US20060247197A1

US20060247197A1 - Method for down-regulating gene expression in fungi

Info

Publication number: US20060247197A1
Application number: US11/396,926
Authority: US
Inventors: Marc van de Craen; Phuay-Yee Goh; Marc Logghe; Yee-Ling Khu; Katherine Mortier; Thierry Bogaert
Original assignee: Devgen NV
Current assignee: Devgen Pte Ltd
Priority date: 2004-10-04
Filing date: 2006-04-03
Publication date: 2006-11-02
Also published as: US20150133523A1; US8865968B2; US20100311819A1

Abstract

The present invention concerns methods for controlling fungus infestation via dsRNA mediated gene silencing, whereby the intact fungus cell(s) are contacted with a double-stranded RNA from outside the fungal cell(s) and whereby the double-stranded RNA is taken up by the intact fungal cell(s). In one particular embodiment, the methods of the invention are used to alleviate plants from fungus pests. Alternatively, the methods are used for treating and/or preventing fungal infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable fungal target genes and fragments thereof, dsRNA constructs, recombinant constructs and compositions are disclosed.

Description

RELATED APPLICATIONS

This application is a continuation-in-part of PCT/IB2005/003495, filed Oct. 4, 2005, which claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application 60/615,695, filed Oct. 4, 2004, the entire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

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.

BACKGROUND TO THE INVENTION

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).
Over the last few years, down-regulation of target genes in multicellular organisms by means of RNA interference (RNAi) has become a well established technique. In general, 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 “target gene”). Reference may be made to International application WO 99/32619 (Carnegie Institute of Washington), International application WO 99/53050 (Benitec), and to Fire et al., Nature, Vol. 391, pp. 806-811, February 1998 for general description of the RNAi technique.
In nematodes, 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. For a description of this so-called “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. Reference may be made to U.S. Pat. No. 6,506,559 (based on WO 99/32619), column 11, line 55 to column 12, line 9 and column 13, line 61 to column 14, line 11), International application WO 00/01846 by the present applicant, page 7, lines 11-8, and International applications WO 01/96584, WO 01/37654 and WO 03/052110 for a description of such techniques.
Elbashir et al. (Nature, 411, 494-498, 2001) have demonstrated effective RNAi-mediated gene silencing in mammalian cells 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 30 bp in length in mammalian cells (Stark G. R. et al., Ann Rev Biochem. 1998, 67: 227-264; Manche, L et al., Mol Cell Biol., 1992, 12: 5238-5248). Thus, 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.
Although the technique of 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.
Kadotani et al., (2003) Mol Plant Microbe Interac. 16: 769-776 describe 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.
Cogoni and Macino, (1999) Nature. 399: 166-169 describe gene silencing by 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. Again, 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.
These studies confirm that RNA interference pathways are active in a number of different species of fungi. However, to date RNAi has only been achieved in fungi by transcription of dsRNA within cells of the fungus, following transformation of fungal cells with a DNA construct or transgene from which the appropriate dsRNA may be transcribed.
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.

DESCRIPTION OF THE INVENTION

It has now been found by the present inventors that 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.
It has surprisingly been found that when intact fungal cells are contacted with double-stranded RNA outside the cell wall, the double-stranded RNA is taken up by the fungal cells in amounts sufficient to specifically cause inhibition of growth. This approach to 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. Hence, 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 limited to, (1) protecting plants against plant pathogenic fungi; (2) pharmaceutical or veterinary use in humans and animals (for example to control, treat or prevent fungal infections in humans); (3) protecting materials against damage caused by fungi; (4) protecting perishable materials (such as foodstuffs, seed, etc.) against damage caused by fungi; (5) functional genomics in fungi to elucidate the gene function of fungal target genes and generally any application wherein fungi need to be controlled and/or wherein damage caused by fungi needs to be prevented.
In accordance with one embodiment 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 expression “complementary to at least part of as used herein means that the nucleotide sequence is fully complementary to the nucleotide sequence of the target over more than two nucleotides, for instance over at least 15, 16, 17, 18, 19, 20, 21, 22 , 23 or 24 contiguous nucleotides.
According to a further embodiment, 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.
According to one embodiment, 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. In addition, 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, or a symbiotic organism (e.g. green algae or cyanbacterium).
According to another embodiment, 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. Preferably, said cell is a plant cell or said organism is a plant.
Preferably, the present invention extends to methods as described herein, wherein said target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75%, 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% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof.
The present invention thus also relates to a method for producing a plant resistant to a plant pathogenic fungus, comprising:

(a) transforming a plant cell with a recombinant construct comprising at least one regulatory sequence operably linked to a sequence complementary to at least part of a nucleotide sequence of a target fungal gene selected from the group consisting of:
- (i) a nucleotide sequence of at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides in length encoding an RNA which is complementary to a fungal target gene comprising 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, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, and
- (ii) a 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; and
(c) growing the transformed plant under conditions suitable for the expression of the recombinant construct, said grown transformed plant resistant to fungi compared to an untransformed plant, hereby producing a fungus-resistant plant.

According to still other embodiments, in the methods of the invention, 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.
In one embodiment of the invention, the fungus may be a mould, or more particularly a filamentous fungus. In other embodiments of the invention, the fungus may be a yeast.
In one embodiment the fungus may be an ascomycetes fungus, i.e. a fungus belonging to the Phylum Ascomycota.
In preferred, but non-limiting, embodiments of the invention the fungal cell is chosen from the group consisting of:
(1) 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. Colletotrichum 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 culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum), Gaeumanomyces spp. (e.g. Gaeumanomyces graminis f.sp. tritici), Gibberella spp. (e.g. Gibberella zeae), Helminthosporium spp. (e.g. Helminthosporium turcicum, Helminthosporium carbonum, Helminthosporium mavdis or Helminthosporium sigmoideum), Leptosphaeria salvinii, Macrophomina spp. (e.g. Macrophomina phaseolina), Magnaportha spp. (e.g. Magnaporthe oryzae), Mycosphaerella spp., 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. tritici, 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. (e.g. Pyricularia oryzae), Pythium spp. (e.g. Pythium ultimum), Rhincosporium secalis, Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis), Rhizopus spp. (e.g. Rhizopus chinensid), Scerotium spp. (e.g. Scerotium rolfsii), Sclerotinia spp. (e.g. Sclerotinia sclerotiorum), Septoria spp. (e.g. Septoria lycopersici, Septoria glycines, Septoria nodorum or Septoria tritici), Thielaviopsis spp. (e.g. Thielaviopsis basicola), Tilletia spp., Trichoderma spp. (e.g. Trichoderma virde), Uncinula spp. (e.g. 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);
(2) 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. capsulatum or Var. duboisii; Sporothrix schenckii; Fusarium spp.; Scopulariopsis brevicaulis; Fonsecaea spp.; Penicillium spp.; or Zygomycetes group of fungi (particularly Absidia corymbifera, Rhizomucor pusillus or Rhizopus arrhizus);
(3) a fungal cell of, or a cell derived from a fungus capable of infesting animals such as, but not limited to Candida spp., Microsporum spp. (particularly Microsporum canis or Microsporum gypseum), Trichophyton mentagrophytes, Aspergillus spp., or Cryptococcus neoforman;
and
(4) a fungal cell of, or a cell derived from a fungus that causes unwanted damage to substrates or materials, such as fungi that attack foodstuffs, seeds, wood, paint, plastic, clothing etc. Examples of such 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) causing e.g. anthracnose in corn; Curvularia spp. causing seedling blight; Diplodia spp. (e.g. Diplodia maydis) causing e.g. ear, kernel and stalk rots in corn; Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum) causing e.g. ear, kernel and stalk rots in corn, fusarium wilt in cotton and Panama disease in banana; Gibberella spp. causing e.g. ear, kernel and stalk rots in corn; Magnaportha spp. (e.g. Magnaporthe oryzae) causing rice blast; Mycosphaerella spp. causing e.g. black and yellow sigatoka in banana; Phakopsora spp. (e.g. Phakopsora pachyrhizi) 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 potato and tomato; Puccinia spp. (e.g. 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. (e.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.
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. 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. In this non-limiting embodiment, 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. Thus, when the fungal cell has a cell wall, 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. As used herein 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.
In cases where fungi reproduce both sexually and asexually, 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. When referring to one of these names in this invention, the “whole” fungus is referred to.
According to one embodiment of the present invention, 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).
According to another embodiment of the present invention, 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. These can be coding or non-coding genes.
The terms “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, or at the level of phenotype. 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. The down-regulation effect of the dsRNA on gene expression may be calculated as being at least 30%, 40%, 50%, 60%, preferably 70%, 80% or even more preferably 90% or 95% when compared with normal gene expression.
Depending on the nature of the target gene, down-regulation or inhibition of gene expression in cells of a fungus can be confirmed by phenotypic analysis of the cell or the whole fungus or by measurement of mRNA or protein expression using molecular techniques such as RNA solution hybridization, PCR, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence activated cell analysis (FACS).
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. For instance if 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.
According to one non-limiting embodiment, 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. Therefore, 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.
According to one embodiment, target genes are conserved genes or fungus-specific genes.
Some preferred, but non-limiting, examples of suitable target genes are listed in Tables 1 and 2.
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.
In addition, any suitable double-stranded RNA or nucleotide 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.
In the methods of the present 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. According to one embodiment of the present invention, an isolated double-stranded RNA is provided 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. According to one embodiment the present invention relates to 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 nucleotide sequence of a fungal target gene, wherein said target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75%%, 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% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859 or the complement thereof, and (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859 or the complement, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 562 to 859, or the complement thereof.
Depending on the assay used to measure gene silencing, 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.
According to another embodiment of the present invention, an isolated double-stranded RNA is provided, 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, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or the RNA equivalent of a 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. Preferably 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. According to another embodiment of the present invention, an isolated double-stranded RNA is provided 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, for use as a medicament. According to another embodiment of the present invention the use as a medicament is provided for the isolated double-stranded RNA as described above.
If the method of the invention is used for controlling growth or infestation of fungus in or on a host cell or host organism, it is preferred that 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. In this context, it is preferred that 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, preferentially transcriptome data, is available for the host organism one may cross-check sequence identity with the double-stranded RNA using standard bioinformatics tools. In one embodiment, there is no sequence identity between the dsRNA and a host sequences over 21 contiguous nucleotides, meaning that in this context, it is preferred that 21 contiguous base pairs of the dsRNA do not occur in the genome of the host organism. In another embodiment, there is less than about 10% or less than about 12.5% sequence identity over 24 contiguous nucleotides of the dsRNA with any nucleotide sequence from a host species.
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.
The expression “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.
It is preferred that (at least part of) the double-stranded RNA will share 100% sequence identity with the target region of the fungal target gene. However, it will be appreciated that 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. However, 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% identical.
The term “complementary” as used herein relates to both DNA-DNA complementarity as to DNA-RNA complementarity. In analogy herewith, the term “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.
Although 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. For example, the dsRNA may contain short non-target regions flanking or inserted into 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.
It has been previously reported that the formation of short interfering RNAs (siRNAs) of about 21 bp is desirable for effective gene silencing. However, in applications of applicant it has been shown that the minimum length of dsRNA preferably is at least about 80-100 bp in order to be efficiently taken up by certain pest organisms. There are indications that in 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.
It has also recently been suggested that synthetic 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. Thus, 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.
Therefore, in one embodiment, the double-stranded RNA fragment (or region) will itself preferably be at least 17 bp in length, preferably 18 or 19 bp in length, more preferably at least 20 bp, 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.
Generally, 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. Preferably 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. For any given target gene and fungus, 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 fungal 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.
Alternatively, 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. Thus, in one embodiment of the invention, 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. According to the invention, 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. Use of such dsRNA constructs in a plant host cell, thus establishes a more potent resistance to a single or to multiple fungal species in the plant.
The terms “hit”, “hits” and “hitting” are alternative wordings to indicate that at least one of the strands-of the dsRNA is complementary to, and as such may bind to, the target gene or nucleotide sequence.
In one embodiment, the double-stranded RNA region comprises multiple copies of the nucleotide sequence that is complementary to the target gene. Alternatively, the dsRNA hits a further target sequence of the same target gene. The invention thus encompasses isolated double-stranded RNA constructs comprising at least two copies of said nucleotide sequence complementary to at least part of a nucleotide sequence of a fungal target. In another embodiment 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, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or comprising at least two copies of the RNA equivalent of a fragment of at least 17 basepairs in length thereof, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof.
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.
The expressions “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.
Accordingly 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, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or comprising at least one 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. Preferably, 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.
Accordingly, 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. According to one preferred embodiment 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. Alternatively, 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.
Accordingly, 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 the RNA equivalents of at least two nucleotide sequences independently chosen from each other. In one embodiment, the dsRNA comprises the RNA equivalents of at least two, preferably at least three, four or five, nucleotide sequences indepently chosen from the sequences represented by any of SEQ ID Nos 99, 100, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or fragments thereof of at least 17 basepairs in length, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 basepairs in length thereof.
The at least two nucleotide sequences may be derived from the target genes herein described. According to one preferred embodiment the dsRNA hits at least one target gene that is essential for viability, growth, development or reproduction of the nematode and hits at least one gene involved in pathogenicity or infectivity as described hereinabove. Alternatively, 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 the same cellular function.
According to a further embodiment, the dsRNA hits at least 2 target genes, which target genes are involved in a different cellular function chosen from those functions as described in Table 1. For example 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 FIG. 3.
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 dsRNA construct, or
- d) the dsRNA regions (targeting a single or different target genes) may be combined in the sense or antisense orientation.

In addition, 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. In the RNA constructs of the invention, 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.
- f) “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. In the RNA constructs of the invention, multiple dsRNA regions targeting a single or different weak gene(s) may be combined to obtain a stronger RNAi effect.
- g) “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).
- i) “conserved pathway” genes encompass genes that are involved in the same biological pathway or cellular process, or encompass genes that have the same functionality in different fungus species resulting in a specific and potent RNAi effect and more efficient fungus control;
- j) alternatively, the 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.

