# US 2013/0160156 A1 · Method for down-regulating gene expression in fungi > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0160156 A1 - Document ID: US20130160156A1 - Status: Published application - Kind: A1 - Application number: 13/702,831 - Filed: 2011-06-08 - Published: 2013-06-20 - Earliest claimed: 2010-06-08 - Assignees: Devgen Private Limited - Inventors: Simone Dorfmueller; Phuay-Yee Goh; Pascale Feldmann - Web page: https://patentgrep.com/patent/US20130160156A1 ## CPC - C12N 15/8282: {for fungal resistance} - A01H 1/06: Processes for producing mutations, e.g. treatment with chemicals or with radiation (specific mutations prepared by genetic engineering on plant cell or plant tissues C12N15/00 {; process for producing transgenic plants C12N15/82}) - A01H 5/04: Stems - A01H 5/10: Seeds - A01N 57/16: containing heterocyclic radicals - C07K 14/375: from Basidiomycetes - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention concerns methods for controlling and/or preventing fungus infestation on a cell, organism, substrate or material via dsRNA mediated gene silencing. The methods of the invention are particularly used to alleviate pathogenic fungal infestation on plants, plant materials or seeds. Suitable fungal target genes and fragments thereof, expression cassettes, dsRNA molecules, host cells expressing the dsRNA, compositions and transgenic plants and plant cells are provided. ## Claims ### Claim 1 A method for preventing and/or controlling pathogenic Rhizoctonia spp. infestation, comprising exposing a pathogenic Rhizoctonia to at least one dsRNA comprising annealed complementary strands, whereby ingestion of said dsRNA by said Rhizoctonia inhibits the expression of a Rhizoctonia target gene, which target gene comprises a nucleotide sequence that is complementary to one of the strands of said dsRNA. ### Claim 2 (depends on 1) The method according to claim 1, wherein at least 20% Rhizoctonia mortality or at least 20% Rhizoctonia control is obtained by silencing of an essential target gene of a pathogenic Rhizoctonia spp. by application of at least one dsRNA comprising annealed complementary strands, one of which comprises at least 21, preferably at least 22, 23 or 24 contiguous nucleotides that are complementary to a Rhizoctonia target gene essential to said Rhizoctonia spp., wherein said Rhizoctonia target gene (i) is selected from the group of genes having a nucleotide sequence comprising any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (ii) is selected from the group of genes having a nucleotide sequence that, when the two sequences are optimally aligned and compared using the BLASTN alignment tool, is at least 75% identical to any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (iii) is selected from the group of genes having a nucleotide sequence encoding an amino acid sequence that, when the two sequences are optimally aligned and compared using the BLASTP alignment tool, is at least 70% identical to any of SEQ ID NOs 47, 49, 51, 2, 4, 6, 24, 80, 99, 101, 103, 137, 139, 141, 143, 145, 147, 180, 182, 208, 210, 212, 214 or 216, as compared to the control Rhizoctonia spp. applied with a dsRNA targeting a non-essential gene or a gene not naturally expressed in said pathogenic Rhizoctonia spp. ### Claim 3 (depends on 1) The method according to claim 1, comprising exposing the pathogenic Rhizoctonia spp. to an agent comprising at least one dsRNA that functions upon uptake by the Rhizoctonia to inhibit the expression of a target gene within said Rhizoctonia, wherein said dsRNA comprises annealed complementary strands, one of which comprises a ribonucleotide sequence that is complementary to the sequence of said target gene, wherein said ribonucleotide sequence is transcribed from: (i) a DNA sequence selected from the group of sequences comprising any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or a fragment thereof, or (ii) a DNA sequence selected from the group of sequences having, when the two sequences are optimally aligned and compared using the BLASTN alignment tool, at least 75% identity with any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (iii) a DNA sequence selected from the group of sequences encoding an amino acid sequence that, when the two sequences are optimally aligned and compared using the BLASTP alignment tool, is at least 70% identical to any of SEQ ID NOs 47, 49, 51, 2, 4, 6, 24, 80, 99, 101, 103, 137, 139, 141, 143, 145, 147, 180, 182, 208, 210, 212, 214 or 216. ### Claim 4 (depends on 1) The method according to claim 1, for preventing and/or controlling the infection of a plant, part of a plant, reproductive plant material, seed or tuber(s) by a pathogenic Rhizoctonia spp., comprising expressing in or applying to said