Modified gene-silencing RNA and uses thereof

Abstract

Methods and means for efficiently downregulating the expression of any gene of interest in eukaryotic cells and organisms are provided. To this end, the invention provides modified antisense and sense RNA molecules, chimeric genes encoding such modified antisense or sense RNA molecules and eukaryotic organisms such as plants, animals or fungi, yeast or molds, comprising the modified antisense and/or sense RNA molecules or the encoding chimeric genes.

1 . A method for downregulating the expression of a target gene in cells of a eukaryotic organism, comprising the steps of:
 providing the cells of the eukaryotic organism with a chimeric RNA molecule, wherein the chimeric RNA molecule comprises   a target gene-specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides, which has at least about 94% sequence identity with the complement of about 19 consecutive nucleotides from the nucleotide sequence of the target gene, operably linked to   a largely double-stranded RNA region; and   identifying those eukaryotic organisms wherein the expression of the target gene is down regulated.   
     
     
         2 . The method according to  claim 1 , wherein the largely double-stranded RNA region comprises a nuclear localization signal from a viroid of the Potato spindle tuber viroid (PSTVd)-type. 
     
     
         3 . The method according to  claim 2 , wherein the nuclear localization signal is from a viroid selected from the group consisting of Potato Spindle tuber viroid, Citrus viroid species III, Citrus viroid species IV, Hop latent viroid, Australian grapevine viroid, Tomato planta macho viroid, Coconut tinangaja viroid, Tomato apical stunt viroid, Coconut cadang-cadang viroid, Citrus exocortis viroid,  Columnea  latent viroid, Hop stunt viroid and Citrus bent leaf viroid. 
     
     
         4 . The method according to  claim 3 , wherein the viroid has a genome nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         5 . The method according to  claim 2 , wherein the nuclear localization signal is from Potato spindle tuber viroid. 
     
     
         6 . The method according to  claim 2 , wherein the nuclear localization signal is from Potato spindle viroid strain RG1. 
     
     
         7 . The method according to  claim 2 , wherein the nuclear localization signal comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         8 . The method according to  claim 2 , wherein the largely double-stranded RNA comprises a viroid genome nucleotide sequence selected from the group consisting of the genome nucleotide sequence of Potato Spindle tuber viroid, the genome nucleotide sequence of Citrus viroid species III, the genome nucleotide sequence of Citrus viroid species IV, the genome nucleotide sequence of Hop latent viroid, the genome nucleotide sequence of Australian grapevine viroid, the genome nucleotide sequence of Tomato planta macho viroid, the genome nucleotide sequence of Coconut tinangaja viroid, the genome nucleotide sequence of Tomato apical stunt viroid, the genome nucleotide sequence of Coconut cadang-cadang viroid, the genome nucleotide sequence of Citrus exocortis viroid, the genome nucleotide sequence of  Columnea  latent viroid, the genome nucleotide sequence of Hop stunt viroid and the genome nucleotide sequence of Citrus bent leaf viroid. 
     
     
         9 . The method according to  claim 8 , wherein the viroid genome nucleotide sequence is selected from group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 
     
     
         10 . The method according to  claim 2 , wherein the largely double stranded RNA region comprises a genomic nucleotide sequence of Potato spindle tuber viroid. 
     
     
         11 . The method according to  claim 10 , wherein the viroid genome nucleotide sequence is the genome nucleotide sequence of Potato spindle tuber viroid strain RG1. 
     
     
         12 . The method of  claim 11 , wherein the genome nucleotide sequence has the nucleotide sequence of SEQ ID NO:3. 
     
     
         13 . The method according to  claim 1 , wherein the largely double stranded RNA region comprises at least about 35 repeats of the trinucleotide CUG. 
     
     
         14 . The method according to  claim 13 , wherein the largely double-stranded RNA region comprises between about 44 and about 2000 repeats of the trinucleotide CUG. 
     
     
         15 . The method according to  claim 1 , wherein the RNA molecule comprises multiple target gene-specific regions. 
     
     
         16 . The method according to  claim 1 , wherein the RNA molecule comprises an intron sequence. 
     
     
         17 . The method according to  claim 16 , wherein the intron sequence is selected from the group consisting of the pdk2 intron, the catalase intron from Castor bean, the Delta12 desaturase intron from cotton, the Delta 12 desaturase intron from  Arabidopsis , the Ubiquitin intron from maize, the Actin intron from rice, the triose phosphate isomerase intron from  Aspergillus  and the intron from SV40. 
     
