
  • Table 4 Overview of cloning details of exons of Magnaporthe grisea target genes using gDNA as template. PCR conditions were as follows: A: Pwo polymerase (Roche) 5' 94 0 C, 30 cycles (30" 94 0 C, 30" 6O 0 C, 1' 72°C) 5' 72 0 C; B: Pwo polymerase (Roche) 5' 94°C, 30 cycles
  • PCRs were performed on the Magnaporthe grisea cDNA or on genomic DNA as a template.
  • the PCR conditions and primers for the target gene are outlined in the tables. Each PCR was performed in duplicate. The resulting two independent PCR products per target gene, were analysed on agarose gel, isolated and cloned into the pTZ57R/T vector (MBI Fermentas). For each PCR product, at least three clones were sequenced. The sequences resulting from the clones were compared to the public database sequences and one or more clones per target gene were selected for further experimentation. Cloning details of the coding sequences of these genes are herein represented in the tables.
  • Genomic DNA of Magnaporthe grisea was prepared using a protocol modified from Naqvi et al. (Molecular Breeding, 1995, 1 : 341-348). About 150 mg of dried mycelium was ground in liquid nitrogen into powder form and extracted for genomic DNA in extraction buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.25 M NaCI, 1% SDS) at 65°C for 30'. Cell debris and proteins were precipitated by adding potassium acetate 5 M pH 4.8 at -20 0 C freezer for 15'.
  • the pellet was removed by centrifugation at 14000 rpm for 10'. The supernatant was transferred to a fresh tube and extracted with phenol/CH 3 CI/isoamyI alcohol mix. The aqueous layer was precipitated with isopropanol. The DNA pellet was washed with 70% ethanol, air-dried and resuspended in TE. This preparation is used as template for amplification of exons by PCR of the following fungal target genes.
  • the amplified exons/PCR products originating from the same target were analyzed on agarose gel, isolated, and ligated to each other in the correct order. This ligation was achieved as follows:
  • Step 1 PCR amplification of each exon using the proof reading enzyme Pwo polymerase (Roche), producing blunt ends.
  • Step 2 Phosphorylation of 2.5 pmols of the second exon using 1 unit of Polynucleotide kinase (PNK, Bangalore Genei) in a 15 ⁇ l reaction with 1mM ATP.
  • Step 3 Blunt end ligation of the two exons.
  • Step 4 PCR amplification of the full-length gene from the ligation mix using gene specific forward and reverse primers. This step ensures selection of only those ligation products in which the exons are ligated in the correct order.
  • target gene MG07031.4 (3) which has 5 exons
  • the first two exons (Exon 1 and 2 which were 70bp and 21 bp respectively) were synthetically made.
  • the last 3 exons were amplified as outlined hereinabove and ligated as follows:
  • the ligation products were cloned into the pTZ57R/T vector (MBI Fermentas). For each ligation product, at least 3 clones were sequenced. The sequences resulting from the clones were compared to the public database sequences and one or more clones per target gene were selected for further experimentation. Cloning details of exons which are joined to form the coding sequences of these target genes are herein represented in Table 4. cDNA's of the other target genes were cloned by one of the above approaches.
  • Seastar AFP (autofluorescent protein) template was an in-house clone pGR37
  • Glutathione S-transferase (GST) was amplified from pGEX4T-1
  • GFP was amplified from an in-house clone, pDW2821.
  • Exons of beta-tubulin genes were amplified directly from gDNA and used as templates for dsRNA synthesis.
  • Example 2 Selection of target nucleotide sequences of the target genes of Magnaporthe grisea for RNAi mediated gene silencing
  • fragments of the target genes herein described were selected for use in further RNAi experiments both in vitro as described in example 3 or in vivo as described in examples 4, 5 and 6. These fragments are listed in Table 5.
  • a person skilled in the art will recognize that other fragments of various lengths may be identified in the Magnaporthe grisea sequences, and that the present invention also extends to these fragments and the use thereof in RNAi mediated silencing of fungal genes. Preferably, such fragments do not have effect on non- target organisms such as rice or humans.
  • Example 3 The effect of silencing a target gene in Maqnaporthe grisea in vitro In vitro assays
  • Germinating conidia have been shown to actively take up materials from the medium by endocytosis.
  • germinating conidia of Magnaporthe grisea were used to demonstrate uptake of dsRNA by fungi in vitro.
  • Conidia were germinated in hydrophilic conditions, mimicking their germination within the leaf after penetration of the fungus. On a hydrophilic surface, the conidia grow vegetatively into mycelia.
  • dsRNA corresponding to target regions of target genes were prepared from genomic
  • DNA or cDNA as follows. Two PCR reactions were set up: one to amplify the sense RNA strand, another for tHe antisense RNA strand. The forward primers of each reaction contain a
  • T7 promoter sequence followed by sequences corresponding to the targeted sequence, while the reverse primer only contains sequence complementary to the target sequence.
  • PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), and subsequently used as template for in vitro transcription to produce double-stranded RNA (T7 RiboMAX Express RNAi System Promega). The dsRNA was precipitated, quantitated and dissolved in RNase-free water.