plant, part of a plant, reproductive plant material, seed or tuber(s) at least one dsRNA comprising annealed complementary strands, one of which comprises at least 21, preferably at least 22, 23 or 24 contiguous nucleotides that are complementary to a Rhizoctonia target gene essential to said Rhizoctonia, wherein said Rhizoctonia target gene (i) is selected from the group of genes having a nucleotide sequence comprising any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (ii) is selected from the group of genes having a nucleotide sequence that, when the two sequences are optimally aligned and compared using the BLASTN alignment tool, is at least 75% identical to any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (iii) is selected from the group of genes having a nucleotide sequence encoding an amino acid sequence that, when the two sequences are optimally aligned and compared using the BLASTP alignment tool, is at least 70% identical to any of SEQ ID NOs 47, 49, 51, 2, 4, 6, 24, 80, 99, 101, 103, 137, 139, 141, 143, 145, 147, 180, 182, 208, 210, 212, 214 or 216. ### Claim 5 (depends on 1) The method according to claim 1, wherein the pathogenic Rhizoctonia spp. is a rice or a potato pathogenic Rhizoctonia spp. ### Claim 6 (depends on 1) The method according to claim 1, wherein the pathogenic Rhizoctonia spp. is a Rhizoctonia strain chosen from the group comprising Rhizoctonia solani spp., Rhizoctonia solani ZG3, anastomosis group AG11, Rhizoctonia solani ZGS, anastomosis group AG2-1 and the rice infecting Rhizoctonia solani, anastomosis group AG1-1A. ### Claim 7 (depends on 1) The method according to claim 1, for preventing and/or controlling the infection of a substrate or material by a Rhizoctonia spp., comprising applying to said substrate or material at least one dsRNA comprising annealed complementary strands, one of which comprises at least 21, preferably at least 22, 23 or 24 contiguous nucleotides that are complementary to a Rhizoctonia target gene essential to said Rhizoctonia, wherein said Rhizoctonia target gene (i) is selected from the group of genes having a nucleotide sequence comprising any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (ii) is selected from the group of genes having a nucleotide sequence that, when the two sequences are optimally aligned and compared using the BLASTN alignment tool, is at least 75% identical to any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (iii) is selected from the group of genes having a nucleotide sequence encoding an amino acid sequence that, when the two sequences are optimally aligned and compared using the BLASTP alignment tool, is at least 70% identical to any of SEQ ID NOs 47, 49, 51, 2, 4, 6, 24, 80, 99, 101, 103, 137, 139, 141, 143, 145, 147, 180, 182, 208, 210, 212, 214 or 216. ### Claim 8 (depends on 1) The method according to claim 1, wherein said dsRNA is expressed by at least one prokaryotic or eukaryotic host cell or host organism. ### Claim 9 (depends on 8) The method of claim 8, wherein the prokaryotic cell is a bacterial cell chosen from the group comprising Gram positive and Gram negative cells comprising Escherichia spp. (such as for instance E. coli), Bacillus spp. (such as for instance B. thuringiensis), Rhizobium spp., Lactobacillus spp., Lactococcus spp., Pseudomonas spp. and Agrobacterium spp. ### Claim 10 (depends on 9) The method of claim 9, wherein the bacterial cell is inactivated by heat or by chemical treatment. ### Claim 11 (depends on 8) The method of claim 8, wherein the eukaryotic cell or organism is a plant cell or a plant. ### Claim 12 A nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule which comprises at least 21, preferably at least 22, 23 or 24 contiguous nucleotides of a nucleotide sequence as represented by any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (ii) a nucleic acid molecule which comprises at least 21, preferably at least 22, 23 or 24 contiguous nucleotides of a nucleotide sequence that, when the two sequences are optimally aligned and compared using the BLASTN alignment tool, is at least 75% identical to any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof, or (iii) a nucleic acid molecule encoding an amino acid sequence that, when the two sequences are optimally aligned and compared using the BLASTP alignment tool, is at least 70% identical to any of SEQ ID NOs 47, 49, 51, 2, 4, 6, 24, 80, 99, 101, 103, 137, 139, 141, 143, 145, 147, 180, 182, 208, 210, 212, 214 or 216. ### Claim 13 (depends on 12) An expression cassette comprising at least one regulatory sequence that directs the expression of at least one nucleic acid molecule selected from: (i) a nucleic acid molecule of claim 12, or (ii) a nucleic acid molecule having a first region containing