According to the invention, 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.
However, provided that one of the double-stranded RNA regions comprises at least one strand that is complementary to a portion of the nucleotide sequence of any one 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).
In one embodiment of the present invention, there is provided an isolated double-stranded RNA or RNA construct for use as a medicament.
According to yet another embodiment of the present invention, there is provided an isolated double-stranded RNA or RNA construct, further comprising at least one additional sequence and optionally a linker. In one embodiment, 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. In one embodiment, 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.
In one embodiment, the multiple dsRNA regions of the double-stranded RNA construct are connected by one or more linkers. In another embodiment, 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.
In another embodiment, the multiple dsRNA regions of the double-stranded RNA construct are connected without linkers.
In a particular embodiment of the invention, the linkers may be used to disconnect smaller dsRNA regions in the pest organism. Advantageously, in this situation 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. Examples of suitable conditionally self-cleaving linkers are RNA sequences that are self-cleaving at high pH conditions. Suitable examples of such RNA sequences are described by Borda et al. (Nucleic Acids Res. 2003 May 15;31(10):2595-600), which document is incorporated herein by reference. This sequence originates from the catalytic core of the hammerhead ribozyme HH16.
In another aspect of the invention, 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.
In one particular embodiment of the invention, 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. One specific example of an aptamer, is an aptamer that binds to a transmembrane protein, for example a transmembrane protein of a fungus. Alternatively, the aptamer may bind a (plant) metabolite or nutrient which is taken up by the fungus.
Alternatively, the linkers are self-cleaving in the endosomes. This may be advantageous when the constructs of the present invention are 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.
The above mentioned 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.
Without wishing to be bound by any particular theory or mechanism, it is thought that long double-stranded 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.
In order to achieve down-regulation of a target gene within a fungal cell 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. However, the invention is not intended to be limited to the use of such siRNAs.
siRNAs may include single-stranded overhangs at one or both ends, flanking the double-stranded portion. In a particularly preferred embodiment the siRNA may contain 3′ overhanging nucleotides, preferably two 3′ overhanging thymidines (dTdT) or uridines (UU). 3′ TT or UU overhangs may be included in the siRNA if the sequence of the target gene immediately upstream of the sequence included in double-stranded part of the dsRNA is AA. This allows the TT or UU overhang in the siRNA to hybridise to the target gene. Although a 3′ TT or UU overhang may also be included at the other end of the siRNA it is not essential for the target sequence downstream of the sequence included in double-stranded part-of the siRNA to have AA. In this context, 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), as discussed above, and also 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. Alternatively, the dsRNA may have a foldback stem-loop or hairpin structure, wherein the two annealed strands of the dsRNA are covalently linked. In this embodiment 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. The features of “hairpin” or “stem-loop” 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). In other embodiments of the invention, 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. For example, 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. In a further embodiment, 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 may be contacted with the double-stranded RNA in any suitable manner, permitting direct uptake of the double-stranded RNA by the fungus. For example, 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. In this embodiment, the fungus may be simply “soaked” with an aqueous solution comprising the double-stranded RNA. In a further embodiment 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.
Alternatively, 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.
In other embodiments 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.
Another aspect of the present invention are target nucleotide sequences of the fungal target genes herein disclosed. Such target nucleotide sequences are particularly important to design the dsRNA constructs according to the present invention. Such 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, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499 and 552 and 562 to 859or the complement thereof, comprising a fragment thereof comprising at least 17, preferably at least 18, 19, 20 or 21; more preferably at least 22, 23 or 24 nucleotides of any of SEQ ID 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499 and 552 and 562 to 859 or the complement thereof.
According to one embodiment, the present invention provides an isolated nucleotide sequence encoding a double-stranded RNA or double-stranded RNA construct as described herein.
According to yet another embodiment, the present invention provides fungal target genes, which comprise a sequence as herein represented by SEQ ID NO 192, 117, 201, 202, 193, 190, 191, 196, 199, 200, 194, 195, 198 and 197, or a fragment thereof of at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides thereof, and which target genes are encompassed by the methods of the present invention.
According to a more specific embodiment, the present invention relates to an isolated nucleic acid sequence consisting of a sequence represented by any of SEQ ID NOs 3, 99, 100, 527, 192, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 201, 202, 5, 101, 102, 528, 193, 1, 97, 98, 526, 184, 185, 190, 191, 37, 124, 9, 106, 531, 188, 189, 196, 13, 109, 534, 199, 200, 33, 126, 23, 119, 35, 127, 7, 103, 104, 105, 529, 186, 187, 194, 195, 29, 118, 17, 108, 533, 198, 25, 121, 19, 125, 31, 123, 11, 107, 532, 197, 27, 122, 21, 120, 15, 110, 535, 458, 530, 460, 539, 540, 462, 541, 464, 542, 466, 543, 468, 544, 470, 545, 472, 546, 474, 547, 476, 548, 478, 549, 480, 550, 482, 551, 484, 552 and 562 to 859, 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.
A person skilled in the art will recognize that homologues of these target genes can be found and that these homologues are also useful in the methods of the present invention.
Protein, or nucleotide sequences are likely to be homologous if they show a “significant” level of, sequence similarity. Truely homologous sequences are related by divergence from a common ancestor gene. Sequence homologues can be of two types:(i) where homologues exist in different species they are known as orthologues. e.g. the α-globin genes in mouse and human are orthologues.(ii) paralogues are homologous genes in within a single species. e.g. the α- and β-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, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof. Methods for determining sequence identity are routine in the art and include use of the Blast software and EMBOSS software (The European Molecular Biology Open Software Suite (2000), Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277). The term “identity” as used herein refers to the relationship between sequences at the nucleotide level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. The reference window is chosen from between at least 10 contiguous nucleotides to about 50, about 100 or to about 150 nucleotides, preferably between about 50 and 150 nucleotides. “% identity” is then calculated by determining the number of nucleotides that are identical between the sequences in the window, dividing the number of identical nucleotides by the number of nucleotides in the window and multiplying by 100.
Other homologues are genes which are alleles of a gene comprising a sequence as represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859. 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, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859.
According to another embodiment, 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.
In one embodiment, the present invention relates to any method described herein wherein a nucleic acid is used encoding a protein with an amino acid sequence which is at least 75%, 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% identical to the amino acid sequence as given in SEQ ID NOs 2, 4, 6, 8, 10, 12, 18, 14, 16, 40, 30, 24, 22, 26, 28, 32, 38, 20, 34, 36, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483 and 485.
In another embodiment, the present invention relates to any method described herein wherein a nucleic acid is used encoding a protein with an amino acid sequence which is at least 75%, at least 80% or 85% similar, preferably at least 90%, 95%, 96%, or more preferably at least 97%, 98% and still more preferably at least 99% similar to the amino acid sequence as given in SEQ ID NOs 2, 4, 6, 8, 10, 12, 18, 14, 16, 40, 30, 24, 22, 26, 28, 32, 38, 20, 34, 36, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483 and 485.
The term “similar” or “similarity” with respect to amino acid sequences allows, for instance, conservative amino acid substitutions to be introduced at one or more positions in the amino acid sequences of target polypeptides. A “conservative amino acid substitution” is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are-also used to define “identity” and “similarity” according to the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389 3402; Altschul (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Inffirmation.
In one embodiment, the present invention relates to a nucleic acid which is degenerated to a nucleic acid encoding a protein as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 18, 14, 16, 40, 30, 24, 22, 26, 28, 32, 38, 20, 34, 36, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483 and 485, as a result of the genetic code.
In another embodiment, the present invention relates to a nucleic acid which is diverging from a nucleic acid encoding a protein as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 18, 14, 16, 40, 30, 24, 22, 26, 28, 32, 38, 20, 34, 36, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483 and 485, as a result of differences in codon usage between organisms.
In yet another embodiment, the present invention relates to a nucleic acid which is diverging from a nucleic acid encoding a protein as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 18, 14, 16, 40, 30, 24, 22, 26, 28, 32, 38, 20, 34, 36, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483 and 485, as a result of differences between alleles.
The invention also encompasses target genes which are fungal orthologues of a gene encoding any of the polypeptides of SEQ ID Nos 2, 4, 6, 8, 10, 12, 18, 14, 16, 40, 30, 24, 22, 26, 28, 32, 38, 20, 34, 36, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483 and 485.
According to another embodiment, the invention encompasses target genes which are fungal orthologues of a gene comprising a nucleotide sequence as represented in any of SEQ ID Nos 562 to 746. A non-limiting list of fungal orthologues genes or sequences comprising at least a fragment of 17 nucleotides of one of the sequences of the invention is given in Table 8. By way of example, orthologues may comprise a nucleotide sequence as represented in any of SEQ ID NOs 562 to 746, or a fragment of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. According to another aspect, the invention thus encompasses any of the methods described herein for controlling fungal growth on a cell or an organism, or for preventing fungal infestation of a cell or an organism susceptible to fungal infection, comprising contacting nematodes with a 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 the nucleotide sequence of a target gene comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 562 to 746, whereby the double-stranded RNA is taken up by the fungus and thereby controls growth or prevents infestation. The invention also relates to fungal-resistant transgenic plants comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 562 to 746. Said fungus may be one of the following non-limiting list of Table 8.
According to another embodiment, the invention encompasses target genes which are nematode orthologues of a gene comprising a nucleotide sequence as represented in any of SEQ ID Nos 747 to 790. A non-limiting list of nematode orthologues genes or sequences comprising at least a fragment of 17 nucleotides of one of the sequences of the invention is given in Table 9. By way of example, orthologues may comprise a nucleotide sequence as represented in any of SEQ ID NOs 747 to 790, or a fragment of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. According to another aspect, the invention thus encompasses any of the methods described herein for controlling nematode growth in an organism, or for preventing nematode infestation of an organism susceptible to nematode infection, comprising contacting nematodes with 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 the nucleotide sequence of a target gene comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 747 to 790, whereby the double-stranded RNA is taken up by the nematode and thereby controls growth or prevents infestation. The invention also relates to nematode-resistant transgenic plants comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 747 to 790. Said nematode may be one of the following non-limiting list of Table 9.
According to another embodiment, the invention encompasses target genes which are insect orthologues of a gene comprising a nucleotide sequence as represented in any of SEQ ID Nos 791 to 859. A non-limiting list of fungal orthologues genes or sequences comprising at least a fragment of 17 nucleotides of one of the sequences of the invention is given in Table 10. By way of example, orthologues may comprise a nucleotide sequence as represented in any of SEQ ID NOs 791 to 859, or a fragment of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides thereof. According to another aspect, the invention thus encompasses any of the methods described herein for controlling-insect growth on a cell or an organism, or for preventing insect infestation of a cell or an organism susceptible to insect infection comprising contacting insects with a 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 the nucleotide sequence of a target gene comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 791 to 859, whereby the double-stranded RNA is taken up by the insect and thereby controls growth or prevents infestation. The invention also relates to insect resistant transgenic plants comprising a fragment of at least 17, 18, 19, 20 or 21 nucleotides of any of the sequences as represented in SEQ ID NOs 791 to 859. Said insect may be one of the following non-limiting list of Table 10.
In one preferred embodiment of the invention 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. In this embodiment 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. Thus, 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. In case the dsRNA hits more than one fungal target gene, 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.
In a preferred embodiment the host organism is a plant and the fungus is a plant pathogenic fungus. In this embodiment 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.
In this context the term “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.
Therefore, in a further aspect 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.
In this embodiment 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.
Accordingly, 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. In a preferred embodiment the plant is rice, corn, soybean, cotton, potato, banana or tomato.
In one embodiment 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. (e.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), Curvularia spp. (e.g. Curvularia oryzae, Curvularia lunatas), Trichoderma spp. (e.g. Trichoderma virde) and 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. (e.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. (e.g. Diplodia maydis) or Puccina spp. (e.g. Puccinia sorgh, Puccinia striiformis (causing yellow rust), Puccinia graminis f.sp. tritici, Puccinia asparag, Puccinia recondita or Puccinia arachidis); in another embodiment the present invention extends to methods as described herein, wherein, the plant is soybean and the target gene is a gene selected from fungus Phakopsora spp. (e.g. Phakopsora pachyrhizi), in another embodiment the present invention extends to methods as described herein, wherein the plant is cotton and the target gene is a gene from a fungus selected from the group consisting of Fusarium spp. (e.g. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum) or Verticillium spp. (e.g. Verticillium dahliae or Verticillium albo-atrum); in another embodiment 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. (e.g. Phytophthora cinnamomi, Phytophthora cactorum, Phytophthora phaseoli, Phytophthora parasitica, Phytophthora citrophthora, Phytophthora megasperma f.sp. soiae or Phytophthora infestans), Rhizoctonia spp. (e.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. Fusarium nivale, Fusarium oxysporum, Fusarium graminearum, Fusarium germinearum, Fusarium culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum); in another embodiment 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.
In a specific embodiment the plant is rice and the fungus is Magnaporthe oryzae causing e.g. rice blast. In another specific embodiment the plant is rice and the fungus is Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis) causing e.g. sheath blight. In yet another embodiment 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), 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) or Rhizopus spp. (e.g. Rhizopus chinensis) causing 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. Cercospora kikuchii or Cercospora zaea-maydis) or Mycosphaerella spp. causing e.g. black and yellow sigatoka. In another specific embodiment 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; in yet another specific embodiment the plant is corn and the fungus is Colletotrichum spp. (e.g. Colletotrichum lindemuthianum) causing e.g. anthracnose. In another specific embodiment the plant is corn and the fungus is Diplodia spp. (e.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. In another specific embodiment the plant is corn and the fungus is Puccinia spp. (e.g. Puccinia sorghi, Puccinia striiformis, Puccinia graminis f.sp. tritici, Puccinia asparagi, Puccinia recondita or Puccinia arachidis) causing e.g. common rust; 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. 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; 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. 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.
In another embodiment the present invention extends to methods as described herein, wherein the plant is rice and wherein said target gene is a gene coding for a fungal orthologue of a protein selected from the group of proteins whose function is given in Table 1.
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 4transgenic plants (or reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell) 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 selected from the group comprising:

- (i) sequences which are at least 75%, preferably 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% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and
- (ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof.

Transgenic,plants according to the invention extend to all plant species specifically described above being resistant to the respective fungal species as specifically described above.
In one embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a rice plant or reproductive or propagation material for a rice plant or a cultured rice plant cell, wherein the target gene is a gene from a fungus selected from the group comprising Magnaporthe spp., Rhizoctonia spp., Fusarium spp., Acremoniella spp., Pythium spp., Curvularia spp., Trichoderma spp. and Rhizopus spp.
In yet another embodiment the transgenic plant (or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell) is a rice, cotton, potato, tomato, corn, tobacco, banana or soybean plant or reproductive or propagation material of such a plant and wherein the target gene is coding for a fungal orthologue of a protein selected from the group of proteins whose function is given in Table 1.
The present invention also encompasses transgenic plants (or reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell) which express or are capable of expressing at least one of the nucleotides of the invention, for instance at least one of the nucleotide sequences represented in any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549; 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859 or the complement thereof, or comprising a fragment thereof comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 nucleotides.
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. Alternatively, 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.
Therefore, according to another embodiment, a recombinant DNA construct is provided comprising the nucleotide sequence encoding the dsRNA or dsRNA construct according to the present invention operably linked to at least one regulatory sequence. Preferably, 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. Preferably the regulatory element is a regulatory element that is active in a plant cell. More preferably, the regulatory element is originating from a plant. The term “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.
Encompassed by the aforementioned term are 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. The term “operably linked” as used herein 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.
By way of example, 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.
Alternatively, 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.
Alternatively, 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.
Furthermore, when using the methods of the present invention for developing transgenic plants resistant against fungi, it might be beneficial to place the nucleic acid encoding the double-stranded RNA according to the present invention under the control of a tissue-specific promoter. In order to improve the transfer of the dsRNA from the plant cell to the pest, the plants could preferably express the dsRNA in a plant part that is first accessed or damaged by the plant pest. In case of a plant pathogenic fungi, 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. Examples of 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 Mol. Biol. 44: 99-106), A (gapA) and B (gapB) subunits of chloroplast glyceraldehyde-3-phosphate dehydrogenase (Conley T. R. et al. 1994 Mol. Cell Biol. 19: 2525-33; Kwon H. B. et al. 1994 Plant Physiol. 105: 357-67), promoter of the Solanum tuberosum gene encoding the leaf and stem specific (ST-LS1) protein (Zaidi M. A. et al., 2005 Transgenic Res. 14:289-98), stem-regulated, defense-inducible genes, such as JAS promoters (patent publication no. 20050034192/US-A1), flower-specific promoters such as chalcone synthase promoter (Faktor O. et al. 1996 Plant Mol. Biol. 32: 849) and fruit-specific specific promoters such as that of RJ39 from strawberry (WO 98 31812).
In yet other embodiments of the present invention, other promoters useful for the expression of dsRNA are used and include, but are not limited to, promoters from an RNA Poll, an RNA Polll, an RNA Pollll, T7 RNA polymerase or SP6 RNA polymerase. These promoters are typically used for in vitro-production of dsRNA, which dsRNA is then included in an 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) contacting an isolated nucleic acid or a recombinant DNA construct of the invention with cell-free components; or (b) introducing (e.g. by transformation, transfection or injection) an isolated nucleic acid or a recombinant DNA construct of the invention in a cell, under conditions that allow transcription of said nucleic acid or recombinant DNA construct to produce the dsRNA or RNA construct.
In one embodiment of the present invention, there is provided a recombinant DNA construct as described herein for use as a medicament.
Accordingly, 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). Preferably said,.cell is a bacterial cell or a plant cell.
Optionally, one or more transcription termination sequences may also be incorporated in the recombinant construct of the invention. The term “transcriptions termination, sequence” encompasses a control sequence at the end of a transcriptional unit, which signals 3′ processing and poly-adenylation of a primary transcript and termination of transcription. Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in the expression construct.
The 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. One example is when 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, f1-ori and colE1 ori.
The recombinant construct may optionally comprise a selectable marker gene. As used herein, the term “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. Examples of 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.
Accordingly, 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. Preferably, 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. Preferably 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). More particularly, methods for expression of 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. 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.
Alternatively, the dsRNA may be secreted by the plant cell and by the plant to the exterior of the plant. As such, the dsRNA may form a protective layer on the surface of the plant.
In a further embodiment, 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. Preferably 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.
In another aspect 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, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859.
The present invention further relates to 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.
The composition may still further contain components which enhance or promote uptake of the dsRNA by the fungal cell. These 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.
The 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.
It is contemplated that the “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. In these embodiments 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, hairpin sequences or compositions described herein.
In one specific embodiment, 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.
Alternatively, 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.
Other conventional pharmaceutical dosage forms may also be produced, including tablets, capsules, pessaries, transdermal patches, suppositories, etc. The chosen form will depend upon the nature of the target fungus and hence the nature of the disease it is desired to treat.
Preferred target human pathogenic and animal pathogenic fungi include, but are not limited to the following:
In humans: 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 group; Blastomyces dermatitidis; Paracoccidioides brasiliensis; Coccidioides immitis; Cryptococcus neoformans; Histoplasma capsulatum Var. capsulatum or Var. duboisii; Sporothrix schenckii; Fusarium spp.; Scopulariopsis brevicaulis and Fonsecaea spp.; Penicillium; Zygomycetes group of fungi, particularly Absidia corymbifera, Rhizomucor pusillus, and Rhizopus arrhizus.;
In animals: Candida spp.; Microsporum spp., particularly Microsporum canis, Microsporum gypseum; Trichophyton mentagrophytes; Aspergillus spp.; or Cryptococcus neoformans.
In one specific embodiment, the composition maybe 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. In this embodiment, 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., Aspergillus spp., Alternaria spp. or Cladosporium 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. For example, 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.
In another embodiment of the invention the compositions are used as a fungicide for a plant or for propagation or reproductive material of a plant, such as on seeds. As an example, 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.
In yet another embodiment, 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.
In another embodiment the invention relates to the use of transiently inserting dsRNA or RNA constructs, or a nucleotide sequence or recombinant DNA constructs encoding the double-stranded RNA or RNA construct described herein, in: plants to treat fungi infested plants or plant fields. The transient transformation can for instance, but not necessarily, be established by modified plant virusses containing the appropriate nucleotide sequences to express fungus derived dsRNA in plants.
In another embodiment 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 kit may be supplied with suitable instructions for use. The instructions may be printed on suitable packaging in which the other components are supplied or may be provided as a separate entity, which may be in the form of a sheet or leaflet for example. The instructions may be rolled or folded for example when in a stored state and may then be unrolled and unfolded to direct use of the remaining components of the kit.
According to a still further embodiment, 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.
In one specific embodiment, 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 3A) (a), MG00884.4.(nt 845-1044, see table 3A) (b), MG07031.4 (nt 251-500, see table 3A) (c), and MG04484.4.(nt 211-409, see table 3A) (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.
FIG. 2: Plasmid map of the plant expression vector pMH115.
FIG. 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.
FIG. 4: List of hairpin sequences. The sequence in bold represents the SI intron (SEQ ID NO 204).
FIG. 5: Magnaporthe grisea strain R67 in the color assay with beta-tubulin dsRNA E7F7 and Seastar dsRNA. Top and side views of four of the wells show the thicker mycelium corresponding in the Seastar dsRNA wells to the darker appearance of the wells.
FIG. 6: Beta-tubulin fragments tested. Three dsRNA fragments of about 200, 400 and 600 bp (SEQ ID Nos 536, 537 and 538) of the regions indicated were tested in the color assay to determine what size gives the best RNAi effect.
FIG. 7: Color assay of negative controls Seastar AFP, GFP and GST compared to tubulin fragment E7F7 (SEQ ID No 537).
FIG. 8: Color assay of some target genes tested. (A) Spores were incubated with various concentrations of dsRNA of targets 1 (MG00170.4), 5 (MG09222.4), 14 (MG02952.4) and 19 (MG05193.4); (B) summarizes results of color assay: “+” indicates the appearance of a light color, “×” indicates a medium color and “−” a dark color for each well of a 4 replica experiment per target, in 0.5, 1, 2 and 4 μM dsRNA used in the treatment.
FIG. 9: Sporulation assay of β-tubulin. Wells of 48-well plates were filled with spores and dsRNA and the plate grown at 26° C. in continuous light for 4-5 days to induce spore formation. The spores were viewed under a Nikon microscope and the pictures taken with a 4× objective at 3 levels of focus ( layers 1, 2, 3). Boxed areas are enlarged to show spores in the Seastar dsRNA-treated mycelium (upper panels) and lack of spores in the β-tubulin dsRNA (E7F7)-treated mycelium (lower panels) (SEQ ID No 537).
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 genes of the budding yeast Saccharomyces cerevisiae are also given, identifiers correspond to gene identifiers of the Saccharomyces Genome Database (SGD™), together with gene function assigned on the basis of yeast data.
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 (A) cDNA's of Magnaporthe grisea target genes and (B) fragments. PCR conditions were as follows: A: Expand High Fidelity PCR system (Roche) 5′ 94° C., 30 cycles (1′ 94° 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° C., 45″ 72°C.), 10′ 72° C.; C: Expand High Fidelity PCR system (Roche) 5′ 94° C., 30 cycles (45″ 94° C., 45″ 58° C., 45″ 72° C.), 10′ 72° C,; D: Expand High Fidelity PCR system (Roche) 5′ 94° C. 30 cycles (1′ 94° C. 1′ 60° C., 3′ 72° C.), 5′ 72° C.; E: Taq Polymerase (Bangalore Genei) 3′ 95° C., 10 cycles (30″ 95° C., 30″ 55° C., 3′ 72), 25 cycles (30″ 95° C., 30″ 55° C., 3′(+5″/cycle)72), 7′ 72° C.; and F: GC Polymerase (BD Biosciences) 5′ 94° C., 30 cycles (1′ 94° C., 1′ 58° C., 3′ 68° C.) 10′ 68° C.; G: RedTaq polymerase (Sigma) 2′ 94° C., 33 cycles (2′ 94° C., 30″ 56° C., 30″ 72° C.) 10′ 72° C.
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° C., 30 cycles (30″ 94° C., 30″ 60° C., 1′ 72° C.) 5′ 72° C.; B: Pwo polymerase (Roche) 5′ 94° C., 30 cycles (30″ 94° C., 30″ 58° C., 30″ 72° C.) 5′ 72° C.; C: Pwo polymerase (Roche) 5′ 94° C., 30 cycles (1′ 94° C., 1′ 61° C., 1′ 30″ 72° C.) 5′ 72° C.; D: Pwo polymerase (Roche) 5′ 94° C. 30 cycles (30″ 94° C., 30″ 60° C., 30″ 72° C.) 5′ 72° C.; E: Pwo polymerase (Roche) 5′ 94° C., 30 cycles (45″ 94° C., 45″ 60° C., 45″ 72° C.) 5′ 72° C.; F: Pwo polymerase (Roche) 10′ 94° C. 30 cycles (1′ 94° C., 1′ 60° C., 3′ 72° C.) 10′ 72° C.
Table 5A and 5B: Selected target nucleotide sequences of the fungal target genes: (IV) stands for experiments in vitro done, (PE) for plant expression experiments and * are ongoing experiments in vitro.
Table 6: Primers used for hairpin construction in pMH115
Table 7: Sporulation phenotype in the presence of 1 or 2 μM dsRNA of targets and negative controls (Seastar and GST) at 4 days. “−” indicates a >90% reduction, “±” indicates a 80-90% reduction, “+” indicates a 75-80% reduction in spore formation. “+++” indicates untreated or negative control dsRNA-treated level of spores formed.
Table 8: Selected sequences* of target genes. Fragments of at least 17 bp of the sequences* are present in any of the orthologues sequences in fungi species (represented by GI number in the right column; several database entry numbers were found but only one is given by way of example).
Table 9: Selected sequences* of target genes. Fragments of at least 17 bp of the sequences* are present in any of the orthologues sequences in nematode species (represented by GI number in the right column; several database entry numbers were found but only one is given by way of example).
Table 10: Selected sequences* of target genes. Fragments of at least 17 bp of the sequences* are present in any of the orthologues sequences in insect species (represented by GI number in the right column; several database entry numbers were found but only one is given by way of example).
The invention will be further understood with reference to the following non-limiting examples.

