# US 7,663,024 B2 · P15 hairpin constructs and use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,663,024 B2 - Document ID: US7663024B2 - Status: Granted - Kind: B2 - Application number: 11/418,384 - Filed: 2006-05-03 - Published: 2006-12-21 - Granted: 2010-02-16 - Earliest claimed: 2006-05-03 - Assignees: SESVanderHave NV - Inventors: Emmanuelle Lauber; Hubert Guilley; Ken Richards; Gerard Jonard; Elodie Klein; David Gilmer - Web page: https://patentgrep.com/patent/US7663024B2 ## CPC - C12N 15/8283: {for virus resistance} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention concerns a method of genetic modification of a TGB-3 wild type viral sequence for reducing or suppressing the possible deleterious effects of the agronomic properties of a transformed plant or plant cell by said TGB-3 viral sequence. The invention further relates to genetically modified TGB-3 viral sequences suitable to induce gene silencing. In particular hairpin constructs based on such sequences proved highly efficient to induce a PTGS mechanism and degradation of the whole of RNA2 thereby. When plants are transformed accordingly the spread of the virus in the plant is significantly reduced or blocked. ## Claims ### Claim 1 A genetically modified TGB-3 viral sequence comprising a sequence selected from the group consisting of (a) a nucleotide sequence comprising SEQ ID NO: 3 and an antisense sequence of SEQ ID NO: 3; and (b) a nucleotide sequence comprising a modified SEQ ID NO: 3 and an antisense sequence of said modified SEQ ID NO: 3, wherein the modification in SEQ ID NO: 3 consists of a modification that alters a start or a stop codon in SEQ ID NO: 3 so as to inhibit translation, wherein said genetically modified TGB-3 viral sequence when transcribed in a cell is capable of forming a double-stranded self-complementing RNA molecule. ### Claim 2 (depends on 1) The TGB-3 viral sequence of claim 1, wherein the sense and antisense sequences are comprised in one nucleic acid sequence. ### Claim 3 (depends on 2) The TGB-3 viral sequence of claim 2, further comprising an intron fragment interspersed between the sense and antisense sequences, wherein the TGB-3 viral sequence when transcribed in a cell is capable of forming a hairpin RNA molecule. ### Claim 4 (depends on 3) The TGB-3 viral sequence of claim 3, wherein the intron fragment is derived from a plant gene. ### Claim 5 (depends on 4) The TGB-3 viral sequence of claim 4, wherein the plant gene is a beet gene. ### Claim 6 (depends on 3) The TGB-3 viral sequence of claim 3, wherein the intron fragment is an intron fragment of highly transcribed genes. ### Claim 7 (depends on 6) The TGB-3 viral sequence of claim 6, wherein the highly transcribed genes are ribosomal RNA genes. ### Claim 8 (depends on 6) The TGB-3 viral sequence of claim 6, wherein the highly transcribed genes are highly transcribed sugar beet genes. ### Claim 9 (depends on 1) The TGB-3 viral sequence of claim 1 comprising SEQ ID NO: 9. ### Claim 10 (depends on 1) The TGB-3 viral sequence of claim 1 consisting of SEQ ID NO: 9. ### Claim 11 (depends on 1) A vector comprising the genetically modified TGB-3 viral sequence according to claim 1. ### Claim 12 (depends on 11) The vector of claim 11 operably linked to one or more regulatory sequence(s) active in a plant cell. ### Claim 13 (depends on 11) A double stranded self-complementary RNA molecule expressed by a vector of claim 11. ### Claim 14 (depends on 1) A transgenic plant or a transgenic plant cell resistant to a virus and comprising a nucleic acid construct having a genetically modified TGB-3 viral sequence according to claim 1 operably linked to one or more regulatory sequence(s) active in a plant or a plant cell. ### Claim 15 (depends on 14) The transgenic plant or transgenic plant cell according to claim 14, wherein the virus is the BNYVV virus. ### Claim 16 (depends on 14) The transgenic plant or transgenic plant cell according to claim 14, wherein said transgenic plant or transgenic plant cell comprises sugar beet. ### Claim 17 (depends on 14) The transgenic plant or transgenic plant cell according to claim 14, wherein the regulatory sequence comprises a promoter sequence or a terminator sequence that are active in a plant. ### Claim 18 (depends on 17) The transgenic plant of claim 17, wherein said promoter is active in the root tissue of