a nucleotide sequence of a nucleic acid molecule of claim 12 identical to at least 21 contiguous nucleotides of a selected target gene, wherein the target gene is pathogenic Rhizoctonia gene, and a second region which is complementary to the first region, said expression cassette being capable of expressing a dsRNA. ### Claim 14 (depends on 12) A dsRNA produced from the expression of a nucleic acid molecule of claim 12. ### Claim 15 (depends on 12) A host cell comprising at least one nucleic acid molecule of claim 12. ### Claim 16 (depends on 15) A host cell according to claim 15, wherein said cell is chosen from a prokaryotic cell, including bacterial cells, or an eukaryotic cell, including yeast cells, plant cells and animal cells. ### Claim 17 (depends on 14) A composition comprising at least one dsRNA of claim 14. ### Claim 18 (depends on 12) A transgenic plant or plant cell comprising at least one nucleic acid molecule of claim 12. ### Claim 19 (depends on 18) A transgenic plant or plant cell according to claim 18, wherein the plant or plant cell is chosen from rice, potato or turfgrass. ### Claim 20 (depends on 18) A transgenic plant or plant cell according to claim 18, (i) wherein the nucleic acid molecule(s) or the expression cassette(s) comprise(s) more than one nucleic acid molecule having a nucleotide sequence corresponding to more than one target gene, or (ii) wherein the at least one dsRNA is produced from the expression of a nucleic acid molecule or an expression cassette that comprises more than one nucleotide sequence corresponding to more than one target gene, wherein the more than one target gene can be a target gene from the same pathogenic Rhizoctonia strain or from different pathogenic Rhizoctonia strains. ### Claim 21 (depends on 12) A method of making a transgenic plant or plant cell which contains at least one dsRNA comprising annealed complementary strands, one of which comprises at least 21 contiguous nucleotides identical to a target gene in a pathogenic Rhizoctonia spp., said method comprising the steps of: a) providing a nucleic acid molecule of claim 12, wherein the nucleic acid molecule is able to form at least one dsRNA once expressed in the plant; wherein the at least one nucleic acid molecule expressed from said expression cassette is able to form at least one dsRNA once expressed in the plant; b) transforming a recipient plant or plant cell with said nucleic acid molecule or with said expression cassette; c) producing one or more offspring of said recipient plant or plant cells; and d) testing the offspring for expression of said dsRNA. ### Claim 22 (depends on 21) A transgenic plant or plant cell obtained by the method of claim 21. ### Claim 23 (depends on 18) A method of producing a plant that is resistant to Rhizoctonia spp. infection comprising: (a) crossing a transgenic plant of claim 18 with another plant, and (b) selecting Rhizoctonia-resistant progeny by analyzing for the presence of a nucleotide sequence that encodes at least one dsRNA comprising annealed complementary strands, one of which comprises at least 21 contiguous nucleotides identical to at least one sequence selected from the group consisting of any of SEQ ID NOs 46, 48, 50, 52, 1, 3, 5, 7, 21, 22, 23, 25, 77, 78, 79, 81, 98, 100, 102, 104, 136, 138, 140, 142, 144, 146, 148, 179, 181, 183, 207, 209, 211, 213, 215, 217 or 250, or the complement thereof. ### Claim 24 (depends on 23) The method of claim 23, wherein the plant is chosen from rice, potato or turfgrass. ### Claim 25 (depends on 21) Seed or tuber(s) produced from the plants obtained by the method of claim 21. ### Claim 26 (depends on 18) Hybrid seed or transgenic tuber(s) produced by crossing a first inbred plant with a second, distinct inbred plant wherein the first or second inbred plant is a transgenic plant of claim 18, and wherein the plant is chosen from rice, potato or turfgrass. ### Claim 27 (depends on 26) A method for producing hybrid seed or transgenic tuber(s) of claim 26, wherein crossing comprises the steps of: (a) planting the seeds or tuber(s) of first and second inbred plants; (b) cultivating the seeds or tuber(s) of said first and second inbred plants into plants that bear flowers; (c) preventing self pollination of at least one of the first or second inbred plant; (d) allowing cross-pollination to occur between the first and second inbred plants; and (e) harvesting seeds or tuber(s) on at least one of the first or second inbred plants, said seeds or tuber(s) resulting from said cross-pollination; wherein the plant is chosen from rice, potato or turfgrass. ### Claim 28 (depends on 26) A hybrid plant produced by growing the seed or tuber(s) of claim 26, wherein the plant is chosen from rice, potato or turfgrass.