EXAMPLES

Example 1

Cloning of Target Genes

The methods for dsRNA mediated gene silencing in fungi as described herein are applicable to combat plant pathogenic fungi with dsRNA corresponding to a plant pathogen target gene. Suitable plant pathogen target genes were identified and isolated from the fungus as follows.
Total RNA was prepared from the fungus Magnaporthe grisea using the RNeasy Mini kit for plants (QIAGEN Cat. No. 74904), from which cDNA was prepared using the Superscript™ Double-Stranded cDNA synthesis kit (Invitrogen). To isolate the coding sequence of the Magnaporthe grisea target genes MG00884.4, MG04484.4, MG07472.4, MG06292.4, MG03668.4, MG05169.4 and MG03872.4, 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.
To isolate fragments of the Magnaporthe grisea target genes, total RNA was extracted from Magnaporthe grisea mycelium (RNeasy Plant Mini kit, Qiagen). A reverse transcription reaction (Superscript II, Invitrogen) was done on the total RNA to produce the first strand cDNA, which was then used to amplify partial or full cDNA of target fragments. The cDNA's were cloned into a TOPO TA vector (Invitrogen), and the constructs were used as templates to synthesize dsRNA from (T7 RiboMAX™ kit, Promega).
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.
Alternative Cloning: Cloning of Exons of cDNA:
For cDNA's that could not be amplified from total RNA, exons were amplified from genomic DNA (gDNA) and the exons ligated together to form the cDNA. 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 NaCl, 1% SDS) at 65° C. for 30′. Cell debris and proteins were precipitated by adding potassium acetate 5 M pH 4.8 at −20° 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/CH₃Cl/isoamyl 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.
To isolate the coding region of the Magnaporthe grisea target genes MG00170.4, MG07031.4, MG02946.4 and MG10192.4, two independent PCRs were performed for each exon of each target gene. The full length genomic gene sequence amplified from gDNA was used as template for amplifying each of the exons to compile the cDNA comprising the coding region. The PCR conditions for each exon of the target genes are outlined in Table 4. Subsequently, to obtain the full length coding sequence, 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 15μl-reaction with 1 mM 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.
In case of target gene MG07031.4 (3), which has 5 exons, the first two exons ( Exon 1 and 2 which were 70 bp and 21 bp respectively) were synthetically made. The last 3 exons were amplified as outlined hereinabove and ligated as follows:
1-Ligation and PCR amplification of Exon 4+5 to obtain Exon 4,5
2-Ligation and PCR amplification of Exon 3+ Exon 4,5 to obtain Exon 3,4,5
3-Ligation of synthetic Exon 1+2 to Exon 3,4,5 followed by amplification to obtain Exon 1,2,3,4,5 (Spliced gene).
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 gens are herein represented in Table 4. cDNA's of the other target genes were cloned by one of the above approaches.

Example 2

Optimization of Fragment Length

The beta-tubulin was used to optimize the size of fragments that give color effect in the color assay (Example 4.3). 200, 400 and 600 bp fragments from the 3′ end of the coding region were tested in the color assay and a 400 bp, E7F7 (FIG. 6), was found to be optimal for causing a lighter color. Subsequently, for all the targets, 400 bp dsRNA was used in the color assay. Spores from two Magnaporthe grisea isolates, R67 (FIG. 5) and B157 (FIGS. 7 & 8) gave a similar color phenotype in this assay.

Example 3

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 4 or in vivo as described in examples 5, 6 and 7. These fragments are listed in Table 5. (PY maybe 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.
These fragments or target nucleotide sequences of fungal target genes were used to produce dsRNA in vitro as is described in example 4, or were cloned in a hairpin construct to produce dsRNA in a plant cell (see example 5).

Example 4

The Effect of Silencing a Target Gene in Magnaporthe grisea in vitro In vitro Assays

Germinating conidia have been shown to actively take up materials from the medium by endocytosis. In the following assay 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. The 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.
4.1. Appressorium Assay on Hydrophobic Surface:
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 10⁴conidia/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 μl in water. As a negative control, dsRNA corresponding to part of a GST was used. After 16-24h incubation at 28° C., the germinated spores were stained with Acid Fuchsin for clearer visualization of cellular structures.
Formation of appressoria on the artificial membrane was observed under a microscope. The inhibition of germ tube and appressorium formation 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 triplicate. The percentage of appressoria formed was calculated by the number of appressoria divided by the number of spores in 3 given fields in the microscope and at least 200 spores were counted per replicate.
4.2. Mycelial Growth Assay on Hydrophilic Surface
Conidia of Magnaporthe grisea were harvested, re-suspended in potato dextrose broth and 1250 conidia (in about 90 μl) were aliquoted in each well of a hydrophilic 96-well plates (Falcon 3072). After 0-2 h pregermination at 28° C., dsRNA was added to a final volume of 100 μl 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 used. After 16-24 h incubation at 28° C., 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 FIG. 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.
4.3. Color Assay
Unless otherwise stated, spores from B157, an Indian isolate of Magnaporthe grisea strain (Naqvi N, personal communication) were used. Spores were generated after exposing mycelium to light for 9-11 days, were harvested and used fresh or frozen. Fresh and frozen spores behaved similarly in this assay (data not shown). For consistency, a single frozen stock was used in all assays reported here. The spores were diluted in PDBM (potato dextrose buffer with 25 mM MES buffer pH 5.5) before aliquoting into the wells of a 96-well plate to give 1250 spores per well.
Various amounts of dsRNA were added to spores in the wells and mixed well. The final volume-was made up to 100 μl with PBDM. The plates were incubated at 28° C. in the dark and the color was recorded with a digital camera at 7 days. Spores from two Magnaporthe grisea isolates, R67 (FIG. 5) and B157 (FIGS. 6 & 7) gave a similar color phenotype in this assay.
4.4. Sporulation Assay
As a separate readout of RNAi effect of contacting dsRNA to spores, the sporulation phenotype was observed. In a similar assay set up as described above, the spores were incubated with dsRNA in 96 or 48 well plates. In contrast to the assay above, the plates were exposed to continuous light to induce spore formation and the plates were incubated at 26° C. At day 4-6, the extent of spore formation is viewed under a Nikon microscope and pictures taken with a Nikon digital camera (FIG. 9). Sporulation results are respresented in Table 7.

Example 5

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 189 bp synthetic intron (SI) from the gene X from Arabidopsis thaliana (SI, 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. 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-SI-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-SI-sense) are represented in FIG. 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.
Alternatively, other vectors are used for transformation and other promoters are used for expression of the hairpin dsRNA for the target genes herein described. For example, such 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 6).

Example 6

Rice Plants Resistant to Magnaporthe grisea

Rice calli were transformed and regenerated into shoots and whole plants as described in literature. The plants were 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 μl water) are inoculated onto the leaves. Infection rate and lesion sizes are compared between test and negative control leaves.
In planta infection of established dsRNA-expressing strains are sprayed with fungal spores of 10⁵/ml density, and then covered with plastic bags perforated with holes. These plants are maintained in environmental chambers (Convirons) until disease symptoms develop. The timing of appearance, size and number of lesions, and the rate of plant wilting are indicators of susceptibility to Magnaporthe grisea infection (Valent B. 1990. Phytopathology 80: 57-67). Fifty to 100 replicates per transformed line are used in each experiment. Resistant rice strains are further tested in Convirons as well as in field conditions.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Furthermore, throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation; many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incorporated by reference in their entirety.

TABLE 1


	SEQ ID
SEQ ID NO	NO	M. grisea	S. cerevisiae
DNA	protein	identifier	identifier	Function (based on yeast database)

1 and 41	2	MG00170.4	YPR103W	20S proteasome beta-type subunit, responsible for the chymotryptic
				activity of the proteasome
3 and 42	4	MG00884.4	YER021W	Essential, non-ATPase regulatory subunit of the 26S proteasome lid,
				similar to the p58 subunit of the human 26S proteasome; temperature-
				sensitive alleles cause metaphase arrest, suggesting a role for the
				proteasome in cell cycle control
5 and 43	6	MG07031.4	YER012W	20S proteasome beta-type subunit; localizes to the nucleus throughout
				the cell cycle
7 and 44	8	MG04484.4	YOL120C	Protein component of the large (60S) ribosomal subunit, identical to
				Rpl18Bp and has similarity to rat L18 ribosomal protein; intron of
				RPL18A pre-mRNA forms stem-loop structures that are a target for
				Rnt1p cleavage leading to degradation
9 and 45	10	MG02946.4	YJL203W	Subunit of the SF3a splicing factor complex, required for spliceosome
				assembly
11 and 46	12	MG07472.4	YAL032C	Ortholog of human transcriptional coactivator SKIP, can activate
				transcription of a reporter gene; interacts with splicing factors Prp22p
				and Prp46p
17 and 49	18	MG06292.4	YBL084C	Subunit of the Anaphase-Promoting Complex/Cyclosome (APC/C),
				which is a ubiquitin-protein ligase required for degradation of anaphase
				inhibitors, including mitotic cyclins, during the metaphase/anaphase
				transition
13 and 47	14	MG03668.4	YLR347C	Karyopherin beta, forms a dimeric complex with Srp1p (Kap60p) that
				mediates nuclear import of cargo proteins via a nuclear localization
				signal (NLS), interacts with nucleoporins to guide transport across the
				nuclear pore complex
15 and 48	16	MG10192.4	YBR079C	Subunit of the core complex of translation initiation factor 3(eIF3),
				essential for translation; part of a subcomplex (Prt1p-Rpg1p-Nip1p)
				that stimulates binding of mRNA and tRNA(i)Met to ribosomes
39 and 60	40	MG00604.4	YFL037W	Beta-tubulin; associates with alpha-tubulin to form tubulin dimer,
				which polymerizes to form microtubules
29 and 55	30	MG05169.4	YJL087C	TRL1 encodes tRNA ligase, which is essential for tRNA splicing. Trl1p
				has phosphodiesterase, polynucleotide kinase, and ligase activities;
				each activity resides in a different domain. In tRNA processing, an
				intron is removed from the tRNA transcript by the multisubunit tRNA
				splicing endonuclease (encoded by SEN2, SEN15, SEN34, and
				SEN54); the resulting half-molecules are joined by Trl1p. Deletion of
				TRL1 is lethal; tRNA splicing intermediates accumulate in cells
				depleted of Trl1p or in trl1 temperature-sensitive mutants at the
				restrictive temperature.
23 and 52	24	MG04056.4	YDR407C	Component of the targeting complex (TRAPP) involved in ER to Golgi
				membrane traffic. Null is inviable.
21 and 51	22	MG08911.4	YJL039C	Essential structural subunit of the nuclear pore complex (NPC),
				localizes to the nuclear periphery of nuclear pores, homologous to
				human p205. Null inviable.
25 and 53	26	MG06314.4	YGR047C	Transcription factor tau (TFIIIC) subunit 131.
27 and 54	28	MG08863.4	YKL033W	The authentic, non-tagged protein was localized to the mitochondria.
31 and 56	32	MG07222.4	YPR175W	Second largest subunit of DNA polymerase II (DNA polymerase
				epsilon), required for normal yeast chromosomal replication;
				expression peaks at the G1/S phase boundary; potential Cdc28p
				substrate. Null mutant is inviable; conditional mutant shows defects in
				DNA replication.
37 and 59	38	MG01760.4	YJL085W	Essential 70 kDa subunit of the exocyst complex (Sec3p, Sec5p,
				Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p), which has the
				essential function of mediating polarized targeting of secretory vesicles
				to active sites of exocytosis.
				Null is also inviable.
19 and 50	20	MG07116.4	YIL129C	Identified in a hunt for mutants that activate OCH1 transcription;
				Transcriptional Activator of OCH1
33 and 57	34	MG03872.4	YJL010C	RNA binding protein involved in RNA processing. Protein required for
				cell viability.
35 and 58	36	MG04185.4	YBL034C	Component of the mitotic spindle that binds to interpolar microtubules
				and plays an essential role in their ability to provide an outward force
				on the spindle poles.
				Null mutant shows defects in spindle assembly.
556 and 486	459	MG09222.5	YGL123W	Protein component of the small (40S) subunit, essential for control of
				translational accuracy; has similarity to E. coli S5 and rat S2 ribosomal
				proteins.
460 and 487	461	MG00961.4	YLR212C	Gamma-tubulin, involved in nucleating microtubules from both the
				cytoplasmic and nuclear faces of the spindle pole body
462 and 488	463	MG02952.5	YBR189W	Ribosomal protein S9
464 and 489	465	MG04095.5	YLR025W	Involved in intracellular protein transport (carrier)
466 and 490	467	contig2.561.g35	YJR145C/YHR203C	Ribosomal protein S4
468 and 491	469	contig2.887.g4	YGL137W	RNA polymerase II 140 kD subunit
470 and 492	471	MG05193.5	YDL126C	Transitional endoplasmatic reticulum ATPase TER94, Golgi
				organization and biogenesis
472 and 493	473	MG09952.5	YLR293C	Putative RAN small monomeric GTPase, cell adhesion
474 and 494	475	MG06910.5	YPR181C	GTPase activator, ER to Golgi protein transport, component of the
				Golgi stack
476 and 495	477	contig2.1499. g3	YDL185W	vATPase
478 and 496	479	MG04829.5	YGL147C	GTPase activator, involved in intracellular protein transport
480 and 497	481	MG5858.4	YFR004W	Proteasome regulatory particle, lid subcomplex, rpn11
482 and 498	483	contig2.1053.g5	YBR084C-	Ribosomal protein L19
			A/ YBL027W
484 and 499	485	contig2.875.g14	YOR151C	COPI vesicle coat; involved in Golgi to ER intracellular protein
				transport

TABLE 2


		Orthologue
		Identifier
	SEQ	(“Accession
M. grisea	ID	nr”.
Identifier	NO	“Version nr”)	Ortholoque Species

MG00170.4	210	XM_387062.1	Gibberella zeae PH-1
	214	XM_745627.1	Aspergillus fumigatus
	229	XM_446439.1	Candida glabrata
	230	DR709833.1	Aspergillus niger
	233	XM_709731.1	Candida albicans
	236	CA582255.1	Paracoccidioides brasiliensis
	243	AJ636229.1	Mycosphaerella graminicola
	245	XM_408069.1	Aspergillus nidulans FGSC A4
	254	DN795961.1	Sclerotinia sclerotiorum
	271	XM_571562.1	Cryptococcus neoformans var.
	272	XM_402498.1	Ustilago maydis
	296	AW790794.1	Blumeria graminis f. sp.
	301	CO136158.1	Aspergillus flavus
	321	CV968020.1	Phytophthora infestans
	324	BI189607.1	Fusarium sporotrichioides
	406	BW644197.1	Fusarium oxysporum f. sp.
	409	XM_369074.1	Magnaporthe grisea 70-15
MG00604.4	208	XM_713903.1	Candida albicans
	215	AL117033.1	Botryotinia fuckeliana
	216	CN239178.1	Paracoccidioides brasiliensis
	217	DR046029.1	Phaeosphaeria nodorum
	219	DR709051.1	Aspergillus niger
	222	DR439037.1	Phytophthora parasitica
	223	CV966689.1	Phytophthora infestans
	224	DR439037.1	Phytophthora parasitica
	231	CO138830.1	Aspergillus flavus
	234	BQ110893.1	Verticillium dahliae
	241	BI191775.1	Fusarium sporotrichioides
	244	BI191775.1	Fusarium sporotrichioides
	249	DN477093.1	Alternaria brassicicola
	325	BQ621948.1	Conidiobolus coronatus
	329	AW789096.1	Blumeria graminis f. sp.
	338	AY337716.1	Fusarium oxysporum f. sp.
	410	AF218256.1	Pythium ultimum
	411	AF257329.1	Leptosphaeria maculans
	412	AY763789.1	Trichophyton rubrum
	413	X81961.1	Erysiphe pisi
	414	XM_368640.1	Magnaporthe grisea 70-15
	415	XM_389706.1	Gibberella zeae PH-1
	416	XM_403443.1	Ustilago maydis
	417	XM_405319.1	Aspergillus nidulans FGSC A4
	418	XM_448766.1	Candida glabrata
	419	XM_569650.1	Cryptococcus neoformans var.
	420	XM_747363.1	Aspergillus fumigatus
MG00884.4	220	AL116400.1	Botryotinia fuckeliana
	239	CO145866.1	Aspergillus flavus
	250	XM_716188.1	Candida albicans
	251	XM_716459.1	Candida albicans
	255	XM_566480.1	Cryptococcus neoformans var.
	256	XM_400867.1	Ustilago maydis
	289	AJ639036.1	Mycosphaerella graminicola
	294	DR706305.1	Aspergillus niger
	312	CN251795.1	Paracoccidioides brasiliensis
	330	CF844359.1	Phytophthora sojae
	366	CV959275.1	Phytophthora infestans
	421	XM_368360.1	Magnaporthe grisea 70-15
	422	XM_386221.1	Gibberella zeae PH-1
	423	XM_406904.1	Aspergillus nidulans FGSC A4
	424	XM_749902.1	Aspergillus fumigatus
MG01760.4	263	XM_750434.1	Aspergillus fumigatus
	267	XM_750434.1	Aspergillus fumigatus
	275	XM_410347.1	Aspergillus nidulans FGSC A4
	335	DR707382.1	Aspergillus niger
	355	CO152071.1	Aspergillus flavus
	367	AW180181.1	Mycosphaerella graminicola
	374	BQ493216.1	Paracoccidioides brasiliensis
	403	XM_399460.1	Ustilago maydis
	404	XM_449482.1	Candida glabrata
	425	XM_363834.1	Magnaporthe grisea 70-15
	426	XM_382095.1	Gibberella zeae PH-1
MG02946.4	207	XM_389109.1	Gibberella zeae PH-1
	225	XM_408897.1	Aspergillus nidulans FGSC A4
	276	DR710305.1	Aspergillus niger
	278	DR710305.1	Aspergillus niger
	287	XM_749869.1	Aspergillus fumigatus
	336	XM_397920.1	Ustilago maydis
	352	CA582353.1	Paracoccidioides brasiliensis
	357	XM_566533.1	Cryptococcus neoformans var.
	389	XM_717783.1	Candida albicans
	405	CV949205.1	Phytophthora infestans
	427	XM_366870.1	Magnaporthe grisea 70-15
MG03668.4	209	XM_570418.1	Cryptococcus neoformans var.
	237	XM_446411.1	Candida glabrata
	252	XM_708682.1	Candida albicans
	268	CN239360.1	Paracoccidioides brasiliensis
	283	BQ110666.1	Verticillium dahliae
	290	DR703485.1	Aspergillus niger
	304	AJ635696.1	Mycosphaerella graminicola
	361	DN478339.1	Alternaria brassicicola
	384	CF843394.1	Phytophthora sojae
	393	CV916642.1	Phytophthora infestans
	428	XM_361125.1	Magnaporthe grisea 70-15
	429	XM_389630.1	Gibberella zeae PH-1
	430	XM_399990.1	Ustilago maydis
	431	XM_405043.1	Aspergillus nidulans FGSC A4
	432	XM_747845.1	Aspergillus fumigatus
MG03872.4	319	XM_387214.1	Gibberella zeae PH-1
	328	XM_749415.1	Aspergillus fumigatus
	377	CO147563.1	Aspergillus flavus
	381	XM_706881.1	Candida albicans
	402	CF645358.1	Ustilago maydis
	433	XM_361398.1	Magnaporthe grisea 70-15
MG04056.4	206	XM_410670.1	Aspergillus nidulans FGSC A4
	212	XM_742501.1	Aspergillus fumigatus
	376	CO151047.1	Aspergillus flavus
	378	DR164852.1	Paracoccidioides brasiliensis
	386	XM_714273.1	Candida albicans
	394	DN477226.1	Alternaria brassicicola
	408	XM_570072.1	Cryptococcus neoformans var.
	434	XM_361582.1	Magnaporthe grisea 70-15
	435	XM_386415.1	Gibberella zeae PH-1
MG04185.4	280	XM_747547.1	Aspergillus fumigatus
	343	XM_405132.1	Aspergillus nidulans FGSC A4
	345	XM_402160.1	Ustilago maydis
	346	XM_708987.1	Candida albicans
	354	AL111352.1	Botryotinia fuckeliana
	370	BI187889.1	Fusarium sporotrichioides
	372	BI187889.1	Fusarium sporotrichioides
	391	XM_569869.1	Cryptococcus neoformans var.
	436	XM_361711.1	Magnaporthe grisea 70-15
	437	XM_386367.1	Gibberella zeae PH-1
MG04484.4	221	XM_362039.1	Magnaporthe grisea 70-15
	257	BU064817.1	Gibberella zeae
	258	XM_390042.1	Gibberella zeae PH-1
	260	CO134330.1	Aspergillus flavus
	261	XM_409937.1	Aspergillus nidulans FGSC A4
	264	CO140779.1	Aspergillus flavus
	265	DR706673.1	Aspergillus niger
	266	XM_749981.1	Aspergillus fumigatus
	269	CD488085.1	Ustilago maydis
	273	CN241271.1	Paracoccidioides brasiliensis
	284	DT932969.1	Leptosphaeria maculans
	288	AL113061.1	Botryotinia fuckeliana
	291	AJ638244.1	Mycosphaerella graminicola
	305	BQ110718.1	Verticillium dahliae
	317	CF845455.1	Phytophthora sojae
	326	CV949997.1	Phytophthora infestans
	333	XM_718100.1	Candida albicans
	337	DN478895.1	Alternaria brassicicola
	341	XM_570672.1	Cryptococcus neoformans var.
	342	DR438661.1	Phytophthora parasitica
	358	BQ283973.1	Blumeria graminis f. sp.
MG05169.4	240	XM_449481.1	Candida glabrata
	297	XM_716256.1	Candida albicans
	308	XM_398486.1	Ustilago maydis
	331	CN239627.1	Paracoccidioides brasiliensis
	349	XM_570638.1	Cryptococcus neoformans var.
	368	AL116927.1	Botryotinia fuckeliana
	438	XM_359608.1	Magnaporthe grisea 70-15
	439	XM_391770.1	Gibberella zeae PH-1
	440	XM_405433.1	Aspergillus nidulans FGSC A4
	441	XM_747243.1	Aspergillus fumigatus
MG06292.4	226	X59269.1	Emericella nidulans
	227	XM_750103.1	Aspergillus fumigatus
	282	XM_566728.1	Cryptococcus neoformans var.
	285	XM_712829.1	Candida albicans
	286	XM_712763.1	Candida albicans
	353	XM_399704.1	Ustilago maydis
	373	CO142798.1	Aspergillus flavus
	399	CN240192.1	Paracoccidioides brasiliensis
	442	XM_369777.1	Magnaporthe grisea 70-15
	443	XM_385668.1	Gibberella zeae PH-1
MG06314.4	293	XM_385552.1	Gibberella zeae PH-1
	295	XM_385552.1	Gibberella zeae PH-1
	320	XM_404544.1	Aspergillus nidulans FGSC A4
	369	XM_745154.1	Aspergillus fumigatus
	383	XM_444925.1	Candida glabrata
	390	CA581516.1	Paracoccidioides brasiliensis
	392	XM_397947.1	Ustilago maydis
	395	XM_567461.1	Cryptococcus neoformans var.
	407	DR439107.1	Phytophthora parasitica
	444	XM_369799.1	Magnaporthe grisea 70-15
MG07031.4	211	XM_367106.1	Magnaporthe grisea 70-15
	238	DR668205.1	Gibberella moniliformis
	242	CN811486.1	Gibberella zeae
	247	DR710271.1	Aspergillus niger
	248	XM_746981.1	Aspergillus fumigatus
	253	AL111548.1	Botryotinia fuckeliana
	259	CN248132.1	Paracoccidioides brasiliensis
	270	CO151671.1	Aspergillus flavus
	298	XM_447214.1	Candida glabrata
	299	XM_408594.1	Aspergillus nidulans FGSC A4
	302	XM_716840.1	Candida albicans
	306	BI201970.1	Fusarium sporotrichioides
	316	XM_401794.1	Ustilago maydis
	332	XM_569789.1	Cryptococcus neoformans var.
	350	CV222840.1	Phanerochaete chrysosporium
	356	DR440327.1	Phytophthora parasitica
	359	BE775707.1	Phytophthora infestans
	360	DR440327.1	Phytophthora parasitica
	379	DN479774.1	Alternaria brassicicola
MG07116.4	303	XM_716554.1	Candida albicans
	363	CA582713.1	Paracoccidioides brasiliensis
	364	XM_397938.1	Ustilago maydis
	445	XM_367191.1	Magnaporthe grisea 70-15
	446	XM_389963.1	Gibberella zeae PH-1
	447	XM_404731.1	Aspergillus nidulans FGSC A4
	448	XM_745894.1	Aspergillus fumigatus
MG07222.4	277	XM_744655.1	Aspergillus fumigatus
	300	XM_411576.1	Aspergillus nidulans FGSC A4
	351	CN247150.1	Paracoccidioides brasiliensis
	385	XM_446786.1	Candida glabrata
	387	XM_400375.1	Ustilago maydis
	396	XM_571676.1	Cryptococcus neoformans var.
	397	XM_714233.1	Candida albicans
	449	XM_367297.1	Magnaporthe grisea 70-15
	450	XM_390075.1	Gibberella zeae PH-1
MG07472.4	218	XM_386292.1	Gibberella zeae PH-1
	279	XM_742945.1	Aspergillus fumigatus
	292	XM_412317.1	Aspergillus nidulans FGSC A4
	307	AL115094.1	Botryotinia fuckeliana
	314	XM_572276.1	Cryptococcus neoformans var.
	318	DR708959.1	Aspergillus niger
	322	CO143369.1	Aspergillus flavus
	334	XM_400233.1	Ustilago maydis
	347	CV957053.1	Phytophthora infestans
	380	CA581803.1	Paracoccidioides brasiliensis
	388	XM_714102.1	Candida albicans
	451	XM_367561.1	Magnaporthe grisea 70-15
MG08863.4	281	XM_385749.1	Gibberella zeae PH-1
	311	XM_750141.1	Aspergillus fumigatus
	339	XM_410205.1	Aspergillus nidulans FGSC A4
	362	AL111561.1	Botryotinia fuckeliana
	375	XM_569100.1	Cryptococcus neoformans var.
	400	BQ499912.1	Paracoccidioides brasiliensis
	401	XM_448856.1	Candida glabrata
	452	XM_364018.1	Magnaporthe grisea 70-15
MG08911.4	213	XM_748293.1	Aspergillus fumigatus
	313	BI191623.1	Fusarium sporotrichioides
	348	CO143726.1	Aspergillus flavus
	382	DR706257.1	Aspergillus niger
	398	XM_447745.1	Candida glabrata
	453	XM_364066.1	Magnaporthe grisea 70-15
	454	XM_385694.1	Gibberella zeae PH-1
MG10192.4	228	XM_403070.1	Ustilago maydis
	232	XM_706539.1	Candida albicans
	235	XM_706539.1	Candida albicans
	246	DR701435.1	Aspergillus niger
	262	BQ110649.1	Verticillium dahliae
	274	AL114181.1	Botryotinia fuckeliana
	309	BI201406.1	Fusarium sporotrichioides
	310	XM_570890.1	Cryptococcus neoformans var.
	315	BW644576.1	Fusarium oxysporum f. sp.
	323	DR045681.1	Phaeosphaeria nodorum
	327	BQ622080.1	Conidiobolus coronatus
	340	BE187972.1	Cladosporium fulvum
	344	CN242658.1	Paracoccidioides brasiliensis
	365	AJ637851.1	Mycosphaerella graminicola
	371	BE776281.1	Phytophthora infestans
	455	XM_365972.1	Magnaporthe grisea 70-15
	456	XM_387413.1	Gibberella zeae PH-1
	457	XM_406880.1	Aspergillus nidulans FGSC A4
	458	XM_745176.1	Aspergillus fumigatus