plants. ### Claim 19 (depends on 14) The transgenic plant or transgenic plant cell according to claim 14, wherein said transgenic plant is sugar beet and said transgenic plant cell is a sugar beet cell. ### Claim 20 (depends on 14) The transgenic plant or transgenic plant cell according to claim 14, wherein the regulatory sequence(s) comprise a promoter sequence which is a constitutive or a foreign vegetal promoter sequence. ### Claim 21 (depends on 20) The transgenic plant or transgenic plant cell according to claim 20, wherein the promoter comprises the 35S Cauliflower Mosaic Virus promoter. ### Claim 22 (depends on 14) A transgenic plant tissue derived from the transgenic plant cell according to claim 14, wherein said tissue comprises said genetically modified TGB-3 viral sequence, and wherein said tissue is selected from the group consisting of fruit, stem, root, tuber, and seed. ### Claim 23 (depends on 14) A transgenic reproducible structure obtained from the transgenic plant cell according to claim 14, wherein said reproducible structure comprises said genetically modified TGB-3 viral sequence, and wherein said reproducible structure is selected from the group consisting of calluses, buds, and embryos. ### Claim 24 A genetically modified TGB-3 viral sequence comprising a sequence selected from the group consisting of: (a) a nucleotide sequence comprising a fragment of SEQ ID NO:3 and an antisense sequence of said fragment of SEQ ID NO:3; and (b) a nucleotide sequence comprising a modified SEQ ID NO: 3 fragment and an antisense sequence of said modified SEQ ID NO: 3 fragment; wherein said fragment of SEQ ID NO:3 is about 25 nucleotides in length with 100% sequence identity to the corresponding part of SEQ ID NO:3, wherein the modification in SEQ ID NO: 3 consists of a modification that alters a start or a stop codon in SEQ ID NO: 3 so as to inhibit translation, wherein said genetically modified TGB-3 viral sequence when transcribed in a cell is capable of forming a double-stranded self-complementing RNA molecule. ### Claim 25 (depends on 24) The TGB-3 viral sequence of claim 24, wherein a nucleic acid sequence comprises the sense and antisense sequences. ### Claim 26 (depends on 25) The TGB-3 viral sequence of claim 25, further comprising an intron fragment interspersed between the sense and antisense sequences, wherein the TGB-3 viral sequence when transcribed in a cell is capable of forming a hairpin RNA molecule. ### Claim 27 (depends on 26) The TGB-3 viral sequence of claim 26, wherein the intron fragment is derived from a plant gene. ### Claim 28 (depends on 27) The TGB-3 viral sequence of claim 27, wherein the plant gene is a beet gene. ### Claim 29 (depends on 26) The TGB-3 viral sequence of claim 26, wherein the intron fragment is an intron fragment of a highly transcribed gene. ### Claim 30 (depends on 29) The TGB-3 viral sequence of claim 29, wherein the highly transcribed gene comprises a ribosomal RNA gene. ### Claim 31 (depends on 29) The TGB-3 viral sequence of claim 29, wherein the highly transcribed gene comprises a highly transcribed sugar beet gene. ### Claim 32 (depends on 24) A vector comprising the genetically modified TGB-3 viral sequence according to claim 24. ### Claim 33 (depends on 32) The vector of claim 32, operably linked to one or more regulatory sequence(s) active in a plant cell. ### Claim 34 (depends on 32) A double stranded self-complementary RNA molecule expressed by a vector of claim 32. ### Claim 35 (depends on 24) A transgenic plant or a transgenic plant cell resistant to a virus and comprising a nucleic acid construct having a genetically modified TGB-3 viral sequence according to claim 24, operably linked to one or more regulatory sequence(s) active in a plant or a plant cell. ### Claim 36 (depends on 35) The transgenic plant or transgenic plant cell according to claim 35, wherein the virus is the BNYVV virus. ### Claim 37 (depends on 35) The transgenic sugar beet plant or transgenic sugar beet plant cell according to claim 35. ### Claim 38 (depends on 35) The transgenic plant or transgenic plant cell according to claim 35, wherein the regulatory sequence comprises a promoter sequence or a terminator sequence. ### Claim 39 (depends on 38) The transgenic plant of claim 38, wherein said promoter is active in the root tissue of a plant. ### Claim 40 (depends