     
         18 . The method according to  claim 1 , wherein the eukaryotic organism is a plant. 
     
     
         19 . The method according to  claim 18 , wherein the plant is selected from the group of  Arabidopsis , alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon. 
     
     
         20 . The method according to  claim 1 , wherein the eukaryotic organism is a fungus, yeast or mold. 
     
     
         21 . The method according to  claim 1 , wherein the eukaryotic organism is an animal. 
     
     
         22 . The method according to  claim 21 , wherein the animal is a human, mammal, bird, fish, cattle, goat, pig, sheep, rodent, hamster, mouse, rat, guinea pig, rabbit, primate, nematode, shellfish, prawn, crab, lobster, insect, fruit fly, Coleopteran insect, Dipteran insect, Lepidopteran insect and Homeopteran insect. 
     
     
         23 . The method according to  claim 1 , wherein the chimeric RNA is produced by transcription from a chimeric DNA molecule. 
     
     
         24 . A chimeric RNA molecule for downregulating the expression of a target gene in a cell of a eukaryotic organism, comprising
 a target-gene specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides, which has at least about 94% sequence identity with the complement of about 19 consecutive nucleotides from the nucleotide sequence of the target gene in the cell of the eukaryotic organism; operably linked to   a largely double stranded RNA region;   
       wherein the chimeric RNA molecule, when provided to cells of the eukaryotic organism, downregulates the expression of the target gene. 
     
     
         25 . The chimeric RNA molecule according to  claim 24 , wherein the largely double-stranded RNA region comprises a nuclear localization signal from a viroid of the Potato spindle tuber viroid (PSTVd) type. 
     
     
         26 . The chimeric RNA molecule according to  claim 25 , wherein the nuclear localization signal is from a viroid selected from the group consisting of Potato Spindle tuber viroid, Citrus viroid species III, Citrus viroid species IV, Hop latent viroid, Australian grapevine viroid, Tomato planta macho viroid, Coconut tinangaja viroid, Tomato apical stunt viroid, Coconut cadang-cadang viroid, Citrus exocortis viroid,  Columnea  latent viroid, Hop stunt viroid and Citrus bent leaf viroid. 
     
     
         27 . The chimeric RNA molecule according to  claim 25 , wherein the viroid has a genome nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 
     
     
         28 . The chimeric RNA molecule according to  claim 25 , wherein the nuclear localization signal is from Potato spindle tuber viroid. 
     
     
         29 . The chimeric RNA molecule according to  claim 25 , wherein the nuclear localization signal is from Potato spindle viroid strain RG1. 
     
     
         30 . The chimeric RNA molecule according to  claim 25 , wherein the nuclear localization signal comprises a nucleotide sequence functioning as a nuclear localization signal selected from the nucleotide sequence of SEQ ID NO:3. 
     
     
         31 . The chimeric RNA molecule according to  claim 25 , wherein the largely double-stranded RNA comprises a viroid genome nucleotide sequence selected from the group consisting of the genome nucleotide sequence of Potato Spindle tuber viroid, the genome nucleotide sequence of Citrus viroid species III, the genome nucleotide sequence of Citrus viroid species IV, the genome nucleotide sequence of Hop latent viroid, the genome nucleotide sequence of Australian grapevine viroid, the genome nucleotide sequence of Tomato planta macho viroid, the genome nucleotide sequence of Coconut tinangaja viroid, the genome nucleotide sequence of Tomato apical stunt viroid, the genome nucleotide sequence of Coconut cadang-cadang viroid, the genome nucleotide sequence of Citrus exocortis viroid, the genome nucleotide sequence of  Columnea  latent viroid, the genome nucleotide sequence of Hop stunt viroid and the genome nucleotide sequence of Citrus bent leaf viroid. 
     
     
         32 . The chimeric RNA molecule according to  claim 31 , wherein the viroid genome nucleotide sequence is selected from group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 
     
     
         33 . The chimeric RNA molecule according to  claim 25 , wherein the largely double stranded RNA region comprises a genomic nucleotide sequence of Potato spindle tuber viroid. 
     
     
         34 . The chimeric RNA molecule according to  claim 33 , wherein the viroid genome nucleotide sequence is the genome nucleotide sequence of Potato spindle tuber viroid strain RG1. 
     