TABLE 3A


Target gene		Specific	Specific
ID	PCR	Primer FW	Primer RV	cDNA clone

MG00884.4	A	(SEQ ID NO: 61)	(SEQ ID NO: 62)	(SEQ ID NO: 3)
		atgaaggacgagacatcacg	ttacatgccctcgaattctcc	MG00884.4TA/02A#01

MG04484.4	B	(SEQ ID NO: 63)	(SEQ ID NO: 64)	(SEQ ID NO: 186)
		atgggtatcgatcttaagaagcacc	ttagaccttgaaaccacgcg	MG04484.4TA/04A#07

MG07472.4	B	(SEQ ID NO: 65)	(SEQ ID NO: 66)	(SEQ ID NO: 11)
		atgcactcgccgccgcggaagttg	tcaatcgtcgtcctccaccc	MG07472.4TA/07A#01

MG06292.4	C	(SEQ ID NO: 67)	(SEQ ID NO: 68)	(SEQ ID NO: 17)
		atggcacctggtagcagtg	tcagccatcaccgttgggtag	MG06292.4TA/08A#03

MG03668.4	D	(SEQ ID NO: 69)	(SEQ ID NO: 70)	(SEQ ID NO: 13)
		atggcgacgtcagacatc	tcaagtctgctgcatgac	MG03668.4TA/09A#01 and
	D	(SEQ ID NO: 71)	(SEQ ID NO: 72)	MG03668.4TA/09B#05
		atggcgacgtcagacatc	tcaagtctgctgcatgac

MG05169.4	E	(SEQ ID NO: 73)	(SEQ ID NO: 74)	(SEQ ID NO: 29)
		taaaatggccaccgatgcccaagggcct	ccctcagaattttgacaggaccgcc	MG05169.4TA/11A#12 and
	E	(SEQ ID NO: 75)	(SEQ ID NO: 76)	MG05169.4TA/11B#15
		taaaatggccaccgatgcccaagggcct	ccctcagaattttgacaggaccgcc

MG03872.4	F	(SEQ ID NO: 77)	(SEQ ID NO: 78)	(SEQ ID NO: 33)
		atgggtaaacgaaagagcgaacag	tcactcatctgcctcctgcttttttc	MG03872.4TA/30B#01

MG00604.4	G	(SEQ ID NO: 557)	(SEQ ID NO: 558)	(SEQ ID NO: 39)
		atgcgtgaaattgttcaccttcag	ctactcctcgccctcaagaggggc	FL b-tub/pBF089

TABLE 3B


Target gene		Specific	Specific
ID	PCR	Primer FW	Primer RV	Fragment

MG09222.5	G	(SEQ ID NO: 500)	(SEQ ID NO: 501)	(SEQ ID NO: 530)
		tgactccgagggccacgttggtct	gctcctgatgagcttggtctcctt	pBF076/TA5

MG00961.4	G	(SEQ ID NO: 559)	(SEQ ID NO: 560)	(SEQ ID NO: 561)
		gtctctactgttatgtctgccagc	tcaccccattctccgatccgtctc	Exon6 g-tub/pBF093

MG02952.4	G	(SEQ ID NO: 502)	(SEQ ID NO: 503)	(SEQ ID NO: 541)
		gtctcaagaacaagcgtgaggtctgg	gccttctttctgaggacacgg	pBF077/TA14

MG04095.4	G	(SEQ ID NO: 504)	(SEQ ID NO: 505)	(SEQ ID NO: 542)
		catcttggggctgcgatcaca	gattgcctcgtttccgagctgat	pBF078/TA15

contig2.561.	G	(SEQ ID NO: 506)	(SEQ ID NO: 507)	(SEQ ID NO: 543)
g35		gcaaggtccgcaccgactcg	gcacaatgttgaagccaccatcg	pBF079/TA17

contig2.887.	G	(SEQ ID NO: 508)	(SEQ ID NO: 509)	(SEQ ID NO: 544)
g4		cctcttgcgtgagcatggatacca	cgatcttggtcttgttcctacatggc	pBF080/TA18

MG05193.4	G	(SEQ ID NO: 510)	(SEQ ID NO: 511)	(SEQ ID NO: 545)
		ggagcaggtccagtacccagtcg	ggtcgctccaataacgaacacgtt	pBF081/TA19

MG09952.4	G	(SEQ ID NO: 512)	(SEQ ID NO: 513)	(SEQ ID NO: 546)
		gccagtgcggtatcatcatgtttg	cttcatcatcatcatcgggcagg	pBF082/TA20

MG06910.4	G	(SEQ ID NO: 514)	(SEQ ID NO: 515)	(SEQ ID NO: 547)
		caggtcggtcttctggagatgaaagg	cagatgagtgctggtaatatgtgagaaattg	pBF083/TA21

contig2.1499.	G	(SEQ ID NO: 516)	(SEQ ID NO: 517)	(SEQ ID NO: 548)
g3		cctgtgactacatcgaccctgtcca	gcctcgtcgtggaaacccatc	pBF084/TA22

MG04829.4	G	(SEQ ID NO: 518)	(SEQ ID NO: 519)	(SEQ ID NO: 549)
		cgttatcggcatcgagattcacca	cgagacgtacataccatccaagaacttacg	pBF085/TA23

MG5858.4	G	(SEQ ID NO: 520)	(SEQ ID NO: 521)	(SEQ ID NO: 550)
		cgagtttgtcgatgacttcacagtacg	caatgcttgaatcgagggtttgttg	pBF086/TA25

contig2.1053.	G	(SEQ ID NO: 522)	(SEQ ID NO: 523)	(SEQ ID NO: 551)
g5		cgtccgttttggactgtggca	gcacggtggatgtgctcaacaag	pBFO87/TA27

contig2.875.	G	(SEQ ID NO: 524)	(SEQ ID NO: 525)	(SEQ ID NO: 552)
g14		ggtgtcctccacttcaatc	gaggaaggtcttgatctggctaatggt	pBF088/TA28

TABLE 4


Target gene			Specific	Specific
ID	Exon	PCR	Primer FW	Primer RV	cDNA clone

MG00170.4	1	A	(SEQ ID NO: 79) 1 FP:	(SEQ ID NO: 80) 1 Ex1	(SEQ ID NO: 184)
			atggacaccctggtagcccgctac	RP: atgtgctactggcggaacc	MG00170.4TA/01A#04
	2	A	(SEQ ID NO: 81) Ex2 FP:	(SEQ ID NO: 82) 1 RP:
			ccctcatcttggctccgca	ctagtccagcgcactcgtaac

MG07031.4	1	synthetic			(SEQ ID NO: 5)
	2	synthetic			MG07031.4TA/03A#05
	3	B	(SEQ ID NO: 83) 3 Ex3 FP:	(SEQ ID NO: 84) 3 Ex3	and
			gttcttttggggatcacgggcaa	RP: tggtgtctccaggctcgc	MG07031.4TA/03B#02
	4	B	(SEQ ID NO: 85) 3 Ex4 FP:	(SEQ ID NO: 86) 3 Ex4
			tccagtttgcggagtaca	RP: tgagcatatccatgcgca
	5	B	(SEQ ID NO: 87) 3 Ex5 FP:	(SEQ ID NO: 88) 3 Ex5
			gtactactgcctctccat	RP: tcaagcactctttacaatcttg

MG02946.4	1	C	(SEQ ID NO: 89) 6 FP:	(SEQ ID NO: 90) 6 Ex1	(SEQ ID NO: 188)
			tttatggcgaccaacggcgat	RP: tcctcatgtgctcctcccactc	MG02946.4TA/06A#1
	2	D	(SEQ ID NO: 91) 6 Ex2	(SEQ ID NO: 92) 6 RP:
			FP: tcgaactgctggatccca	ccgttattcctttttgtctcc

MG10192.4	2	E	(SEQ ID NO: 93)	10 FP:	(SEQ ID NO: 94) 10 Ex2	(SEQ ID NO: 15)
			atgcctcctccaccgcatc	RP: ctctatcgtgccgacatttgtg	MG10192.4TA/10A-EXON2#H1
	3	F	(SEQ ID NO: 95)	10 Ex3	(SEQ ID NO: 96) 10 RP:	and
			FP: cttgttttgaaaaaattcat	ctagttcccggcattctcacg	MG10192.4TA/10B-EXON3#09
			cgagctcgccgc

TABLE 5A


		In vitro
		(IV) or
Target gene		Plant
(ID)	Fragment of the target gene	expressed (PE)

MG00170.4	(SEQ ID NO:97)	PE
(nt 11-306)	tggtagcccgctacagccgcccggcttaccagcagaacgagacattcacagaagatgat
	cagcaagacctttgcgattccgtcccaagtctttcactcaagtttgcggttccgccagt
	agcacatccctcatcttggctccgcacagcaacggacgaccacgcaaacccaaactgcc
	ccatcaagatcgcacacggaacgacgacgctcgctttcaggtttcagggaggcatcatc
	gttgcgaccgactctcgtgccaccgccggcaactggattgcttcgcagacggtcaagaa
	g (296 bp)

MG00170.4	(SEQ ID NO: 98)	PE
(nt 1-835)	atggacaccctggtagcccgctacagccgcccggcttaccagcagaacgagacattcac
	agaagatgatcagcaagacctttgcgattccgtcccaagtctttcactcaagtttgcgg
	ttccgccagtagcacatccctcatcttggctccgcacagcaacggacgaccacgcaaac
	ccaaactgccccatcaagatcgcacacggaacgacgacgctcgctttcaggtttcaggg
	aggcatcatcgttgcgaccgactctcgtgccaccgccggcaactggattgcttcgcaga
	cggtcaagaaggtcatcgagatcaactgttgcctgctcggcaccatggccggcggtgcc
	gcagactgccagtactggctcgcctggctgggcatgcagtgccgtctgcacgagctccg
	ccacaagcgccgcatctcggtcgccgccgcctccaagattctcgccaacctcgtctacc
	agtacaagggcatgggcctcagcatgggtaccatgtgcgccggcgtcaccaaggaggag
	ggtcccgccctgtactacatcgacagcgacggcaccaggcttgccggcaatctgttctg
	tgtgggatccggtcagacctttgcctatggtgtgctggatgccgagtacaagtacgacc
	tgtcggatgaggatgcgctcgagctcggccgcaggagcattctcgccgccacccacagg
	gatgcctactccggtggttttatcaacttgtaccacgtcaaggaggacggttgggtcaa
	gcacggattcaacgacacgaaccctatcttctggaagactaagctggagaagggcgagt
	ttaccaacg (835 bp)

MG00170.4	(SEQ ID NO: 526)	IV
(nt 1-400)	atggacaccctggtagcccgctacagccgcccggcttaccagcagaacgagacattcac
	agaagatgatcagcaagacctttgcgattccgtcccaagtctttcactcaagtttgcgg
	ttccgccagtagcacatccctcatcttggctccgcacagcaacggacgaccacgcaaac
	ccaaactgccccatcaagatcgcacacggaacgacgacgctcgctttcaggtttcaggg
	aggcatcatcgttgcgaccgactctcgtgccaccgccggcaactggattgcttcgcaga
	cggtcaagaaggtcatcgagatcaactcttgcctgctcggcaccatggccggcggtgcc
	gcagactgccagtactggctcgcctggctgggcatgcagtgccgtctgcacgagctccg
	ccacaagcgccgcatctcggtcgccgccgcctccaagattctcgccaacctcgtctacc
	agtacaagggcatgggcctcagcatggg (500 bp)

MG00884.4	(SEQ ID NO: 99)	PE
(nt 852-	cgacctgctcgtctcacatacacagttccccgagaatgctgcaaacaaccaagttgcgc
1139)	ggtatctctactaccttggtcggattcgcgccatacagctgcgctacacggaggcacac
	gagcacctgacggctgcgacgcggaaggcaccgagctctaactgcgcggttggtttctc
	gcagacggccaccaagcttttgctggtggttgagcttcttatgggcgatattcctgatc
	gcgcgaccttccgccaaccgaccctcgaggaggcactgcacccttacttcct
	(288 bp)

MG00884.4	(SEQ ID NO: 100)	IV: 33%
(nt 845-	accaggccgacctgctcgtctcacatacacagttccccgagaatgctgcaaacaaccaa	(see FIG. 1b)
1044)	gttgcgcggtatctctactaccttggtcggattcgcgccatacagctgcgctacacgga
	ggcacacgagcacctgacggctgcgacgcggaaggcaccgagctctaactgcgcggttg
	gtttctcgcagacggccaccaag (200 bp)

MG00884.4	(SEQ ID NO: 527)	IV
(nt 845-	accaggccgacctgctcgtctcacatacacagttccccgagaatgctgcaaacaaccaa
1244)	gttgcgcggtatctctactaccttggtcggattcgcgccatacagctgcgctacacgga
	ggcacacgagcacctgacggctgcgacgcggaaggcaccgagctctaactgcgcggttg
	gtttctcgcagacggccaccaagcttttgctggtggttgagcttcttatgggcgatatt
	cctgatcgcgcgaccttccgccaaccgaccctcgaggaggcactgcacccttacttcct
	gctagtccaggctgttcgggtcggtaacctcgacaacttcgagacgaccattgcggacc
	acgccgatactttccgcaaggacggaacctacaccctcatcctccg (400 bp)

MG07031.4	(SEQ ID NO: 101)	PE
(nt 261-543)	ggagtacatcatggcaaacgcgaagctctattcgatgcgtaacgagaccgatctttcac
	cgtccgcattggcgcattacgtccgtggcgagcttgcctcgagcttgaggtcgcggaag
	ccatacacagtcaacctcctcttgggcggtgtcgaccccatcacacacaagccgagcct
	ctactggctggactaccttgcggctctggcgccggtcccatatgctgcgcatggatatg
	ctcagtactactgcctctccatcctcgacaaacatcaccatcccgat (283 bp)