  • Conidia (asexual spores) were generated by exposing fungal mycelia to light for 7-10 days. Freshly harvested, hydrated conidia were re-suspended in water at a density of 10 4 conidia/ml, and inoculated on the hydrophobic surface of an artificial membrane (GelBond film, Cambrex). DsRNA corresponding to the respective fungal target genes (see Table 5) were tested individually at concentrations ranging from 0.1-1 mg/ml in a final volume of 20 ⁇ l in water. As a negative control, dsRNA corresponding to part of a GST was used. After 16-24 h incubation at 28 0 C, the germinated spores were stained with Acid Fuchsin for clearer visualization of cellular structures.
  • dsRNA Conidia of Magnaporthe grisea were harvested, re-suspended in potato dextrose broth and 1250 conidia (in about 90 ⁇ l) were aliquoted in each well of a hydrophilic 96-well plates (Falcon 3072). After 0-2 h pregermination at 28 0 C, dsRNA was added to a final volume of 100 ⁇ l and to a final concentration of 0.1 or 0.5 mg/ml. dsRNA fragments of about 200 bp in length corresponding to distinct target regions of the target genes were tested. As negative controls, dsRNA corresponding to a part of GST, Seastar AFP or GFP were us.ed.
  • the growth of mycelia in the wells was quantified by optical density reading of the 96-well plates at wavelength 595 nm, in a plate reader (GENios Tecan, Austria).
  • the fungus showed growth inhibition in the presence of target dsRNA fragments compared to controls (see Table 5 and Figure 1).
  • the growth inhibition phenotype was a direct indication of inhibition of target gene expression by RNAi due to uptake of dsRNA by the intact fungus. Soaking experiments were performed in quadruplicate and percentage inhibition was calculated with reference to control-soaked samples. At least two other negative controls were included in each assay to ensure consistency of inhibition results.
  • Statistical analyses were performed using Analyse-it software.
  • Example 4 Cloning of hairpin constructs and plant expression vectors for dsRNA production in plant cells. Since the mechanism of RNA interference operates through dsRNA fragments, the target nucleotide sequences of the target genes as selected above and indicated in Table 5 were cloned in anti-sense and sense orientation, separated by the 189bp synthetic intron (Sl) from the gene X from Arabidopsis thaliana (Sl 1 SEQ ID NO: 204), to form a dsRNA hairpin construct. These hairpin constructs were cloned into multiple cloning sites of the plant expression vector pMH115 (SEQ ID NO: 205), comprising the double CaMV35S promoter and the CaMV35S 3' element.
  • Sl 189bp synthetic intron
  • the cDNA clones as described above were used as templates for the PCRs. These cloning experiments resulted in a hairpin construct for each target gene, having the structure promoter-sense-SI-antisense or more preferably, promoter-antisense- Sl-sense, wherein the sense fragments are given in Table 5, and wherein the promoter is any plant operable promoter, preferably a strong constitutive promoter, such as the CaMV35S promoter.
  • the complete sequences of several hairpin constructs (antisense- Sl- sense) are represented in Figure 4.
  • the hairpin constructs as described above were embedded in the binary vector pMH115 with the double CaMV35S promoter, which vector is suitable for transformation into A. tumefaciens, transformation into rice, and expression of the hairpin in rice.
  • promoters are selected from strong constitutive promoters including strong constitutive promoters such as CaMV35S promoter, doubled CaMV35S promoter, ubiquitin promoter, actin promoter, rubisco promoter, GOS2 promoter and FMV promoter.
  • the plant expression vectors comprising the Magnaporthe grisea hairpins were subsequently transformed into in Agrobacterium tumefaciens (see example 5).
  • Example 5 Rice plants resistant to Magnaporthe grisea
  • Rice calli are transformed and regenerated into shoots and whole plants as described in literature.
  • the plants are transferred to a greenhouse and cultivated to reach maturity and to set seeds.
  • Genomic PCR and/or Southern blotting is performed on leaf tissue of T1 plants to determine the homozygosity/heterozygosity of the integrated locus and the number of inserted copies of transgene.
  • Transgene-positive plants are further analyzed by Northern blotting and/or RT-PCR to detect expression of dsRNA and siRNA. Homozygous lines showing expression of dsRNA and/or siRNA are established and used for fungal infection studies.
  • Explants (15-20 replicates each) from T1 plants (both heterozygous and homozygous integrants) are used for initial analysis of resistance to rice blast infection.
  • the leaves of 20 day-old plants are cut and the ends of the leaves are inserted into kinetin agar plates.
  • a small drop of Magnaporthe grisea spores (200-1000 spores in 20 ⁇ l water) are inoculated onto the leaves. Infection rate and lesion sizes are compared between test and negative control leaves.

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Abstract

Cette invention concerne des procédés permettant de lutter contre les infections fongiques au moyen du silençage génique médié par des ARN double brin, la/les cellule(s) fongique(s) intactes étant mises en contact avec un ARN double brin depuis l'extérieur de la/les cellule(s) fongique(s) et l'ARN double brin étant absorbé par la/les cellule(s) fongique(s). Dans un mode de réalisation particulier, on utilise les procédés de l'invention pour éliminer les parasites fongiques des plantes. En variante, on utilise les procédés pour traiter et/ou empêcher les infections fongiques sur un substrat ou sur un sujet nécessitant un tel traitement et/ou une telle prévention. L'invention concerne également des gènes cibles fongiques appropriés et leurs fragments, des constructions d'ARN double brin et des constructions recombinantes.