     
         35 . The chimeric RNA molecule of  claim 34 , wherein the genome nucleotide sequence has the nucleotide sequence of SEQ ID NO:3. 
     
     
         36 . The chimeric RNA molecule according to  claim 24 , wherein the largely double stranded RNA region comprises at least about 35 repeats of the trinucleotide CUG. 
     
     
         37 . The chimeric RNA molecule according to  claim 36 , wherein the largely double stranded RNA region comprises between about 44 and about 2000 repeats of the trinucleotide CUG. 
     
     
         38 . The chimeric RNA molecule according to  claim 24 , wherein the RNA molecule comprises multiple target gene-specific regions. 
     
     
         39 . The chimeric RNA molecule according to  claim 24 , wherein the RNA molecule comprises an intron sequence. 
     
     
         40 . The chimeric RNA molecule according to  claim 39 , wherein the intron sequence is selected from the group consisting of the pdk2 intron, the catalase intron from Castor bean, the Delta12 desaturase intron from cotton, the Delta 12 desaturase intron from  Arabidopsis , the Ubiquitin intron from maize, the Actin intron from rice, the triose phosphate isomerase intron from  Aspergillus  and the intron from SV40. 
     
     
         41 . A chimeric DNA molecule for reduction of the expression of a target gene in a cell of a eukaryotic organism, comprising
 a promoter or promoter region capable of being recognized by RNA polymerases in the cells of the eukaryotic organism, operably linked to   a DNA region that, when transcribed, yields an RNA molecule comprising a target gene-specific RNA region comprising   a nucleotide sequence of at least about 19 consecutive nucleotides having at least about 94% sequence identity with the complement of 19 consecutive nucleotides from the nucleotide sequence of the target gene in the cell of the eukaryotic organism, operably linked to   a largely double-stranded RNA region;   
       wherein the chimeric DNA molecule, when provided to cells of the eukaryotic organism, reduces the expression of the target gene. 
     
     
         42 . The chimeric DNA molecule according to  claim 41 , wherein the largely double-stranded RNA region comprises a nuclear localization signal from a viroid of the potato spindle tuber viroid type. 
     
     
         43 . The chimeric DNA molecule according to  claim 42 , wherein the nuclear localization signal is from a viroid selected from the group consisting of Potato Spindle tuber viroid, Citrus viroid species III, Citrus viroid species IV, Hop latent viroid, Australian grapevine viroid, Tomato planta macho viroid, Coconut tinangaja viroid, Tomato apical stunt viroid, Coconut cadang-cadang viroid, Citrus exocortis viroid,  Columnea  latent viroid, Hop stunt viroid and Citrus bent leaf viroid. 
     
     
         44 . The chimeric DNA molecule according to  claim 42 , wherein the viroid has a genome nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         45 . The chimeric DNA molecule according to  claim 42 , wherein the nuclear localization signal is from Potato spindle tuber viroid. 
     
     
         46 . The chimeric DNA molecule according to  claim 45 , wherein the nuclear localization signal is from Potato spindle viroid strain RG1. 
     
     
         47 . The chimeric DNA molecule according to  claim 42 , wherein the nuclear localization signal comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         48 . The chimeric DNA molecule according to  claim 42 , wherein the largely double-stranded RNA comprises a viroid genome nucleotide sequence selected from the group consisting of the genome nucleotide sequence of Potato Spindle tuber viroid, the genome nucleotide sequence of Citrus viroid species III, the genome nucleotide sequence of Citrus viroid species IV, the genome nucleotide sequence of Hop latent viroid, the genome nucleotide sequence of Australian grapevine viroid, the genome nucleotide sequence of Tomato planta macho viroid, the genome nucleotide sequence of Coconut tinangaja viroid, the genome nucleotide sequence of Tomato apical stunt viroid, the genome nucleotide sequence of Coconut cadang-cadang viroid, the genome nucleotide sequence of Citrus exocortis viroid, the genome nucleotide sequence of  Columnea  latent viroid, the genome nucleotide sequence of Hop stunt viroid and the genome nucleotide sequence of Citrus bent leaf viroid. 
     
     
         49 . The chimeric DNA molecule according to  claim 48 , wherein the viroid genome nucleotide sequence is selected from group consisting of SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         50 . The chimeric DNA molecule according to  claim 42 , wherein the largely double-stranded RNA region comprises a genomic nucleotide sequence of Potato spindle tuber viroid. 
     