MG07031.4	(SEQ ID NO: 102)	IV: 43%
(nt 251-500)	tccagtttgcggagtacatcatggcaaacgcgaagctctattcgatgcgtaacgagacc	(see FIG. 1c)
	gatctttcaccgtccgcattggcgcattacgtccgtggcgagcttgcctcgagcttgag
	gtcgcggaagccatacacagtcaacctcctcttgggcggtgtcgaccccatcacacaca
	agccgagcctctactggctggactaccttgcggctctggcgccggtcccatatgctgcg
	catggatatgctca (250 bp)

MG07031.4	(SEQ ID NO: 528)	IV
(nt 251-650)	tccagtttgcggagtacatcatggcaaacgcgaagctctattcgatgcgtaacgagacc
	gatctttcaccgtccgcattggcgcattacgtccgtggcgagcttgcctcgagcttgag
	gtcgcggaagccatacacagtcaacctcctcttgggcggtgtcgaccccatcacacaca
	agccgagcctctactggctggactaccttgcggctctggcgccggtcccatatgctgcg
	catggatatgctcagtactactgcctctccatcctcgacaaacatcaccatcccgatat
	caccctgcaccagggtatcaagatcctgactatgtgcaccgacgagctgaagaggagat
	taccgattgacttcaaaggaatggtggtgaaggcggtgacaaagga (400 bp)

MG04484.4	(SEQ ID NO: 103)	PE
(nt 7-276)	atcgatcttaagaagcaccacgtgcgcagcacgcaccgcaaggcccccaagagcgacaa
	tgtctacctcaagctcttggtgaagctctaccgcttcctggcccgcaggaccgactcca
	gcttcaacaggttgtccttcgccgcctctttatgtcgcgcaccaaccgccctcccgtct
	ccctgtcgcgcatcgcaggcaacctgaagaacggcaacgagaagaagaccgttgtcgtt
	gtcggcaccgtcaccgacgacaaccgcctggtt (270 bp)

MG04484.4	(SEQ ID NO: 104)	PE
(nt 1-555)	atgggtatcgatcttaagaagcaccacgtgcgcagcacgcaccgcaaggcccccaagag
	cgacaatgtctacctcaagctcttggtgaagctctaccgcttcctggcccgcaggaccg
	actccagcttcaacaaggttgtccttcgccgcctctttatgtcgcgcaccaaccgccct
	cccgtctccctgtcgcgcatcgcaggcaacctgaagaacggcaacgagaagaagaccgt
	tgtcgttgtcggcaccgtcaccgacgacaaccgcctggttgagtgccccaaggcccagg
	tcgctgcccttcgcttcaccgccaccgcccgtgcccgcatcgttgccgccggcggccag
	gccatcacccttgaccagctggctcttgagaagcccactggtgccaacaccctcctcct
	ccccaagaacgcccgtgaggctgtcaagcacttcggcttcggtccccacaagcacaaga
	agccttacgtccagtccaagggccgcaagttcgagaaggcccgtggtcgcagacgatcg
	cgtggtttcaaggtctaa (555 bp)

MG04484.4	(SEQ ID NO: 105)	IV: 30%
(nt 211-409)	aagaacggcaacgagaagaagaccgttgtcgttgtcggcaccgtcaccgacgacaaccg	(see FIG. 1d)
	cctggttgagtgccccaaggcccaggtcgctgcccttcgcttcaccgccaccgcccgtg
	cccgcatcgttgccgccggcggccaggccatcacccttgaccagctggctcttgagaag
	cccactggtgccaacaccctcc (199 bp)

MG04484.4	(SEQ ID NO: 529)	IV
(nt 1-400)	atgggtatcgatcttaagaagcaccacgtgcgcagcacgcaccgcaaggcccccaagag
	cgacaatgtctacctcaagctcttggtgaagctctaccgcttcctggcccgcaggaccg
	actccagcttcaacaaggttgtccttcgccgcctctttatgtcgcgcaccaaccgccct
	cccgtctccctgtcgcgcatcgcaggcaacctgaagaacggcaacgagaagaagaccgt
	tgtcgttgtcggcaccgtcaccgacgacaaccgcctggttgagtgccccaaggcccagg
	tcgctgcccttcgcttcaccgccaccgcccgtgcccgcatcgttgccgccggcggccag
	gccatcacccttgaccagctggctcttgagaagcccactggtgcca (400 bp)

MG02946.4	(SEQ ID NO: 106)	PE
(nt 58-333)	actgcggccgcggcacagctcgacggcctcaggcctcccgagggcgtcatcattccgcc
	acccggtgagatccgcgaggtgattgaaaagacggccgggtacgtggcgcgtggtggac
	tgggcatcgagcagcgcctacgcgagaaccacagcgggaacccaaagttcagcttcgtc
	acgagccagagcgacgcgtacaatccgtactacgagtggcgcaaggccgagtacaaggc
	cgggcgtggcacggcgctggcggctggtcgggcggatgcg (276 bp)

MG02946.4	(SEQ ID NO: 531)	IV
(nt 1-400)	atggcgaccaacggcgatgctgctcccccggcgccggcggcagagacggcagctgcgac
	tgcggccgcggcacagctcgacggcctcaggcctcccgagggcgtcatcattccgccac
	ccggtgagatccgcgaggtgattgaaaagacggccgggtacgtggcgcgtggtggactg
	ggcatcgagcagcgcctacgcgagaaccacagcgggaacccaaagttcagcttcgtcac
	gagccagagcgacgcgtacaatccgtactacgagtggcgcaaggccgagtacaaggccg
	ggcgtggcacggcgctggcggctggtcgggcggatgcggccgcgttggcggccgccaag
	ctggcagagcagaacaaggagccccagggcccgcccaagccgccgg (400 bp)

MG07472.4	(SEQ ID NO: 107)	PE
(nt 603-886)	ccaatcatctggtttcgagggcggcatcaacgaggacaacccttatgacaagccgcttt
	ttgctgttcaggacgccatcagtagtatctaccggccccgtgcgaacaacgatgatgag
	gacgaggcggctggcgatgcggagatggccaagatacaaaaggcgagcagatatggtga
	ggtgctaggccgaggtacattctccggtgccggggatgtggaagcccgagaaggtccag
	tgcagttcgaaaaggatgctgcaggcgcggaccctttcaatgtggaca (284 bp)

MG07472.2	(SEQ ID NO: 532)	*
(nt 551-940)	tcgccaagcccacgcagtcgtcagaaggcatgtacgattctcgacttttcaaccaatca
	tctggtttcgagggcggcatcaacgaggacaacccttatgacaagccgctttttgctgt
	tcaggacgccatcagtagtatctaccggccccgtgcgaacaacgatgatgaggacgagg
	cggctggcgatgcggagatggccaagatacaaaaggcgagcagatatggtgaggtgcta
	ggccgaggtacattctccggtgccggggatgtggaagcccgagaaggtccagtgcagtt
	cgaaaaggatgctgcaggcgcggaccctttcaatgtggacaagttcctgtccgaggtcg
	aacagaccgcgtcatcaaagaggggttatggtctgcaagactcgag (400 bp)

MG06292.4	(SEQ ID NO: 108)	PE
(nt 1684-	tatgctgaagcagaggcctccttcaagcgcttaagaacattggcaccgaatcggctgga
1978)	ggacatggaggtctattccacagtactttggcacctgaagaaggaaacagaggcatcgt
	ttctggcacacgagcttgttgacatagcgtggcattcaccgcacgcctggtgcgccttg
	ggcaatgcatggtcgctggcatcagatcgcgaacaagcgctgcggtgcttcaagcgtgc
	tacgcagctcgacgcaaaatttgcatatgcctacacacttcaagggcatgagcactttg
	(295 bp)

MG06292.4	(SEQ ID NO: 533)	IV
(nt 1601-	ctttcagcacgataccgcgagctcatgtcgacaccccgtgggttttagctcacatcgga
2000)	cgcgctcagtatgaacagaccaagtatgctgaagcagaggcctccttcaagcgcttaag
	aacattggcaccgaatcggctggaggacatggaggtctattccacagtactttggcacc
	tgaagaaggaaacagaggcatcgtttctggcacacgagcttgttgacatagcgtggcat
	tcaccgcacgcctggtgcgccttgggcaatgcatggtcgctggcatcagatcgcgaaca
	agcgctgcggtgcttcaagcgtgctacgcagctcgacgcaaaatttgcatatgcctaca
	cacttcaagggcatgagcactttgttagtgaagaatacgataaggc (400 bp)

MG03668.4	(SEQ ID NO: 109)	PE
(nt 1325-	cctgctccgaggctattgaccccagccagcacctcgagcctctgatccgctcgttgttt
1614)	gctggcctgctaaacactcccaagatggccgcctcgtgctgttgggctctgatgaacct
	ggcggaacgttttgccggtgagcccggagcgccccagaatgccatcactgcctacttca
	acgacagtgtgcgcagcctcctggacgtcaccgccaagaacgattgcgactcggctgtc
	aggacggccgcgtatgaggttctcaacgctttcatcgtcaatgcggcaaacgatt
	(290 bp)

MG03668.4	(SEQ ID NO: 534)	IV
(1301-1700)	acgctcttggtcgcatcaccgaggcctgctccgaggctattgaccccagccagcacctc
	gagcctctgatccgctcgttgtttgctggcctgctaaacactcccaagatggccgcctc
	gtgctgttgggctctgatgaacctggcggaacgttttgccggtgagcccggagcgcccc
	agaatgccatcactgcctacttcaacgacagtgtgcgcagcctcctggacgtcaccgcc
	aagaacgattgcgactcggctgtcaggacggccgcgtatgaggttctcaacgctttcat
	cgtcaatgcggcaaacgatagtctgcaggctgttgccactctttcggatgttatcatca
	agcgcctagaggagacgataccactacagacccaggttgtcagcgt (400 bp)

MG10192.4	(SEQ ID NO: 110)	PE
(nt 2825-	gtgacagggtcccatcttcaaacgtcggagccgctccctcggagcgttctgagcgtccc
3103)	tcggagcgccctgctcctgcccgaactggtacccccatggagcgcactgattcaaacga
	tcgtgctggtggtccgccccggcttaaccttgctcgtgctgacggtgccaagcccagct
	ggcgtgaacgtgagcaggccaaggctagcggtggccctgagcgggatcttcctcccagc
	agggcggcttctggaaggggcccaccgatgcacaggaccgatt (279 bp)

MG10192.4	(SEQ ID NO: 535)	IV
(nt 2751-	tgtcagggcctcggagtctgctggcggcccgccttcgcctcaacttggctggtgcaggt
3150)	ggcaagcccagctggcgtgacagggtcccatcttcaaacgtcggagccgctccctcgga
	gcgttctgagcgtccctcggagcgccctgctcctgcccgaactggtacccccatggagc
	gcactgattcaaacgatcgtgctggtggtccgccccggcttaaccttgctcgtgctgac
	ggtgccaagcccagctggcgtgaacgtgagcaggccaaggctagcggtggccctgagcg
	ggatcttcctcccagcagggcggcttctggaaggggcccaccgatgcacaggaccgatt
	cgggccgtggagagaacggcagggacgagtcgccggcgccccccaga (400 bp)

MG00604.4	(SEQ ID NO: 111)	IV
(nt 160-759)	gcctccggcaacaagcatgttccccgtgctgtcctcgtcgatctcgagcccggcaccat
	ggacgccgtccgtgccggtccttttggccagctcttccgccccgacaacttcgtcttcg
	gtcagtctggtgctggaaacaactgggccaagggtcactacactgagggtgccgagctt
	gtcgaccaggtccttgacgtcgtccgtcgtgaggctgagggctgtgactgcctccaggg
	tttccagatcacccactccctgggtggtggtaccggtgccggtatgggtactctgctga
	tctccaagatccgcgaggagttccccgaccgtatgatggccaccttctcggtcgttccc
	tcgcccaaggtttccgacaccgtcgttgagccctacaacgctaccctctcggtccacca
	gctggtcgagaactctgacgagaccttctgcattgacaacgaggctctgtacgacatct
	gcatgcgcaccctgaagctgtcgaacccctcatacggtgacctgaactacctggtttcg
	gccgtcatgtctggcgtcaccacctgcttgcgtttccccggccagctcaactctgatct
	ccgcaagctt (600 bp)

MG00604.4	(SEQ ID NO: 112)	IV
(nt 361-950)	cgtcgtgaggctgagggctgtgactgcctccagggtttccagatcacccactccctggg
	tggtggtaccggtgccggtatgggtactctgctgatctccaagatccgcgaggagttcc
	ccgaccgtatgatggccaccttctcggtcgttccctcgcccaaggtttccgacaccgtc
	gttgagccctacaacgctaccctctcggtccaccagctggtcgagaactctgacgagac
	cttctgcattgacaacgaggctctgtacgacatctgcatgcgcaccctgaagctgtcga
	acccctcatacggtgacctgaactacctggtttcggccgtcatgtctggcgtcaccacc
	tgcttgcgtttccccggccagctcaactctgatctcaccgttcccgagttgacccagca
	gatgttcgaccccaagaacatgatggctgcttctgacttcaggaatggtcgttacctga
	cctgctctgccatctt (590 bp)

MG00604.4	(SEQ ID NO: 113)	IV
(nt 952-	cgtggaaaggtttccatgaaggaggtcgaggaccagatgcgcaacgtccagaacaagaa
1344)	ctcgtcgtacttcgtcgagtggatccccaacaacatccagaccgctctctgctctatcc
	cgccccgcggcctcaagatgtcgtcgactttcatcggaaactcgaccgccatccaggag
	ctgttcaagcgtgtcggtgagcagttcactgccatgttcaggcgcaaggctttcttgca
	ttggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatga
	acgatcttgtttccgagtaccagcagtaccaggatgctggtgttgacgaggaggaagag
	gagtacgaggaggaggcccctcttgagggcgaggagtag (393 bp)

MG00604.4	(SEQ ID NO: 114)	IV: 10-50%
(nt 160-359)	gcctccggcaacaagcatgttccccgtgctgtcctcgtcgatctcgagcccggcaccat
	ggacgccgtccgtgccggtccttttggccagctcttccgccccgacaacttcgtcttcg
	gtcagtctggtgctggaaacaactgggccaagggtcactacactgagggtgccgagctt
	gtcgaccaggtccttgacgtcgt (200 bp)

MG00604.4	(SEQ ID NO: 115)	IV: 20-50%
(nt 760-950)	gccgtcaacatggttcccttccctcgtctgcacttcttcatggttggcttcgctccttt
	gaccagccgtggtgcccactctttccgcgctgtcaccgttcccgagttgacccagcaga
	tgttcgaccccaagaacatgatggctgcttctgacttcaggaatggtcgttacctgacc
	tgctctgccatctt (191 bp)

MG00604.4	(SEQ ID NO: 116 and 536)	IV: 50-90%
(nt 1151-	agttcactgccatgttcaggcgcaaggctttcttgcattggtacactggtgagggtatg	see FIG. 1a;
1344)	gacgagatggagttcactgaggccgagtccaacatgaacgatcttgtttccgagtacca	PE
	gcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggaggaggcccctc
	ttgagggcgaggagtag (194 bp)

MG00604.4	(SEQ ID NO: 117)	IV
(3X nt 1151-	agttcactgccatgttcaggcgcaaggctttcttgcattggtacactggtgagggtatg
1344)	gacgagatggagttcactgaggccgagtccaacatgaacgatcttgtttccgagtacca
	gcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggaggaggcccctc
	ttgagggcgaggagtagagttcactgccatgttcaggcgcaaggctttcttgcattggt
	acactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgat
	cttgtttccgagtaccagcagtaccaggatgctggtgttgacgaggaggaagaggagta
	cgaggaggaggcccctcttgagggcgaggagtagagttcactgccatgttcaggcgcaa
	ggctttcttgcattggtacactggtgagggtatggacgagatggagttcactgaggccg
	agtccaacatgaacgatcttgtttccgagtaccagcagtaccaggatgctggtgttgac
	gaggaggaagaggagtacgaggaggaggcccctcttgaggcgaggagtag (582 bp)

MG00604.4	(SEQ ID NO: 537)	IV
(nt 951-	ccgtggaaaggtttccatgaaggaggtcgaggaccagatgcgcaacgtccagaacaaga
1344)	actcgtcgtacttcgtcgagtggatccccaacaacatccagaccgctctctgctctatc
	ccgccccgcggcctcaagatgtcgtcgactttcatcggaaactcgaccgccatccagga
	gctgttcaagcgtgtcggtgagcagttcactgccatgttcaggcgcaaggctttcttgc
	attggtacactggtgagggtatggacgagatggagttcactgaggccgagtccaacatg
	aacgatcttgtttccgagtaccagcagtaccaggatgctggtgttgacgaggaggaaga
	ggagtacgaggaggaggcccctcttgagggcgaggagtag (394 bp)

MG00604.4	(SEQ ID NO: 538)	IV
(nt 745-	gctgtcaccgttcccgagttgacccagcagatgttcgaccccaagaacatgatggctgc
1344)	ttctgacttoaggaatggtcgttacctgacctgctctgccatcttccgtggaaaggttt
	ccatgaaggaggtcgaggaccagatgcgcaacgtccagaacaagaactcgtcgtacttc
	gtcgagtggatccccaacaacatccagaccgctctctgctctatcccgccccgcggcct
	caagatgtcgtcgactttcatcggaaactcgaccgccatccaggagctgttcaagcgtg
	tcggtgagcagttcactgccatgttcaggcgcaaggctttcttgcattggtacactggt
	gagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatcttgtttc
	cgagtaccagcagtaccaggatgctggtgttgacgaggaggaagaggagtacgaggagg
	aggcccctcggtgagcagttcactgccatgttcaggcgcaaggctttcttgcattggta
	cactggtgagggtatggacgagatggagttcactgaggccgagtccaacatgaacgatc
	ttgtttccgagtaccagcagtaccaggatgctggtgttgacgaggaggaagaggagtac
	gaggaggaggcccctcttgagggcgaggagtag (600 bp)

MG05169.4	(SEQ ID NO: 118)	IV
(nt 31-	ctcgggaacaaggcagccgcctcagatggcatgccaaatatggtgatgccatatgaac
330)	ctcaggaccccgctgtcatcgcagagatggtccgcgtgctcgatgagcatacaaaggg
	tggcgccaagggtcgtttccgtatcaagaagaccaagtttgccgttactggttctcct
	tccaaggtcactgtagattcctggaagctacaggactgggactacaagaaacctgggc
	tgcctacatatgcccgtggcctcttcaccacgcgcctcccaaataatgtaccagaaat
	tgccgtcaga (300 bp)

MG04056.4	(SEQ ID NO: 119)	IV
(nt 2377-	gcaggtgcatcgacggcgcgcgaatcccaggacgttgatcctctaattcacaacccgt
2676)	tcctgaagccgcccgacaaggcagcagtagaaagtaccctggttgcgttcgagcccgc
	gactttcaaattgactttacaaaatccgtatgaaatcgaggttgaaattgaaagcgtg
	cggttagacacggaaggggtcgagtttgaatcctctgtcgacagcgccctcattggcc
	catatcgcacgcagatacttcgtctagtcggcataccgaaggctgcggggtctctaaa
	gattaccggc (300 bp)

MG08911.4	(SEQ ID NO: 120) IV
(nt 2448-	ggtgatggaatggatgttcaacggccaggttgtatttatgccatatttgaaacgatac
2747)	accaacagtccagcgatatcggaagcgtttcgcccgattcgcccttgatattgggcat
	tatacgagctgttgaggtggtgtcgaaagttttggagcttcaggacacatatgttgat
	cttgtgcggcaagtcataaaacagcacacaggacaacgccatagacatgtgcctcacg
	cttcctacgcctccttcgaagaagggtttgctcatcacctcgaggtcgtcgcagatct
	cggccgatattg (300 bp)

MG06314.4	(SEQ ID NO: 121)	IV
(nt 551-850)	ggcggcacgcgctcgaggcaaaacgtatccaggcctcactcaccaagggcgacacaca
	gctttggcttggtactgcagacttggctttgcacatggtagatggcgcctatcaggat
	tcacccgaggaaatcgacaagactttgaaagtagctatggactgctacaggagcgtat
	tacagatcgacaagacaaaccccgttgctcgcctgggcaaagccgacatcctcgcgga
	cttgggacagtccagcaaggccgttgctgcttatctcgactacctcaaacagaaacct
	tataatcttc (300 bp)