PCT/IB2005/003495 2004-10-04 2005-10-04 Procede de regulation de l'expression genetique dans des champignons WO2006070227A2 (fr)

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WO2013050024A3 (fr) * 2011-10-06 2013-07-11 Kws Saat Ag Plante transgénique de l'espèce bêta vulgaris présentant une résistance accrue au cercospora
DE102012016009A1 (de) 2012-08-08 2014-02-13 Kws Saat Ag Transgene Pflanze der Art Solanum tuberosum mit Resistenz gegenüber Phytophthora
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WO2016170547A1 (fr) * 2015-04-21 2016-10-27 Metahelix Life Sciences Ltd. Nouvelle composition d'adn de recombinaison et procédés de lutte contre des agents phytopathogènes
EP3101135A1 (fr) 2015-06-01 2016-12-07 Universität Hamburg Procédé permettant de conférer une résistance contre une maladie de plante de fusarium
US10167484B2 (en) 2004-04-09 2019-01-01 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
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US10167484B2 (en) 2004-04-09 2019-01-01 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US11685930B2 (en) 2004-04-09 2023-06-27 Monsanto Technology, Llc Compositions and methods for control of insect infestations in plants
US11492638B2 (en) 2004-04-09 2022-11-08 Monsanto Technology, Llc Compositions and methods for control of insect infestations in plants
US10787680B2 (en) 2004-04-09 2020-09-29 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US9121034B2 (en) 2004-10-21 2015-09-01 Venganza Inc Methods and materials for conferring resistance to pests and pathogens of corn
US8461416B2 (en) 2004-10-21 2013-06-11 Venganza, Inc. Methods and materials for conferring resistance to pests and pathogens of plants
US8581039B2 (en) 2004-10-21 2013-11-12 Venganza, Inc. Methods and materials for conferring resistance to pests and pathogens of plants
US11312975B2 (en) 2005-09-16 2022-04-26 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US8759611B2 (en) 2005-09-16 2014-06-24 Monsanto Technology Llc Methods for genetic control of insect infestation in plants and compositions thereof
US10538783B2 (en) 2005-09-16 2020-01-21 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US11939589B2 (en) 2005-09-16 2024-03-26 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US9695439B2 (en) 2005-09-16 2017-07-04 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
WO2007104570A2 (fr) * 2006-03-16 2007-09-20 Devgen N.V. Lutte contre les nématodes
WO2007104570A3 (fr) * 2006-03-16 2008-04-17 Devgen Nv Lutte contre les nématodes
WO2011154465A1 (fr) 2010-06-08 2011-12-15 Devgen Private Limited Procédé pour réguler négativement l'expression de gènes dans des champignons
EA028662B1 (ru) * 2011-10-04 2017-12-29 Байер Интеллекчуал Проперти Гмбх Рнк-интерференция для борьбы с грибами и оомицетами путем ингибирования гена сахаропиндегидрогеназы
AU2012320554B2 (en) * 2011-10-04 2017-11-09 Bayer Intellectual Property Gmbh RNAi for the control of fungi and oomycetes by inhibiting saccharopine dehydrogenase gene
WO2013050410A1 (fr) * 2011-10-04 2013-04-11 Bayer Intellectual Property Gmbh Arni pour la lutte contre des champignons et oomycètes par inhibition du gène de la saccharopine déshydrogénase
WO2013050024A3 (fr) * 2011-10-06 2013-07-11 Kws Saat Ag Plante transgénique de l'espèce bêta vulgaris présentant une résistance accrue au cercospora
US10030251B2 (en) 2012-08-08 2018-07-24 Kws Saat Se Transgenic plant of the species solanum tuberosum with resistance to phytophthora
WO2014023285A2 (fr) 2012-08-08 2014-02-13 Kws Saat Ag Plante transgénique de l'espèce solanum tuberosum présentant une résistance à phytophthora
DE102012016009A1 (de) 2012-08-08 2014-02-13 Kws Saat Ag Transgene Pflanze der Art Solanum tuberosum mit Resistenz gegenüber Phytophthora
US10760094B2 (en) 2012-08-08 2020-09-01 KWS SAAT SE & Co. KGaA Transgenic plant of the species Solanum tuberosum with resistance to Phytophthora
US10174387B2 (en) 2013-03-12 2019-01-08 Dow Agrosciences Llc Soybean markers linked to phytophthora resistance
WO2016170547A1 (fr) * 2015-04-21 2016-10-27 Metahelix Life Sciences Ltd. Nouvelle composition d'adn de recombinaison et procédés de lutte contre des agents phytopathogènes
EP3101135A1 (fr) 2015-06-01 2016-12-07 Universität Hamburg Procédé permettant de conférer une résistance contre une maladie de plante de fusarium

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