     
         51 . The chimeric DNA molecule according to  claim 50 , wherein the viroid genome nucleotide sequence is the genome nucleotide sequence of Potato spindle tuber viroid strain RG1. 
     
     
         52 . The chimeric DNA molecule of  claim 51 , wherein the genome nucleotide sequence has the nucleotide sequence of SEQ ID NO: 3. 
     
     
         53 . The chimeric DNA molecule according to  claim 41 , wherein the largely double-stranded RNA region comprises at least about 35 repeats of the trinucleotide CUG. 
     
     
         54 . The chimeric DNA molecule according to  claim 53 , wherein the largely double-stranded RNA region comprises between about 44 and about 2000 repeats of the trinucleotide CUG. 
     
     
         55 . The chimeric DNA molecule according to  claim 41 , wherein the RNA molecule comprises multiple target gene-specific regions. 
     
     
         56 . The chimeric DNA molecule according to  claim 41 , wherein the RNA molecule comprises an intron sequence. 
     
     
         57 . The chimeric DNA molecule according to  claim 56 , wherein the intron sequence is selected from the group consisting of the pdk2 intron, the catalase intron from Castor bean, the Delta12 desaturase intron from cotton, the Delta 12 desaturase intron from  Arabidopsis , the Ubiquitin intron from maize, the Actin intron from rice, the triose phosphate isomerase intron from  Aspergillus  and the intron from SV40. 
     
     
         58 . The chimeric DNA molecule according to  claim 41 , further comprising a transcription termination and polyadenylation signal operably linked to the DNA region encoding the RNA molecule. 
     
     
         59 . The chimeric DNA molecule according to  claim 41 , wherein the promoter or promoter region is plant-expressible. 
     
     
         60 . The chimeric DNA molecule according to  claim 41 , wherein the promoter or promoter region functions in animals. 
     
     
         61 . The chimeric DNA molecule according to  claim 41 , wherein the promoter or promoter region functions in yeast, fungi or molds. 
     
     
         62 . The chimeric DNA molecule according to  claim 41 , wherein the promoter or promoter region is recognized by a single subunit bacteriophage RNA polymerase. 
     
     
         63 . A cell from a eukaryotic organism comprising a chimeric DNA molecule according to  claim 41 . 
     
     
         64 . A eukaryotic cell comprising a chimeric RNA molecule according to  claim 24 . 
     
     
         65 . The cell according to  claim 63 , wherein the eukaryotic organism is a plant. 
     
     
         66 . The cell according to  claim 64 , wherein the eukaryotic organism is a plant. 
     
     
         67 . The cell according to  claim 65 , wherein the plant is selected from the group of  Arabidopsis , alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon. 
     
     
         68 . The cell according to  claim 63 , wherein the eukaryotic organism is a fungus, yeast or mold. 
     
     
         69 . The cell according to  claim 63 , wherein the eukaryotic organism is an animal. 
     
     
         70 . The cell according to  claim 64 , wherein the eukaryotic organism is an animal. 
     
     
         71 . The cell according to  claim 69 , wherein the animal is a human, mammal, bird, fish, cattle, goat, pig, sheep, rodent, hamster, mouse, rat, guinea pig, rabbit, primate, nematode, shellfish, prawn, crab, lobster, insect, fruit fly, Coleopteran insect, Dipteran insect, Lepidopteran insect and Homeopteran insect. 
     
     
         72 . A eukaryotic organism comprising in its cells a chimeric DNA molecule according to  claim 41 . 
     
     
         73 . A eukaryotic organism, comprising in its cells a chimeric RNA molecule according to  claim 24 . 
     
     
         74 . The eukaryotic organism according to  claim 72 , wherein the eukaryotic organism is a plant. 
     
     
         75 . The eukaryotic organism according to  claim 73 , wherein the eukaryotic organism is a plant. 
     
     
         76 . The cell according to  claim 74 , wherein the plant is selected from the group of  Arabidopsis , alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon. 
     
     
         77 . The eukaryotic organism according to  claim 72 , wherein the eukaryotic organism is a fungus, yeast or mold. 
     
     
         78 . The non-human eukaryotic organism according to  claim 72 , wherein the eukaryotic organism is an animal. 
     
     
         79 . The eukaryotic organism according to  claim 73 , wherein the eukaryotic organism is an animal. 
     