MG08863.4	(SEQ ID NO: 122)	IV
(nt 1419-	cgtggccgacgctgaatttgaaggggagatgagtgtcaaccccacagacttggttaca
1718)	cggccgggtgaaacgcgaacattcaagcccatcatgctggcgcatgggagtcaagcag
	gtacacgtgatgccttcctcaggttggtttccggtattggctcgccatctcagcaggc
	agatctggctaccgacatgatcagtctggcaagggatttgtcaggaatagatcaggtc
	acagccttatggctagcgtttgagttgctgaaagtagcactagccaagaacgagcagg
	acgagcagtt (300 bp)

MG07222.4	(SEQ ID NO: 123)	IV
(nt 161-	ccgctcacacactgcggcccttcaacccgcaaaagcccgccggagcagccatcctccc
460)	gatcctcttaccgccagcaacactccgcccattggccttccggacctttaccaaaaaa
	cacagcctgaccctcacatcgtctgctctacaagagctggcgtcctttgtcggccgac
	actgcggctcaggatggcgtgaggaagggctcgccgagcgtgtgctagaggaggtggc
	aaaatcgtggaaggctggcaatggcggggtgatagtcgatggggccagccccatcctg
	aaggacatcc (300 bp)

MG01760.4	(SEQ ID NO: 124)	IV
(nt 1241-	gtggcggtggcgatgccttaccaagcctggcctcctttgacgttggagcgaatggcca
1540)	ggagatctttgcggattactgcagtgatactatcgacattcttttgctctcgctggat
	ggcaaagcacggatgatgaatggaaagaagccggttgtcggtgtcttcattgcaaaca
	gtattgccatcaccgagcggtcgattcgcgagtctgatctggcgcctttgatggagac
	acgcctgggaatattggaaacataccgaaagaaggcgaagctttattacacagagcct
	tgcaaggatg (300 bp)

MG07116.4	(SEQ ID NO: 125)	IV
(nt 959-	tgatgttgtggaggaagagcaaaagcgatgccgcgagtgacgcgcgcaagcagctgca
1258)	aatgtccaaatccgtaccgcccagcggtcctctcctgaggagaaacacagagccactg
	cagttgatgtcaggccctaatcctcaagaccagaacggttccatatcgccatcgcctc
	tcgcagcgaagcaggagtatgttgtgcaaacagaaagacggtcaagcgtctccaccta
	tctgctatgccgtgtcctcctcgaggtcatcagccaaagcagtttgagtctgatcact
	cctgaaatgg (300 bp)

MG03872.4	(SEQ ID NO: 126)	IV
(nt 1125-	gagcgaagagacctcggaagcggcaaggtttgtcagtggcacgatctacgactcgata
1424)	ggatcacgcctgctcgaaacactcgtaagccattgcccaggcaagatattcaaaggcc
	tgtggagtcacatcattgggcctcggtttgagacactcctccgtaacgagacggcaac
	atatgtagcaatgaaggcattggtcaggctgagtcgagaggacttggcggacgccgtg
	cttaagacgataccgaaagtggaaatgctagtgtcgaaaggccgcttcaatatcttga
	cgctgctgtt (300 bp)

MG04185.4	(SEQ ID NO: 127)	IV
(nt 2637-	actggtgactataggtgacccttcggaaatggtcgtagttctgacacaggccctgcag
2936)	ggtgtcgagatggacgcaaggggttgtcggtcgctcaacatgggcttgcacgttctga
	gggaagttgtcgatgcgcgccggattttcatccgaccgaatctgaacttgccgccctg
	agcgggttggccgcgcgctgccttgaaagccatgagtctggcgtgcggatggatgcag
	tccaattgtgtgttgccctgcatgcccgtgtgggcgatacacgcttctgggacaacat
	caagggtgt (300 bp)

Seastar#2	(SEQ ID NO: 128)	PE
AFP (nt 162-	cgatatcttgtcaaacgcgttcgcgtacggaaacagagcattgaccaaatacccagac
453)	gatatagcagactatttcaagcagtcgtttcccgagggatattcctgggaaagaagca
	tgacttttgaagacaaaggcattgtcaccgtggaaactgacgtcagcatggaagacga
	ctgctttatctataaaattcgttttgatggggtgaactttcctcccaatggtccagta
	atgcagaagaaaactttgaggtgggaaccatccactgagattatgtacgttcgtgatg
	gc (292 bp)

Seastar#2	(SEQ ID NO: 129)	IV: Taken as
AFP (nt 161-	acgatatcttgtcaaacgcgttcgcgtacggaaacagagcattgaccaaatacccaga	100%, negative
360)	cgatatagcagactatttcaagcagtcgtttcccgagggatattcctgggaaagaagc	control
	atgacttttgaagacaaaggcattgtcaccgtggaaactgacgtcagcatggaagacg
	actgctttatctataaaattcgtttt (200 bp)

GST	(SEQ ID NO: 130)	IV: Taken as
(nt 1-200)	atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttc	100%, negative
	ttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtga	control
	taaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattat
	attgatggtgatgttaaattaacaca (200 bp)

GFP	(SEQ ID NO: 131)	IV: Taken as
	ttatggtgttcaatgcttttcaagatacccagatcatatgaaacggcatgacttttca	100%, negative
	agagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgg	control
	gaactacaagacacgtaagtttaaacagttcggtactaactaaccatacatatttaaa
	ttttcaggtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaa
	aaggtattgattttaaagaagatggaaacattcttggacacaaattg (279 bp)

TABLE 6


Target gene M.	primer		sense (S)/	Restriction
grisea identifier	name	primer sequence	antisense (AS)	enzyme

MG00170.4	oBG109	(SEQ ID NO: 132) GAATTCATGGACACCCTGGTAGCC	S	ECoRI
	oBG110	(SEQ ID NO: 133) CTGCAGCGTTGGTAAACTCGCCCT	S	PstI
	oBG107	(SEQ ID NO: 134) CCCGGGATGGACACCCTGGTAGCC	AS	XmaI
	oBG108	(SEQ ID NO: 135) AGATCTCGTTGGTAAACTCGCCCT	AS	BgIII

MG00884.4	oBG33	(SEQ ID NO: 136) GAATTCCGACCTGCTCGTCTCACATA	S	ECoRI
	oBG34	(SEQ ID NO: 137) CTGCAGAGGAAGTAAGGGTGCAGTGC	S	PstI
	oBG35	(SEQ ID NO: 138) CCCGGGCGACCTGCTCGTCTCACATA	AS	XmaI
	oBG36	(SEQ ID NO: 139) CCATGGAGGAAGTAAGGGTGCAGTGC	AS	NCoI

MG07031.4	oBG37	(SEQ ID NO: 140) GAATTCGGAGTACATCATGGCAAACG	S	ECoRI
	oBG38	(SEQ ID NO: 141) CTGCAGATCGGGATGGTGATGTTTG	S	PstI
	oBG39	(SEQ ID NO: 142) CCCGGGGGAGTACATCATGGCAAACG	AS	XmaI
	oBG40	(SEQ ID NO: 143) CCATGGATCGGGATGGTGATGTTTG	AS	NcoI

MG04484.4	oBG087	(SEQ ID NO: 144) GAATTCATGGGTATCGATCTTAAGAAGCACCACG	S	ECoRI
	oBG088	(SEQ ID NO: 145) CTGCAGTTAGACCTTGAAACCACGCGATCGTCT	S	PstI
	oBG089	(SEQ ID NO: 146) CCCGGGATGGGTATCGATCTTAAGAAGCACCACG	AS	XmaI
	oBG090	(SEQ ID NO: 147) CCATGGTTAGACCTTGAAACCACGCGATCGTCT	AS	NCoI

MG02946.4	oBG49	(SEQ ID NO: 148) GAATTCACTGCGGCCGCGGCACAG	S	ECoRI
	oBG50	(SEQ ID NO: 149) CTGCAGCGCATCCGCCCGACCAGC	S	PstI
	oBG51	(SEQ ID NO: 150) CCCGGGACTGCGGCCGCGGCACAG	AS	XmaI
	oBG52	(SEQ ID NO: 151) CCATGGCGCATCCGCCCGACCAGC	AS	NCoI

MG07472.4	oBG53	(SEQ ID NO: 152) GAATTCCCAATCATCTGGTTTCGAGG	S	ECoRI
	oBG54	(SEQ ID NO: 153) ACGCGTTGTCCACATTGAAAGGGTCC	S	MluI
	oBG55	(SEQ ID NO: 154) CCCGGGCCAATCATCTGGTTTCGAGG	AS	XmaI
	oBG56	(SEQ ID NO: 155) CCATGGTGTCCACATTGAAAGGGTCC	AS	NCoI

MG06292.4	oBG57	(SEQ ID NO: 156) GAATTCTATGCTGAAGCAGAGGCCTC	S	ECoRI
	oBG58	(SEQ ID NO: 157) CTGCAGCAAAGTGCTCATGCCCTTG	S	PstI
	oBG59	(SEQ ID NO: 158) CCCGGGTATGCTGAAGCAGAGGCCTC	AS	XmaI
	oBG60	(SEQ ID NO: 159) CCATGGCAAAGTGCTCATGCCCTTG	AS	NCoI

MG03668.4	oBG61	(SEQ ID NO: 160) GAATTCCCTGCTCCGAGGCTATTG	S	ECoRI
	oBG62	(SEQ ID NO: 161) CTGCAGATCGTTTGCCGCATTGAC	S	PstI
	oBG63	(SEQ ID NO: 162) CCCGGGCCTGCTCCGAGGCTATTG	AS	XmaI
	oBG64	(SEQ ID NO: 163) CCATGGATCGTTTGCCGCATTGAC	AS	NCoI

MG10192.4	oBG65	(SEQ ID NO: 164) GAATTCGTGACAGGGTCCCATCTTCA	S	ECoRI
	oBG66	(SEQ ID NO: 165) CTGCAGAATCGGTCCTGTGCATCG	S	PstI
	oBG67	(SEQ ID NO: 166) CCCGGGGTGACAGGGTCCCATCTTCA	AS	XmaI
	oBG68	(SEQ ID NO: 167) AGATCTAATCGGTCCTGTC3CATCG	AS	BgIII

MG00604.4	oBG093	(SEQ ID NO: 168) GAATTCGCCTCCGGCAACAAGCATGTTC	S	ECoRI
(11.1)	oBG094	(SEQ ID NO: 169) CTGCAGAAGCTTGCGGAGATCAGAGTTGAGCTGG	S	PstI
	oBG091	(SEQ ID NO: 170) CCCGGGGCCTCCGGCAACAAGCATGTTC	AS	XmaI
	oBG092	(SEQ ID NO: 171) AGATCTAAGCTTGCGGAGATCAGAGTTGAGCTGGA	S	BgIII

MG00604.4	oBG097	(SEQ ID NO: 172) GAATTCCCGTCGTGAGGCTGAGGGCTGTG	S	ECoRI
(11.2)	oBG098	(SEQ ID NO: 173) CTGCAGAAGATGGCAGAGCAGGTCAGGTAACGA	S	PstI
	oBG095	(SEQ ID NO: 174) CCCGGGCCGTCGTGAGGCTGAGGGCTGTG	AS	XmaI
	oBG096	(SEQ ID NO: 175) CCATGGAAGATGGCAGAGCAGGTCAGGTAACGA	AS	NCoI

MG00604.4	oBG101	(SEQ ID NO: 176) GAATTCCCGTGGAAAGGTTTCCATGAAGGAGG	S	ECoRI
(11.3)	oBG102	(SEQ ID NO: 177) CTGCAGCTACTCCTCGCCCTCAAGAGGG	S	PStI
	oBG099	(SEQ ID NO: 178) CCCGGGCCGTGGAAAGGTTTCCATGAAGGAGG	AS	XmaI
	oBG100	(SEQ ID NO: 179) CCATGGCTACTCCTCGCCCTCAAGAGGG	AS	NCoI

MG00604.4	oBG105	(SEQ ID NO: 180) GAATTCAGTTCACTGCCATGTTCAG	S	ECoRI
(11.4)	oBG106	(SEQ ID NO: 181) CTGCAGCTACTCCTCGCCCTCAAG	S	PStI
	oBG103	(SEQ ID NO: 182) CCCGGGAGTTCACTGCCATGTTCAG	AS	XmaI
	oBG104	(SEQ ID NO: 183) CCATGGCTACTCCTCGCCCTCAAG	AS	NCoI

	TABLE 7


	Sporulation

	1 μM	2 μM

MG02946.4	+	−
MG03668.4	+	−
MG00604.4	+/−	−
Seastar	+++	+++
GST	+++	+++

TABLE 8


	SEQ
Target ID	ID No	Sequence*	Example Gi-number and species

MG00170.4	562	GCAGACTGCCAGTACTGGCTCGCCTGGCT	14663286 (Fusarium sporotrichioldes)
	563	TACAAGGGCATGGGCCTCAGCATGG	14663286 (Fusarium sporotrichioldes)

MG07031.4	564	CTTTTTTTTTTTTTTTTGCTG	58096826 (Phytophthora infestans)
	565	GAGACTTTTTTTTTTTTTTTT	60673784 (Alternaria brassicicola)
	566	GCTCTGAATCGTCGTCCGAGC	67522041 (Aspergillus nidulans FGSC A4)
	567	TACTACTGCCTCTCCATCCTCGACAA	48689269 (Gibberella zeae)
	568	TGAAGAGGAGATTACCGATTG	58264247 (Cryptococcus neoformans var.)
	569	TTTTTTTTTTTTTTTTGCTGCGG	58102240 (Phytophthora infestans)

MG04484.4	570	ACCGACTCCAGCTTCAACAAGGTTGTCCT	46347545 (Paracoccidioldes brasiliensis)
	571	AGAGCGACAATGTCTACCTCAAGCTCTTG	70711195 (Gibberelia moniliformis)
		GTGAAGCTCTACCGCTTCCTGGCCCGC
	572	CAACCGCCCTCCCGTCTCCCTGTC	70711195 (Gibberella moniliformis)
	573	CGGCACCGTCACCGACGACAAC	70816217 (Aspergilius niger)
	574	GCCCCCAAGAGCGACAATGTCTACCTCAA	22509454 (Gibberella zeae)
		GCTCTTGGTGAAGCTCTACCGCTTCCTGGC
	575	TTCACCGCCACCGCCCGTGCCCGCATC	22509454 (Gibberella zeae)

MGG09222.5	576	ACCAAGACCCTCGAGAACACCCTGAAGGCCAC	29427674 (Verticillium dahliae)
	577	AGCGTCATCCCCGTCCGTCGTGGTTACTGGGG	22503034 (Gibberella zeae)
		TACCAACCTTGGT
	578	CCCAACCTGTGGAAGGAGACCAAGCTCATC	29427674 (Verticillium dahliae)
	579	CCCGCTGTCAAGCGTCTCCTCCAGCTTGC	70982515 (Aspergillus fumigatus)
	580	GGTATCAAGACCTCCAAGGAGGTTGCCAC	31371647 (Gibberella zeae)
	581	TTCCTTACCCCCAACCTGTGGAAGGAGAC	21906277 (Colletotrichum trifolli)
		CAAGCTCATCAGGAG

MG00604.4	582	CAGAACAAGAACTCGTCGTACTTCGTCGA	68417116 (Phytophthora parasitica)
		GTGGATCCCCAACAACATC
	583	CAGTTCACTGCCATGTTCAGGCGCAAGGC	66909865 (Phaeosphaeria nodorum)
		TTTCTTGCATTGGTACACTGGTGAGGGTAT
		GGACGAGATGGAGTTCACTGAGGC
	584	CCATCCAGGAGCTGTTCAAGCGTGTCGGTGA	70825342 (Aspergillus niger)
	585	CCCAACAACATCCAGACCGCTCTCTGCTC	67518028 (Aspergillus nidulans FGSC A4)
	586	CCGTTCCCGAGTTGACCCAGCAGATGTTCGA	67518028 (Aspergillus nidulans FGSC A4)
		CCCCAAGAACATGATGGCTGC
	587	GAGATGGAGTTCACTGAGGCCGAGTCCAACA	46136028 (Gibberella zeae PH-1)
		TGAACGATCTTGT
	588	GAGGAGGAAGAGGAGTACGAGGAGGAG	70713442 (Gibberella moniliformis)
	589	GAGGGTATGGACGAGATGGAGTTCACTGAGGCCGAG	70995399 (Aspergillus fumigatus)
	590	GAGTACCAGCAGTAGCAGGATGC	68417116 (Phytophthora parasitica)
	591	GAGTTGACCCAGCAGATGTTCGACCCCAAGA	70995399 (Aspergillus fumigatus)
		ACATGATGGCTGCTTC
	592	GCCGTCAACATGGTTCCCTTCCCTCGTCTGCACT	70995399 (Aspergilius fumigatus)
		TCTTCATGGT
	593	GGTCGTTACCTGACCTGCTCTGCCATCTTCCGTGG	46136028 (GibberelIa zeae PH-1)
	594	GGTGAGCAGTTCACTGCCATGTTC	70713442 (Gibberella moniliformis)
	595	GTCAACATGGTTCCCTTCCCTCGTCTGCACTTCT	49079639 (Ustilago maydis)
		TCATGGTTGG
	596	TACCAGCAGTACCAGGATGCTGGT	70713442 (Gibberella moniliformis)
	597	TACTTCGTCGAGTGGATCCCCAACAACATCCAGAC	70825342 (Aspergillus niger)
		CGC
	598	TCCATGAAGGAGGTCGAGGACCAGATGCGCAACG	66909865 (Phaeosphaeria nodorum)
		TCCAGAACAAGAACTCGTC
	599	TCCGAGTACCAGCAGTACCAGGA	67518028 (Aspergilius nidulans FGSC A4)

MGG02952.5	600	AACAAGCGTGAGGTCTGGCGTGTCCAGCTCAC	29426916 (Verticillium dahliae)
	601	AAGTCCATCCACCACGCCCGTGTCCTCAT	21907821 (Colletotrichum trifolli)
	602	CAAGCTTGGTCTCGCCAAGTCC	33513606 (Cryptococcus neoformans var.)
	603	CAGAAGCACATCGACTTCGCCCTTACCTCGCCATT	47031693 (Mycosphaerella graminicola)
	604	CAGACCTGCGTCTACAAGCTTGG	2844032 (Emericella nidulans)
	605	CGCATGAAGCTCGATTACGTT	5826473 (Botryotinia fuckeliana)
	606	CGCCAGCGCCACATCCGCGTCGGCAAGCAGAT	21907821 (ColletotriChum trifolii)
		CGTCAACGT
	607	CGTCGTGCTGCCCGTCAGCTTCTTACCCTCGAC	70734819 (Gibberella moniliformis)
		GAGAAGGACCCCAAGCG
	608	CTCATTCGCCAGCGCCACATCCGCGTCGGCAAGCAGATCGT	29426916 (Verticillium dahliae)
	609	CTCGCCAAGTCCATCCACCACGCCCGTGT	29426916 (Verticillium dahliae)
	610	GACGAGAAGGACCCCAAGCGCCT	21907821 (Colletotrichum trifolii)
	611	GACTCCCAGAAGCACATCGACTTCGCCCT	70823211 (Aspergillus niger)
	612	GAGAAGGACCCCAAGCGCCTGTTCGAGGGTAACGCC	34332427 (Ustilago maydis)
	613	GCCAAGTCCATCCACCACGCCCGTGTCCT	2844032 (Emericella nidulans)
	614	GGCGGTGGCCGTCCCGGCCGTGTCCGCAGAAAGAAGG	70694169 (Gibberella moniliformis)
	615	GTCGGCAAGCAGATCGTCAACGTCCCCTC	70823211 (Aspergillus niger)
	616	TCCAAGATTCGTCGTGCTGCCCGTCAGCT	21907821 (Colletotrichum trifolii)
	617	TCGTCCGTCTCGACTCCCAGAAGCACATCGACTTCGC	70694169 (Gibbereila moniliformis)