on 35) The transgenic plant or transgenic plant cell according to claim 35, wherein said transgenic plant is sugar beet and said transgenic plant cell is a sugar beet cell. ### Claim 41 (depends on 35) The transgenic plant, or transgenic plant cell according to claim 35, wherein the regulatory sequence(s) comprise a promoter sequence which is a constitutive or a foreign vegetal promoter sequence. ### Claim 42 (depends on 41) The transgenic plant or transgenic plant cell according to claim 41, wherein the promoter comprises the 35S Cauliflower Mosaic Virus promoter. ### Claim 43 (depends on 35) A transgenic plant tissue derived from the transgenic plant cell according to claim 35, wherein said tissue comprises said genetically modified TGB-3 viral sequence, and wherein said tissue is selected from the group consisting of fruit, stem, root, tuber, and seed. ### Claim 44 (depends on 14) A transgenic reproducible structure obtained from the transgenic plant cell according to claim 14, wherein said reproducible structure comprises said genetically modified TGB-3 viral sequence, and wherein said reproducible structure is selected from the group consisting of calluses, buds, and embryos. ### Claim 45 (depends on 9) A vector comprising the genetically modified TGB-3 viral sequence according to claim 9. ### Claim 46 (depends on 45) The vector of claim 45 operably linked to one or more regulatory sequence(s) active in a plant cell. ### Claim 47 (depends on 45) A double stranded self-complementary RNA molecule expressed by a vector of claim 45. ### Claim 48 (depends on 9) A transgenic plant or a transgenic plant cell resistant to a virus and comprising a nucleic acid construct having a genetically modified TGB-3 viral sequence according to claim 9 operably linked to one or more regulatory sequence(s) active in a plant or a plant cell. ### Claim 49 (depends on 48) The transgenic plant or transgenic plant cell according to claim 48, wherein the virus is the BNYVV virus. ### Claim 50 (depends on 48) The transgenic plant or transgenic plant cell according to claim 48, wherein said transgenic plant or transgenic plant cell comprises sugar beet. ### Claim 51 (depends on 48) The transgenic plant or transgenic plant cell according to claim 48, wherein the regulatory sequence comprises a promoter sequence or a terminator sequence that is active in a plant. ### Claim 52 (depends on 48) The transgenic plant or transgenic plant cell according to claim 48, wherein said transgenic plant is sugar beet and said transgenic plant cell is a sugar beet cell. ### Claim 53 (depends on 48) The transgenic plant or transgenic plant cell according to claim 48, wherein the regulatory sequence(s) comprise a promoter sequence which is a constitutive or a foreign vegetal promoter sequence. ### Claim 54 (depends on 53) The transgenic plant or transgenic plant cell according to claim 53, wherein the promoter comprises the 35S Cauliflower Mosaic Virus promoter. ### Claim 55 (depends on 9, 48) A transgenic plant tissue comprising the genetically modified TGB-3 sequence of claim 9, wherein said tissue is derived from the transgenic plant cell according to claim 48, and wherein said tissue is selected from the group consisting of fruit, stem, root, tuber, and seed. ### Claim 56 (depends on 9, 48) A transgenic reproducible structure comprising a genetically modified TGB-3 sequence of claim 9, wherein said genetically modified TGB-3 viral sequence when transcribed in a cell is capable of forming a double-stranded self-complementing RNA molecule, wherein said reproducible structure is obtained from the transgenic plant cell according to claim 48, and wherein said reproducible structure is selected from the group consisting of calluses, buds, and embryos. ### Claim 57 (depends on 10) A vector comprising the genetically modified TGB-3 viral sequence according to claim 10. ### Claim 58 A nucleotide sequence comprising a modified SEQ ID NO: 3 and an antisense sequence of said modified SEQ ID NO: 3, wherein the modifications in SEQ ID NO: 3 consist of a modification which alters a start codon in SEQ ID NO: 3 so as to inhibit translation and a modification which alters a stop codon in SEQ ID NO: 3 so as to inhibit translation, wherein the nucleotide sequence when transcribed in a cell is capable of forming a double-stranded self-complementing RNA molecule.