     
         80 . The non-human eukaryotic organism according to  claim 78 , wherein the animal is a human, mammal, bird, fish, cattle, goat, pig, sheep, rodent, hamster, mouse, rat, guinea pig, rabbit, primate, nematode, shellfish, prawn, crab, lobster, insect, fruit fly, Coleopteran insect, Dipteran insect, Lepidopteran insect and Homeopteran insect. 
     
     
         81 . A method for making a transgenic eukaryotic organism in which expression of a target gene in cells of the organism is reduced, the method comprising the steps of:
 providing a chimeric DNA molecule according to  claim 41  to a cell or cells of the organism to make a transgenic cell or cells; and   growing or regenerating a transgenic eukaryotic organism from the transgenic cell or cells.   
     
     
         82 . A method for downregulating the expression of a target gene in cells of a eukaryotic organism, comprising the steps of
 providing the cells of the eukaryotic organism with a first and second chimeric RNA molecule, wherein   the first chimeric RNA molecule comprises an antisense target gene-specific RNA region, comprising a nucleotide sequence of at least about 19 consecutive nucleotides, which has at least about 94% sequence identity with the complement of about 19 consecutive nucleotides from the nucleotide sequence of the target gene;   the second chimeric RNA molecule comprises a sense target gene-specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides, which has at least about 94% sequence identity to the complement of the first chimeric RNA molecule;   the first and second chimeric RNA are capable of basepairing at least between the about 19 consecutive nucleotides of the first chimeric RNA and the about 19 consecutive nucleotides of the second chimeric RNA; and   either the first or the second chimeric RNA molecule comprises a largely double-stranded RNA region operably linked to the antisense target-specific RNA region or to the sense target-specific RNA region; and   identifying those eukaryotic organisms wherein the expression of the target gene is down regulated.   
     
     
         83 . The method according to  claim 82 , wherein the first and the second chimeric RNA molecule comprise a largely double-stranded RNA region. 
     
     
         84 . The method according to  claim 83 , wherein the first and the second chimeric RNA molecule comprise the same largely double-stranded RNA region. 
     
     
         85 . The method according to  claim 82 , wherein the largely double-stranded RNA region comprises a nuclear localization signal from a viroid of the Potato spindle tuber viroid (PSTVd)-type. 
     
     
         86 . The method according to  claim 85 , wherein the nuclear localization signal is from a viroid selected from the group consisting of Potato Spindle tuber viroid, Citrus viroid species III, Citrus viroid species IV, Hop latent viroid, Australian grapevine viroid, Tomato planta macho viroid, Coconut tinangaja viroid, Tomato apical stunt viroid, Coconut cadang-cadang viroid, Citrus exocortis viroid,  Columnea  latent viroid, Hop stunt viroid and Citrus bent leaf viroid. 
     
     
         87 . The method according to  claim 85 , wherein the viroid has a genome nucleotide sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         88 . The method according to  claim 85 , wherein the nuclear localization signal is from Potato spindle tuber viroid. 
     
     
         89 . The method according to  claims 85 , wherein the nuclear localization signal is from Potato spindle viroid strain RG1. 
     
     
         90 . The method according to  claim 85 , wherein the nuclear localization signal comprises a nucleotide sequence functioning as a nuclear localization signal selected from the nucleotide sequence of SEQ ID NO: 3. 
     
     
         91 . The method according to  claim 85 , wherein the largely double-stranded RNA comprises a viroid genome nucleotide sequence selected from the group consisting of the genome nucleotide sequence of Potato Spindle tuber viroid, the genome nucleotide sequence of Citrus viroid species III, the genome nucleotide sequence of Citrus viroid species IV, the genome nucleotide sequence of Hop latent viroid, the genome nucleotide sequence of Australian grapevine viroid, the genome nucleotide sequence of Tomato planta macho viroid, the genome nucleotide sequence of Coconut tinangaja viroid, the genome nucleotide sequence of Tomato apical stunt viroid, the genome nucleotide sequence of Coconut cadang-cadang viroid, the genome nucleotide sequence of Citrus exocortis viroid, the genome nucleotide sequence of  Columnea  latent viroid, the genome nucleotide sequence of Hop stunt viroid and the genome nucleotide sequence of Citrus bent leaf viroid. 
     