MGG04095.5	618	AACATGGGTCGTGTTGGTGTTATCAC	70678182 (Gibberella moniliformis)
	619	AACTTCCGTCTCGTCTACGACACCAAGGG	46343134 (Paracoccidioides brasiliensis)
	620	ACCCACCGTGAGCGCCACGATGGTGG	70823112 (Aspergilius niger)
	621	AGAACCATCCGCTACCCCGACCCT	48901692 (Aspergillus flavus)
	622	ATCCCATTCTTGGTTACGCATGA	47032032 (Mycosphaerella graminicola)
	623	ATCGAGAAGACTGGCGAGAACTTCCGTCTC	49094939 (Aspergillus nidulans FGSC A4)
	624	ATGGAACGTGATTGCATGATTGC	34331251 (Ustilago maydis)
	625	CAACATGGGTCGTGTTGGTGT	46128672 (Gibberella zeae PH-1)
	626	CACTGGCAAGATCACCGACTTCATCAAGTTCGACAC	46128672 (Gibberella zeae PH-1)
	627	CCCCGCCGGCTTCATGGACGT	45244151 (Phytophthora nicotianae)
	628	CGAGGAGGCCGAGTACAAGCT	46128672 (Gibberella zeae PH-1)
	629	CGCCACGATGGTGGCTTCAACATTGT	24455690 (Paracoccidioides brasiliensis)
	630	CGCTTCACCGTCCACCGCATCCAGGC	70823112 (Aspergillus niger)
	631	GACTTCATCAAGTTCGACACTGG	70678182 (Gibberella moniliformis)
	632	GAGAAATGGAACGTGATTGCA	58118641 (Phytophthora infestans)
	633	GAGAAGACTGGCGAGAACTTCCGTCTCGTCTACGA	46128672 (Gibberella zeae PH-1)
	634	GAGGAGGCCGAGTACAAGCTC	70823112 (Aspergillus niger)
	635	GAGGAGGCCGAGTACAAGCTCGGCAAGGTCAAGCGTGT	29426157 (Verticililum dahliae)
	636	GCAAGGTCAAGCGTGTTCAGCTCGGC	70823112 (Aspergilius niger)
	637	GCAAGGTCCGCACCGACTCGAC	60673542 (Alternaria brassicicola)
	638	GGCGGGATCCCATTCTTGGTTACGCA	70823112 (Aspergillus niger)
	639	TACCCCGACCCTCTGATCAAGGTCAACGACACTGTCAAGAT	46128672 (Gibberella zeae PH-1)
	640	TTCGGAGAAATGGAACGTGATTG	70710347 (Gibberella moniliformis)

MGG05193.5	641	AGCAAACTTTATCTCCGTCAAGGG	41543841 (Cryptococcus neoformans var.)
	642	AGCAACATTCGTGATATCTTCGACAA	25129989 (Paracoccidioides brasiliensis)
	643	ATGTTGGCCAAGGCCGTTGCCAACGAGTGTGC	70683086 (Gibberella moniliformis)
	644	CTCAGCATGTGGTTTGGAGAGTC	47029850 (Mycosphaerella graminicola)
	645	GAGAAGTTCCTCAAGTTCGGTCT	47029546 (Mycosphaerella graminicola)
	646	GAGATGGACGGTATGACTTCCAAGAAGAACGT	46122304 (Gibberella zeae PH-1)
	647	GTCGACCACCCCGAGAAGTTCC	70823844 (Aspergillus niger)

MGG09952.5	648	AACAAGGTCGACGTCAAGGAGCG	24447584 (Paracoccidioides brasiliensis)
	649	AACTACAACTTCGAGAAGCCTTTCCTGTGGCTCG	70767614 (Gibberelia moniliformis)
	650	AAGTCGAACTACAACTTCGAGAAGCC	48899092 (Aspergillus flavus)
	651	ACCTACAAGAACGTCCCCAACTGGCACCGTGA	70699127 (Gibbereila moniliformis)
	652	ATCACCTACAAGAACGTCCCCAACTGGCACCGTGA	48884377 (Aspergillus flavus)
	653	ATCACCTACAAGAACGTCCCCAACTGGCACCGTGA	10181839 (Aspergillus niger)
	654	ATTGTTCTCTGCGGTAACAAGGTCGA	22506497 (Gibberella zeae)
	655	GCCAGTGCGGTATCATCATGTT	48899092 (Aspergillus flavus)
	656	TACAAGAACGTCCCCAACTGGCACCGTGAC	70992714 (Aspergillus fumigatus)

MGG06910.5	657	ATCTGGAAGACAGAATGGATGATG	70756653 (Gibberella moniliformis)
	658	CACTTCCCGTCCATCAACACTTC	46343356 (Paracoccidioides brasiliensis)
	659	CAGCGCAAGCACTTCCCGTCC	9834135 (Phytophthora sojae)
	660	CAGGTCTTCTGGGGTCTCGACAAGAA	70756653 (Gibberella moniliformis)
	661	TTCTGGGGTCTCGACAAGAAA	71004203 (Ustilago maydis)

MGG04829.5	662	AACAAGGATATCCGTAAGTTCTTGGATGGTAT	50286950 (Candida glabrata CBS 138)
	663	AGCGCCGCCGATATCCAGCAGATCTGC	34332067 (Ustilago maydis)
	664	CAAGTACAAGATGCGTTACGTCTACGCCCAT	29426217 (Verticillium dahliae)
		TTCCCCATCAACGTCAAC
	665	CCCAGAAGGATGAGCTCATCCTG	70700474 (Gibberella moniliformis)
	666	GAGATCCGAAACTTCATCGGCGAGAAGCTCGT	70702890 (Gibberella moniliformis)
	667	TACGTCTACGCCCATTTCCCCATCAACGTCAACGT	70702890 (Gibberella monhliformis)
	668	TGATCATTGGTGTCACCAAGGGCT	70690968 (Gibberella moniliformis)

MG5858.4	669	TTTGCCATGCCACAAAGCGGTAC	70712617 (Gibberella monhliformis)

Contig2.1053.	670	TGCAAGAGCTTGGACGAGCGGGAGATT	70996393 (Aspergillus fumigatus)
g5	671	AGGAAGGCCAAGCAGCTGGAGATGCA	70823132 (Aspergillus niger)
	672	CTCGACGAGCTCTACATGGTTCTCATCAC	62926964 (Fusarium oxysporum f. sp.)
	673	CAGGATATCGACTCCCTGCACCT	46122116 (Gibberella zeae PH-1)
	674	TACGAGGCTGAGAAGAACAAGTC	46122116 (Gibberella zeae PH-1)

Contig2.1499.	675	ATCGCCAACACATCGAACATGCC	70985197 (Aspergillus fumigatus)
g3	676	ATCATGAAGCGTACCACCCTT	41565642 (Cryptococcus neoformans var.)
	677	ATCTGGAAGACAGAGTGGATGATG	70675344 (Gibberelia moniliformis)
	678	CAGGTCTTCTGGGGTCTCGACAAGAA	70712111 (Gibberella moniliformis)
	679	GAGCGAGGCAATGAGATGGCTGAAGT	70712110 (Gibberelia moniliformis)
	680	TGGAAGACAGAGTGGATGATG	70742555 (Gibberella moniliformis)
	681	ATGCCTGCTGATCAGGGTTTCCC	46125252 (Gibberella zeae PH-1)
	682	GACCAGGTCGTGCAGCTTGTCGG	46125252 (Gibberella zeae PH-1)
	683	CACTTCCCGTCCATCAACACTTC	46343356 (Paracoccidioides brasiliensis)
	684	CAGCGCAAGCACTTCCCGTCC	38064229 (Phytophthora sojae)
	685	CAGCGCAAGCACTTCCCGTCC	38098094 (Phytophthora sojae)
	686	GCCGAGTACTTCCGTGACCAGGGCATGAACGT	38098094 (Phytophthora sojae)
	687	TTCTGGGGTCTCGACAAGAAA	49067893 (Ustilago maydis)
	688	AGCATTGTCGGTGCCGTCAGCCCGCCCGGTGGTGATTTCTC	29426867 (Verticillium dahliae)

Contig2.561.	689	ATCGAGAAGACTGGCGAGAACTTCCG	60673542 (Alternaria brassicicola)
g35	690	CCTCGCCCGGTCCTCACAAGC	60673542 (Alternaria brassicicola)
	691	GTCAAGGTTGACGGCAAGGTCCGCACCGACTCGAC	60673542 (Alternaria brassicicola)
	692	AACATGGGTCGTGTTGGTGTT	48901692 (Aspergillus flavus)
	693	AGAACCATCCGCTACCCCGACCCT	48901692 (Aspergilius flavus)
	694	CCATCGCTGAGGAGCGTGACCGC	48901692 (Aspergillus flavus)
	695	GCAAGGTCAAGCGTGTTCAGCTCGGC	48901692 (Aspergillus flavus)
	696	GGCGGGATCCCATTCTTGGTTACGCA	48901692 (Aspergillus flavus)
	697	AACATGGGTCGTGTTGGTGTT	49094939 (Aspergillus nidulans FGSC A4)
	698	ACTGGCAAGATCACCGACTTC	49094939 (Aspergilius nidulans FGSC A4)
	699	ATCGAGAAGACTGGCGAGAACTTCCGTCTC	49094939 (Aspergillus nidulans FGSC A4)
	700	CCCAAGGGCAAGGGTGTCAAGCTC	49094939 (Aspergillus nidulans FGSC A4)
	701	GGCGGGATCCCATTCTTGGTTACGCA	49094939 (Aspergilius nidulans FGSC A4)
	702	AAGCCCTGGATCTCCCTGCCCAAGGGCAAGGGTGTCAAGCTC	70814642 (Aspergilius niger)
	703	ACCCACCGTGAGCGCCACGATGGTGG	70814642 (Aspergilius niger)
	704	CCATCGCTGAGGAGCGTGACCGC	70814642 (Aspergillus niger)
	705	CGCTTCACCGTCCACCGCATCCAGGC	70823112 (Aspergillus niger)
	706	GACGGCAAGGTCCGCACCGAC	70823112 (Aspergillus niger)
	707	GAGGAGGCCGAGTACAAGCTC	70823112 (Aspergillus niger)
	708	GCAAGGTCAAGCGTGTTCAGCTCGGC	10181989 (Aspergillus niger)
	709	GCAAGGTCAAGCGTGTTCAGCTCGGC	70823112 (Aspergillus niger)
	710	TAAGAAGCACCAGAAGCGCCTT	70823112 (Aspergillus niger)
	711	AAGATCACCGACTTCATCAAGTTCGACACTGG	5831532 (Botryotinia fuckeliana)
	712	CGTGAGAGCAACGTTTTCGTCATCGG	5831532 (Botryotinia fuckeliana)
	713	ATCAAGGTCAACGACACTGTCAAG	41561006 (Cryptococcus neoformans var.)
	714	CTGCCCAAGGGCAAGGGTGTCAAGCTCACCATCGCTGAGGAG	14664568 (Fusarium sporotrichioides)
		CGTGACCGC
	715	AACATGGGTCGTGTTGGTGTTATCAC	70741411 (Gibberelia moniliformis)
	716	CCATCGCTGAGGAGCGTGACCGC	70741411 (Gibberella moniliformis)
	717	CGTGAGAGCAACGTTTTCGTCATCGGC	70662858 (Gibberella moniliformis)
	718	CGTGAGAGCAACGTTTTCGTCATCGGC	70685009 (Gibberella moniliformis)
	719	CGTGAGAGCAACGTTTTCGTCATCGGC	70741411 (Gibberella moniliformis)
	720	CTGCCCAAGGGCAAGGGTGTCAAGCTCACCATCG	70685009 (Gibberelia moniliformis)
	721	CTGCCCAAGGGCAAGGGTGTCAAGCTCACCATCGCTGAGGAG	70745388 (Gibberella moniliformis)
		CGTG
	722	GGGTGTCAAGCTCACCATCGCTGA	70748989 (Gibberella moniliformis)
	723	TGCCCAAGGGCAAGGGTGTCAAGCTC	70741411 (Gibberella moniliformis)
	724	ACAACATGGGTCGTGTTGGTGT	22500577 (Gibberella zeae)
	725	CAAGGCCATCCTGATGCAGCG	47836630 (Gibberella zeae)
	726	CGAGGAGGCCGAGTACAAGCT	22501231 (Gibberella zeae)
	727	ATCCCATTCTTGGTTACGCATGA	47032032 (Mycosphaerella graminicola)
	728	CGAGGAGGCCGAGTACAAGCT	47032032 (Mycosphaerella graminicola)
	729	AACTTCCGTCTCGTCTACGACACCAAGGG	46345858 (Paracoccidioides brasiliensis)
	730	CGCCACGATGGTGGCTTCAACATTGT	24455928 (Paracoccidioides brasiliensis)
	731	CCCCGCCGGCTTCATGGACGT	40545328 (Phytophthora nicotianae)
	732	CCCCGCCGGCTTCATGGACGT	68416641 (Phytophthora parasitica)
	733	AAGATCACCGACTTCATCAAGTTCGACAC	40545852 (Sclerotinia sclerotiorum)
	734	CGTGAGAGCAACGTTTTCGTCATCGG	40545852 (Sclerotinia sclerotiorum)
	735	CTCATCGTCTTCATCCGCAACCG	40545852 (Sclerotinia sclerotiorum)
	736	ATCAAGGTCAACGACACTGTC	37404210 (Ustilago maydis)
	737	GACCCTCTGATCAAGGTCAACGACAC	29426157 (Verticillium dahliae)
	738	GAGGAGGCCGAGTACAAGCTCGGCAAGGTCAAGCGTGT	29426157 (Verticillium dahliae)
	739	GGCAAGATCACCGACTTCATCAAGTTCGACAC	29426157 (Verticillium dahliae)
	740	GTCAAGGTTGACGGCAAGGTCCGCACCGAC	29426157 (Verticillium dahliae)

Contig2.875.	741	CCTCGCGGTGCCATGATCTTCT	71000869 (Aspergillus fumigatus)
g14	742	AACAAGTATTCCGAGGGTTACCCCGG	49091467 (Aspergillus nidulans FGSC A4)
	743	CGCGGTGCCATGATCTTCTTC	70682910 (Gibberella moniliformis)
	744	TCCGTCTTCCCCGGTCACCAGGG	70682910 (Gibberella moniliformis)
	745	TCCGTCTTCCCCGGTCACCAGGGCGG	70685149 (Gibberella moniliformis)
	746	TACGACCTTGAGAACCCCATCAAC	46123824 (Gibberella zeae PH-1)

TABLE 9


	SEQ ID
Target ID	No	Sequence*	Example Gi-number and species

MGG09222.5	747	CGTCATCCCCGTCCGTCGTGGTTACTGGGGTA	17972115 (Nippostrongylus brasiliensis)

MG00604.4	748	AACTCGACCGCCATCCAGGAGCTGTTCAAGCG	28706250 (Heterodera glycines)
	749	ATCCAGGAGCTGTTCAAGCGT	159160 (Haemonchus contortus)
	750	CAACATGAACGATCTTGTTTC	72003699 (Caenorhabditis elegans)
	751	CAGAACAAGAACTCGTCGTACTTCGT	23260428 (Ascaris lumbricoides)
	752	CATTGGTACACTGGTGAGGGTATGGACGAGATGGAGTTC	6081554 (Pristionchus pacificus)
	753	CCCAAGAACATGATGGCTGCTT	71184729 (Caenorhabditis remanel)
	754	GAGGCCGAGTCCAACATGAACGATCTTGT	28706250 (Heterodera glycines)
	755	GAGGGTATGGACGAGATGGAGTTCAC	28706250 (Heterodera glycines)
	756	GATCTCCGCAAGCTTGCCGTCAACATGGTTCCC	47663797 (Caenorhabditis elegans)
	757	GCCGAGTCCAACATGAACGAT	27428224 (Heterodera glycines)
	758	GCCGTCAACATGGTTCCCTTCCC	6067860 (Pristionchus pacificus)
	759	GCTTTCTTGCATTGGTACACTGG	53809993 (Xiphinema index)
	760	GGTATGGACGAGATGGAGTTCACTGA	53748584 (Cyathostomum catinatum)
	761	GTCGAGTGGATCCCCAACAAC	28185390 (Ancylostoma ceylanicum)
	762	TCCGAGTACCAGCAGTACCAGGA	6081554 (Pristionchus pacificus)
	763	TCCGAGTACCAGCAGTACCAGGATGC	28706250 (Heterodera glycines)
	764	TGAGCAGTTCACTGCCATGTTC	3046904 (Onchocerca volvulus)
	765	TTCTTGCATTGGTACACTGGTGAGGGTATGGA	68303302 (Ancylostoma caninum)
	766	GAGGCTGAGGGCTGTGACTGCCT	21393141 (Nippostrongylus brasiliensis)
	767	GACTGCCTCCAGGGTTTCCAG	27926935 (Ascaris suum)
	768	GACGAGACCTTCTGCATTGACAACGAGGC	6067860 (Pristionchus pacificus)
	769	GGAAACAACTGGGCCAAGGGTCACTACAC	71983644 (Caenorhabditis elegans)
	770	TTCTGCATTGACAACGAGGCTCT	71983644 (Caenorhabditis elegans)
	771	TGGTGCTGGAAACAACTGGGCCA	754758 (Caenorhabditis briggsae)

MG5858.4	772	ATTCAGTCCGTCAAAGGAAAGGTTGT	28559556 (Ancylostoma ceylanicum)
	773	TTGATGTTTTTGCCATGCCACAA	30165647 (Meloidogyne chitwoodi)

MGG02952.5	774	CTCAAGAACAAGCGTGAGGTCTGGCGTGTC	71988689 (Caenorhabditis elegans)
	775	CTCGCCAAGTCCATCCACCACGCCCGT	18090904 (Parastrongyloides trichosuri)
	776	GACTCCCAGAAGCACATCGACTTC	71988689 (Caenorhabditis elegans)
	777	GTCTCAAGAACAAGCGTGAGGT	20133278 (Ostertagia ostertagi)

MGG04095.5	778	ATCAAGGTCAACGACACTGTC	15666921 (Ancylostoma ceylanicum)
	779	GCCGAGGAGGCCGAGTACAAGCTC	32320500 (Heterodera glycines)
	780	TTCGGAGAAATGGAACGTGAT	6067677 (Pristionchus pacificus)

MGG04829.5	781	AACAAGGATATCCGTAAGTTCTTGGA	45213836 (Wuchereria bancrofti)
	782	TACGCCCATTTCCCCATCAACGTCA	15498642 (Haemonchus contortus)

MGG06910.5	783	CAGGTCTTCTGGGGTCTCGACAAGAAACT	17994314 (Ascaris suum)

MGG09952.5	784	ACCTACAAGAACGTCCGCAACTGGCAC	18826906 (Pristionchus pacificus)
	785	GTGAAGGCCAAGACCATCACCTTCCAC	47598490 (Caenorhabditis elegans)
	786	TACAACTTCGAGAAGCCTTTCCT	32183546 (Meloidogyne chitwoodi)
	787	TTCGAGAAGCCTTTCCTGTGG	60292774 (Angiostrongylus cantonensis)

Contig2.1499.	788	CAGGTCTTCTGGGGTCTCGACAAGAAACT	17994314 (Ascaris suum)
g3	789	GCCGAGTACTTCCGTGACCAGG	31246711 (Caenorhabditis elegans)

MG04484.4	790	TCCAAGGGCCGCAAGTTCGAG	29053280 (Heterodera glycines)