     
         92 . The method according to  claim 91 , wherein the viroid genome nucleotide sequence is selected from group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         93 . The method according to  claim 85 , wherein the largely double-stranded RNA region comprises a genomic nucleotide sequence of Potato spindle tuber viroid. 
     
     
         94 . The method according to  claim 93 , wherein the viroid genome nucleotide sequence is the genome nucleotide sequence of Potato spindle tuber viroid strain RG1. 
     
     
         95 . The method of  claim 94 , wherein the genome nucleotide sequence has the nucleotide sequence of SEQ ID NO: 3. 
     
     
         96 . The method according to  claim 82 , wherein the largely double stranded RNA region comprises at least about 35 repeats of the trinucleotide CUG. 
     
     
         97 . The method according to  claim 96 , wherein the largely double stranded RNA region comprises between about 44 and about 2000 repeats of the trinucleotide CUG. 
     
     
         98 . The method according to  claim 82 , wherein the RNA molecule comprises multiple target gene-specific regions. 
     
     
         99 . The method according to  claim 82 , wherein the RNA molecule comprises an intron sequence. 
     
     
         100 . The method according to  claim 99 , wherein the intron sequence is selected from the group consisting of the pdk2 intron, the catalase intron from Castor bean, the Delta12 desaturase intron from cotton, the Delta 12 desaturase intron from  Arabidopsis , the Ubiquitin intron from maize, the Actin intron from rice, the triose phosphate isomerase intron from  Aspergillus , and the intron from SV40. 
     
     
         101 . The method according to  claim 82 , wherein the first chimeric RNA and the second chimeric RNA are transcribed from a first and second chimeric gene. 
     
     
         102 . A cell from a eukaryotic organism comprising a first and second chimeric RNA molecule, wherein
 the first chimeric RNA molecule comprises an antisense target gene-specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides having at least about 94% sequence identity with the complement of about 19 consecutive nucleotides from the nucleotide sequence of the target gene;   the second chimeric RNA molecule comprises a sense target gene-specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides having at least about 94% sequence identity to the complement of the first chimeric RNA molecule;   the first and second chimeric RNA are capable of basepairing at least between the about 19 consecutive nucleotides of the first chimeric RNA and the about 19 consecutive nucleotides of the second chimeric RNA; and   either the first or the second chimeric RNA molecule comprises a largely double-stranded RNA region operably linked to the antisense target-specific RNA region or to the sense target-specific RNA region.   
     
     
         103 . The cell according to  claim 102 , wherein the first and the second chimeric RNA molecule comprise a largely double-stranded RNA region. 
     
     
         104 . The cell according to  claim 103 , wherein the first and the second chimeric RNA molecule comprise the same largely double-stranded RNA region. 
     
     
         105 . The cell according to  claim 102 , wherein the largely double-stranded RNA region comprises a nuclear localization signal from a viroid of the Potato spindle tuber viroid (PSTVd)-type. 
     
     
         106 . The cell according to  claim 105 , wherein the nuclear localization signal is from a viroid selected from the group consisting of Potato Spindle tuber viroid, Citrus viroid species III, Citrus viroid species IV, Hop latent viroid, Australian grapevine viroid, Tomato planta macho viroid, Coconut tinangaja viroid, Tomato apical stunt viroid, Coconut cadang-cadang viroid, Citrus exocortis viroid,  Columnea  latent viroid, Hop stunt viroid and Citrus bent leaf viroid. 
     
     
         107 . The cell according to  claim 105 , wherein the viroid has a genome nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         108 . The cell according to  claim 105 , wherein the nuclear localization signal is from Potato spindle tuber viroid. 
     
     
         109 . The cell according to  claim 105 , wherein the nuclear localization signal is from Potato spindle viroid strain RG1. 
     
     
         110 . The cell according to  claim 107 , wherein the nuclear localization signal comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         111 . The cell according to  claim 105 , wherein the largely double-stranded RNA comprises a viroid genome nucleotide sequence selected from the group consisting of the genome nucleotide sequence of Potato Spindle tuber viroid, the genome nucleotide sequence of Citrus viroid species III, the genome nucleotide sequence of Citrus viroid species IV, the genome nucleotide sequence of Hop latent viroid, the genome nucleotide sequence of Australian grapevine viroid, the genome nucleotide sequence of Tomato planta macho viroid, the genome nucleotide sequence of Coconut tinangaja viroid, the genome nucleotide sequence of Tomato apical stunt viroid, the genome nucleotide sequence of Coconut cadang-cadang viroid, the genome nucleotide sequence of Citrus exocortis viroid, the genome nucleotide sequence of  Columnea  latent viroid, the genome nucleotide sequence of Hop stunt viroid and the genome nucleotide sequence of Citrus bent leaf viroid. 
     