TABLE 10


	SEQ ID
Target ID	No	Sequence*	Example Gi-number and species

MG00604.4	791	AAGGCTTTCTTGCATTGGTACACTGG	77881122 (Aedes aegypti)
	792	ACCGCCATCCAGGAGCTGTTCAAGCG	4163124 (Bombyx mon)
	793	ATGGACGAGATGGAGTTCACTGAGGCCGAG	67888072 (Drosophila pseudoobscura)
	794	ATGTTCAGGCGCAAGGCTTTCTTGCATTGGTACAC	4163124 (Bombyx mon)
	795	GAGGGTATGGACGAGATGGAGTTCAC	77676133 (Aedes aegypti)
	796	GCCATCCAGGAGCTGTTCAAGCG	33552986 (Anopheles gambiae)
	797	GCTTTCTTGCATTGGTACACTGGTGA	47515641 (Acyrthosiphon pisum)
	798	GTCGAGTGGATCCCCAACAAC	61951569 (Tnibolium castaneum)
	799	GTTTCCGAGTACCAGCAGTACCAGGA	75465871 (Tnibolium castaneum)
	800	TCCGAGTACCAGCAGTACCAGGA	4419807 (Drosophila melanogaster)
	801	TGGTACACTGGTGAGGGTATGGA	40935930 (Bombyx mon)
	802	GCTGGAAACAACTGGGCCAAGGGTCA	47518574 (Acyrthosiphon pisum)
	803	CTCGAGCCCGGCACCATGGACGCCGT	6910199 (Bombyx mon)
	804	CTGCATTGACAACGAGGCTCT	29535524 (Bombyx mon)
	805	CTGCATTGACAACGAGGCTCTGTACGACATCTGC	40918504 (Bombyx mon)
	806	GACAACTTCGTCTTCGGTCAGTC	6910199 (Bombyx mon)
	807	GGTGCTGGAAACAACTGGGCCAA	34787982 (Callosobruchus maculatus)
	808	CATGTCTGGCGTCACCACCTGC	25957081 (Carabus granulatus)
	809	AACAACTGGGCCAAGGGTCACTACACTGA	158744 (Drosophila hydel)
	810	GACGAGACCTTCGCATTGACAACGAGGCTCT	67885077 (Drosophila pseudoobscura)
	811	GGAAACAACTGGGCCAAGGGTCACTACAC	67875471 (Drosophila pseudoobscura)
	812	ATCTCCAAGATCCGCGAGGAGT	78052349 (Heliconius erato)
	813	CTTCCGCCCCGACAACTTCGT	78050966 (Heliconius erato)
	814	TCTTCCGCCCCGACAACTTCGT	78052742 (Heliconius erato)
	815	CCGACAACTTCGTCTTCGGTCAG	55911790 (Locusta migratoria)
	816	GAGGGTGCCGAGCTTGTCGAC	55911790 (Locusta migratoria)
	817	GACAACTTCGTCTTCGGTCAGTC	58371315 (Lonomia obliqua)
	818	CTCGAGCCCGGCACCATGGAC	10763921 (Manduca sexta)
	819	CCCCGACAACTTCGTCTTCGG	75714350 (Tribolium castaneum)
	820	TTCCGCCCCGACAACTTCGTCTTCGG	75711493 (Tribolium castaneum)

MG00961.4	821	GGTCTCATGTTAGCCAACCACACCA	55889923 (Locusta migratoria)

MG02946.4	822	CGGCAGCTGCGACTGCGGCCG	58395387 (Anopheies gambiae str. PEST)

MG04484.4	823	CTCTTGGTGAAGCTCTACCGCTT	56154926 (Rhynchosciara americana)
	824	GTTGTCGGCACCGTCACCGACGA	4245179 (Drosophila melanogaster)

MG5858.4	825	CATCCCGGTTTCGGCTGCTGG	17945075 (Drosophila melanogaster)
	826	GTGGATATCAACACTCAGCAG	77871215 (Aedes aegypti)

MGG02952.5	827	AACAAGCGTGAGGTCTGGCGTGTC	61675474 (Aedes aegypti)
	828	CGCATGAAGCTCGATTACGTTCT	3946433 (Drosophila melanogaster)
	829	CTCGACTCCCAGAAGCACATCGACTTC	49209929 (Drosophila melanogaster)
	830	GACGAGTCGCGCATGAAGCTCGATTACGT	56772258 (Drosophila virilis)
	831	TGGTCTCGCCAAGTCCATCCACCACGCCCG	49532931 (Plutella xylostella)

MGG04095.5	832	ACTTACCCCGCCGGCTTCATGGA	58371410 (Lonomia obliqua)

MGG04829.5	833	AACAAGGATATCCGTAAGTTCTTGGA	42766318 (Armigeres subalbatus)
	834	CCACCCTCCGTACCGTCCGCAC	77778750 (Aedes aegypti)
	835	GAACAAGGATATCCGTAAGTTC	15046304 (Drosophila melanogaster)
	836	GATATCCGTAAGTTCTTGGATGGT	60298646 (Diaphorina citri)
	837	TACGCCCATTTCCCCATCAAC	15046304 (Drosophila melanogaster)

MGG05193.5	838	AACCAGCTGCTGACGGAGATGGACGG	58388417 (Anopheles gambiae str. PEST)

MGG06910.5	839	CAGCGCAAGCACTTCCCGTCCATCAAC	22474258 (Helicoverpa armigera)
	840	TTCTGGGGTCTCGACAAGAAACT	55915724 (Locusta migratoria)

MGG09222.5	841	CGGCAAGTGCGGTTCCGTCAC	60313286 (Sphodromantis centralis)

MGG09952.5	842	AAGAACCTGCAGTATTACGACATCTC	25959135 (Meladema coriacea)
	843	CAGTATTACGACATCTCGGCCAAGTC	77843224 (Aedes aegypti)
	844	TCACCTACAAGAACGTCCCCAACTGGCAC	77843224 (Aedes aegypti)

Contig2.1053.	845	CAATTTCGCCATCGATATGGCC	13773184 (Drosophila melanogaster)
g5

Contig2.1499.	846	GCCGAGTACTTCCGTGACCAG	48719395 (Anopheles funestus)
g3	847	CAGCGCAAGCACTTCCCGTCCATCAAC	22474258 (Helicoverpa armigera)
	848	TTCTGGGGTCTCGACAAGAAACT	55915722 (LoCusta migratoria)
	849	AAGGAAGCAGAGGAAGAAGAAA	75717792 (Tribolium castaneum)

Contig2.561.	850	GACGGCAAGGTCCGCACCGAC	77724691 (Aedes aegypti)
g35	C
	851	CGAGGAGGCCGAGTACAAGCT	27616676 (Anopheles gambiae)
	852	ATCTCCCTGCCCAAGGGCAAGGGTGTCAAGCT	67882036 (Drosophila pseudoobscura)
	853	TCAAGGTTGACGGCAAGGTCCGCAC	48927129 (Hydropsyche sp.)
	854	ACTTACCCCGCCGGCTTCATGGA	58371411 (Lonomia obliqua)

MG00170.4	855	CGGTTGGGTCAAGCACGGATT	40946757 (Bombyx mon)
	856	CAGGGATGCCTACTCCGGTGGT	17862097 (Drosophila melanogaster)

MG04484.4	857	AAGGGCCGCAAGTTCGAGAAGGC	27569966 (Anopheles gambiae)
	858	GTTGTCGGCACCGTCACCGACGA	4245179 (Drosophila melanogaster)
	859	CTCTTGGTGAAGCTCTACCGCTT	56154926 (Rhynchosciara americana)

Claims

1. A method for controlling fungal growth 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.

2. A method for down-regulating expression of a target gene in a fungus, comprising contacting fungal cell(s) with a double-stranded RNA 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.

3. A method according to claim 1 or 2 wherein said fungal cell(s) is contacted with a composition comprising said double-stranded RNA.

4. A method according to claim 1 or 2, wherein said fungal cell(s) is contacted with double-stranded RNA which is expressed by a prokaryotic (such as a bacterial cell) or eukaryotic (such as a yeast cell) host cell or host organism.

5. A method according to claim 1 wherein said double-stranded RNA is expressed by said cell or organism infested with or susceptible to infestation by said fungus.

6. A method according to claim 5 wherein said cell is a plant cell or wherein said organism is a plant.

7. A method according to any of claims 1 or 2, wherein said 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.

8. A method according to any of claims 1 or 2, wherein said fungal target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and (ii) sequences comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof.

9. A method for producing a plant resistant against a plant pathogenic fungus, comprising:

(a) transforming a plant cell with a recombinant construct comprising at least one regulatory sequence operably linked to a sequence complementary to at least part of a nucleotide sequence of a target fungal gene selected from the group consisting of:

(i) a nucleotide sequence of at least 17 nucleotides in length encoding an RNA which is complementary to a fungal target gene comprising a sequence which is at least 75% identical to a sequence selected from the group of sequences represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1,41, 97,98, 526, 184, 185, 37, 59, 124, 9,45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, and

(ii) a 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; and

(c) growing the transformed plant under conditions suitable for the expression of the recombinant construct, said grown transformed plant resistant to fungi compared to an untransformed plant, hereby producing a fungus-resistant plant.

10. A method according to any of claims 1 or 9 wherein said fungal cell(s) is a cell(s) of a plant pathogenic fungus selected from the group consisting of 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. Colletotrichum 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 culmorum, Fusarium solani, Fusarium moniliforme or Fusarium roseum), Gaeumanomyces spp. (e.g. Gaeumanomyces graminis f.sp. tritici), Gibberella spp. (e.g. Gibberella zeae), Helminthosporium spp. (e.g. Helminthosporium turcicum, Helminthosporium carbonum, Helminthosporium mavdis or Helminthosporium sigmoideum), Leptosphaeria salvinii, Macrophomina spp. (e.g. Macrophomina phaseolina), Magnaportha spp. (e.g Magnaporthe oryzae), Mycosphaerella spp., 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. tritici, 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. (e.g. Pyricularia oryzae), Pythium spp. (e.g. Pythium ultimum), Rhincosporium secalis, Rhizoctonia spp. (e.g. Rhizoctonia solani, Rhizoctonia oryzae or Rhizoctonia cerealis), Rhizopus spp. (e.g. Rhizopus chinensid), Scerotium spp. (e.g. Scerotium rolfsi), Sclerotinia spp. (e.g. Sclerotinia sclerotiorum), Septoria spp. (e.g. Septoria lycopersici, Septoria glycines, Septoria nodorum or Septoria tritici), Thielaviopsis spp. (e.g. Thielaviopsis basicola), Tilletia spp., Trichoderma spp. (e.g. Trichoderma virde), Uncinula spp. (e.g. Uncinula necator), Ustilago maydis (e.g. corn smut), Venturia spp. (e.g. Venturia inaequalis or Venturia pirina) and Verticillium spp. (e.g. Verticillium dahliae or Verticillium albo-atrum).

11. (canceled)

12. A method according to any of claims 6 or 9 wherein said plant is chosen from the group consisting of rice, corn, soybean, cotton, potato, banana, tomato, wheat, sorghum, millet, beans, groundnuts, rapeseed, sunflower and sugarcane; preferably chosen from rice, corn, soybean, cotton, potato, banana and tomato.

13. A method according to any of claims 6 or 9 wherein said plant is rice and wherein said target gene is a gene from a fungus selected from the group consisting of Magnaporthe spp., Rhizoctonia spp., Acremoniella spp., Phytium spp., Curvularia spp., Trichoderma spp., Fusarium spp. and Rhizopus spp.

14. A method according to any of claims 6 or 9 wherein said plant is corn and wherein said target gene is a gene from a fungus selected from the group consisting of Colletotrichum spp., Gibberella spp., Fusarium spp., Diplodia spp. and Puccina spp.

15. A method according to any of claims 6 or 9 wherein said plant is soybean and wherein said target gene is a gene selected from the group of Phakopsora spp.

16. A method according to any of claims 6 or 9 wherein said plant is cotton and wherein said target gene is a gene from a fungus selected from the group consisting of Fusarium spp. and Verticillium spp.

17. A method according to any of claims 6 or 9 wherein said plant is potato and wherein said target gene is a gene from a fungus selected from the group consisting of Phytophthora spp., Rhizoctonia spp. and fungal species that cause wilt, rot or scurf.

18. A method according to any of claims 6 or 9 wherein said plant is banana and wherein said target gene is a gene from a fungus selected from the group consisting of Mycosphaerella spp., Cercospora spp. and Fusarium spp.

19. A method according to any of claims 6 or 9 wherein said plant is tomato and wherein said target gene is a gene from a fungus selected from the group consisting of Phytophthora spp. and fungal species that cause foliar disease, wilt or fruit rot.

20. A method according to any of claims 1 or 9, 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.

21. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell, which expresses or is capable of expressing at least one double-stranded RNA, wherein said 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 wherein said double-stranded RNA is taken up by a fungal cell upon plant-fungus interaction.

22. A transgenic plant, reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell according to claim 21, 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.

23. A transgenic plant, reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell according to claim 21, wherein said fungal target gene comprises a sequence which is selected from the group comprising:

(i) sequences which are at least 75%, preferably 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% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and

(ii) sequences comprising at least 17, preferably at least 18, 19, 20 or 21, more preferably at least 22, 23 or 24 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said fungal target gene is a fungal orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof.

24. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a rice plant or reproductive or propagation material for a rice plant or a cultured rice plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of Magnaporthe spp., Rhizoctonia spp., Acremoniella spp., Phytium spp., Curvularia spp., Trichoderma spp., Fusarium spp., and Rhizopus spp.

25. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a corn plant or reproductive or propagation material for a corn plant or a cultured corn plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of Colletotrichum spp., Gibberella spp., Fusarium spp., Diplodia spp. and Puccina spp.

26. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a soybean plant or reproductive or propagation material for a soybean plant or a cultured soybean plant cell, wherein the target gene is a gene from the group of Phakopsora spp.

27. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a cotton plant or reproductive or propagation material for a cotton plant or a cultured cotton plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of Fusarium spp. and Verticillium spp.

28. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a potato plant or reproductive or propagation material for a potato plant or a cultured potato plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of Phytophthora spp., Rhizoctonia spp. and fungal species that cause wilt, rot or scurf.

29. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a banana plant or reproductive or propagation material for a banana plant or a cultured banana plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of Mycosphaerella spp., Cercospora spp. and Fusarium spp.

30. A transgenic plant, or reproductive or propagation material for a transgenic plant or a cultured transgenic plant cell according to any of claims 21 or 23 which is a tomato plant or reproductive or propagation material for a tomato plant or a cultured tomato plant cell, wherein the target gene is a gene from a fungus selected from the group consisting of Phytophthora spp. and species that cause foliar disease, wilt or fruit rot.

31. 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 nucleotide sequence of a fungal target, 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.

32. An isolated double-stranded RNA according to claim 31 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 target gene comprises a sequence which is selected from the group comprising: (i) sequences which are at least 75% identical to a sequence represented by any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, and (ii) sequences comprising at least 17 contiguous nucleotides of any of SEQ ID Nos 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof, or wherein said target gene is an orthologue of a gene comprising at least 17 contiguous nucleotides of any of SEQ ID NOs 3, 42, 99, 100, 527, 39, 60, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 5, 43, 101, 102, 528, 1, 41, 97, 98, 526, 184, 185, 37, 59, 124, 9, 45, 106, 531, 188, 189, 13, 47, 109, 534, 33, 57, 126, 23, 52, 119, 35, 58, 127, 7, 44, 103, 104, 105, 529, 186, 187, 29, 55, 118, 17, 49, 108, 533, 25, 53, 121, 19, 50, 125, 31, 56, 123, 11, 46, 107, 532, 27, 54, 122, 21, 51, 120, 15, 48, 110, 535, 458, 486, 530, 460, 487, 539, 540, 462, 488, 541, 464, 489, 542, 466, 490, 543, 468, 491, 544, 470, 492, 545, 472, 493, 546, 474, 494, 547, 476, 496, 548, 478, 556, 549, 480, 497, 550, 482, 498, 551, 484, 499, 552 and 562 to 859, or the complement thereof

33. An isolated double-stranded RNA according to claim 32, 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, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or the RNA equivalent of a fragment of at least 17 basepairs in length thereof.

34. An isolated double-stranded RNA according to claim 33 wherein the nucleotide sequences comprises at least one of the sequences represented by any of SEQ ID NOs 192, 201, 202, 193, 190, 191, 196, 199, 260, 194, 195, 198 and 197, or a double-stranded fragment of at least 17 basepairs in length thereof.

35. (canceled)

36. (canceled)

37. An isolated double-stranded RNA construct according to claim 32 comprising at least two copies of said nucleotide sequence complementary to at least part of a nucleotide sequence of a fungal target.

38. An isolated double-stranded RNA construct according to claim 37 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, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or comprising at least two copies of the RNA equivalent of a fragment of at least 17 basepairs in length thereof.

39. An isolated double-stranded RNA construct according to claim 37 comprising at least two copies of the RNA equivalent of the nucleotide sequence as represented in SEQ ID NO 117.

40. An isolated double-stranded RNA or RNA construct comprising the RNA equivalents of at least two nucleotide sequences independently chosen from the sequences represented by any of SEQ ID NOs 99, 100, 527, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 101, 102, 528, 97, 98, 526, 124, 106, 531, 109, 534, 126, 119, 127, 103, 104, 105, 529, 118, 108, 533, 121, 125, 123, 107, 532, 122, 120, 110, 535, 530, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and 562 to 859, or fragments thereof of at least 17 basepairs in length.

41. An isolated double-stranded RNA or RNA construct according to claim 32, which comprises at least one additional dsRNA region, at least one strand thereof comprising a nucleotide sequence which is complementary to at least part of a nucleotide sequence of at least one other fungal target gene.

42. An isolated double-stranded RNA or RNA construct according to claim 32, further comprising at least one additional sequence and optionally a linker.

43. An isolated double-stranded RNA or RNA construct according to claim 42 wherein said 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) one or more additional sequences to catalyze processing of dsRNA regions.

44. (canceled)

45. An isolated nucleotide sequence encoding a double-stranded RNA according to claim 32.

46. (canceled)

47. An isolated nucleotide sequence consisting of a sequence represented by any of SEQ ID NOs 3, 99, 100, 527, 192, 39, 111, 112, 113, 114, 115, 116, 117, 536, 537, 538, 201, 202, 5, 101, 102, 528, 193, 1, 97, 98, 526, 184, 185, 190, 191, 37, 124, 9, 106, 531, 188, 189, 196, 13, 109, 534, 199, 200, 33, 126, 23, 119, 35, 127, 7, 103, 104, 105, 529, 186, 187, 194, 195, 29, 118, 17, 108, 533, 198, 25, 121, 19, 125, 31, 123, 11, 107, 532, 197, 27, 122, 21, 120, 15, 110, 535, 458, 530, 460, 539, 540, 462, 541, 464, 542, 466, 543, 468, 544, 470, 545, 472, 546, 474, 547, 476, 548, 478, 549, 480, 550, 482, 551, 484, 552 and 562 to 859, or the complement thereof, 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, or the complement thereof.

48. A recombinant DNA construct comprising the nucleotide sequence of claim 47 operably linked to at least one regulatory sequence.

49. A recombinant DNA construct according to claim 48, wherein said regulatory sequence is selected from the group comprising constitutive promoters 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., or tissue specific promoters such as any selected from the group comprising root specific promoters of genes encoding PsMTA Class III Chitinase, photosynthetic tissue-specific promoters such as promoters of cab1 and cab2, rbcS, gapA, gapB and ST-LS1 proteins, JAS promoters, chalcone synthase promoter and promoter of RJ39 from strawberry.

50. (canceled)

51. A composition comprising at least one double-stranded RNA, wherein said 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 further comprising at least one suitable carrier, excipient or diluent.

52. A composition comprising at least one double-stranded RNA according to claim 32 and further comprising at least one suitable carrier, excipient or diluent.

53. A composition comprising at least one nucleotide sequence according to claim 47; and further comprising at least one suitable carrier, excipient or diluent.

54. (canceled)

55. (canceled)

56. (canceled)

57. (canceled)

58. (canceled)

59. A cell comprising the nucleotide sequence of claim 47.

60. The cell of claim 59 wherein said cell is a prokaryotic cell or an eukaryotic cell.

61. The cell of claim 60 wherein said cell is a bacterial cell.

62. The cell of claim 60 wherein said cell is a plant cell.

63. A plant comprising at least one double-stranded RNA according to claim 32, at least one nucleotide sequence according to claim 47, or at least one plant cell of claim 62.

64. The plant of claim 61 wherein said plant is chosen from the group comprising rice, barley, rye, wheat, miller, lovegrass and crabgrass.

65. The plant of claim 63 wherein said plant is rice.

66. A seed comprising at least one nucleotide sequence according to claim 47.

67. The seed of claim 66 wherein said seed is of a plant chosen from the group comprising rice, barley, rye, wheat, miller, lovegrass and crabgrass.

68. The seed of claim 66 wherein said seed is a rice seed.

69. A method for treating and/or preventing fungal infestation on a substrate comprising applying an effective amount of a composition according to claim 52 to said substrate.

70. A method for treating and/or preventing fungal growth and/or fungal infestation of a plant or propagative or reproductive material of a plant comprising applying an effective amount of a composition according to claim 52 to a plant or to propagation or reproductive material of a plant.

71. 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, a composition according to claim 52, 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.

72. (canceled)

73. (canceled)

74. A kit comprising a double-stranded RNA according to claim 32, or a nucleotide sequence according to claim 47, or a cell according to claim 60, or a composition according to claim 52 for treating fungal infection of plants.

75. A method for increasing plant yield comprising introducing in a plant a nucleotide sequence according to claim 47 in an expressible format.

76. A method according to claim 75 wherein said plant is chosen from the group comprising rice, barley, rye, wheat, miller, lovegrass and crabgrass.

77. A method according to claim 75 wherein said plant is rice.