     
         112 . The cell according to  claim 111 , wherein the viroid genome nucleotide sequence is selected from group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         113 . The cell according to  claim 105 , wherein the largely double stranded RNA region comprises a genomic nucleotide sequence of Potato spindle tuber viroid. 
     
     
         114 . The cell according to  claim 113 , wherein the viroid genome nucleotide sequence is the genome nucleotide sequence of Potato spindle tuber viroid strain RG1. 
     
     
         115 . The cell of  claim 114 , wherein the genome nucleotide sequence has the nucleotide sequence of SEQ ID NO: 3. 
     
     
         116 . The cell according to  claim 102 , wherein the largely double-stranded RNA region comprises at least about 35 repeats of the trinucleotide CUG. 
     
     
         117 . The cell according to  claim 116 , wherein the largely double-stranded RNA region comprises between about 44 and about 2000 repeats of the trinucleotide CUG. 
     
     
         118 . The cell according to  claim 102 , wherein the RNA molecule comprises multiple target gene-specific regions. 
     
     
         119 . The cell according to  claim 102 , wherein the RNA molecule comprises an intron sequence. 
     
     
         120 . The cell according to  claim 119 , wherein the intron sequence is selected from the group consisting of the pdk2 intron, the catalase intron from Castor bean, the Delta12 desaturase intron from cotton, the Delta 12 desaturase intron from  Arabidopsis , the Ubiquitin intron from maize, the Actin intron from rice, the triose phosphate isomerase intron from  Aspergillus  and the intron from SV40. 
     
     
         121 . The cell according to  claim 102  wherein the first and second chimeric RNA are transcribed from a first and second chimeric gene. 
     
     
         122 . A eukaryotic organism comprising the cell according to  claim 102 . 
     
     
         123 . A chimeric sense RNA molecule for reduction of expression of a target gene in a cell of a eukaryotic organism in cooperation with a chimeric antisense RNA molecule, the chimeric sense RNA molecule comprising
 a sense target gene-specific RNA region comprising a nucleotide sequence of at least about 19 consecutive nucleotides, which has at least about 94% sequence identity to the nucleotide of the target gene; operably linked to   a largely double-stranded RNA region.   
     
     
         124 . The chimeric RNA molecule according to  claim 123 , wherein the largely double-stranded RNA region comprises a nuclear localization signal from a viroid of the Potato spindle tuber viroid (PSTVd)-type. 
     
     
         125 . The chimeric RNA molecule according to  claim 124 , wherein the nuclear localization signal is from a viroid selected from the group consisting of Potato Spindle tuber viroid, Citrus viroid species III, Citrus viroid species IV, Hop latent viroid, Australian grapevine viroid, Tomato planta macho viroid, Coconut tinangaja viroid, Tomato apical stunt viroid, Coconut cadang-cadang viroid, Citrus exocortis viroid,  Columnea  latent viroid, Hop stunt viroid and Citrus bent leaf viroid. 
     
     
         126 . The chimeric RNA molecule according to  claim 125 , wherein the viroid has a genome nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         127 . The chimeric RNA molecule according to  claim 124 , wherein the nuclear localization signal is from Potato spindle tuber viroid. 
     
     
         128 . The chimeric RNA molecule according to  claim 124 , wherein the nuclear localization signal is from Potato spindle viroid strain RG1. 
     
     
         129 . The chimeric RNA molecule according to  claim 124 , wherein the nuclear localization signal comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         130 . The chimeric RNA molecule according to  claim 124 , wherein the largely double-stranded RNA comprises a viroid genome nucleotide sequence selected from the group consisting of the genome nucleotide sequence of Potato Spindle tuber viroid, the genome nucleotide sequence of Citrus viroid species III, the genome nucleotide sequence of Citrus viroid species IV, the genome nucleotide sequence of Hop latent viroid, the genome nucleotide sequence of Australian grapevine viroid, the genome nucleotide sequence of Tomato planta macho viroid, the genome nucleotide sequence of Coconut tinangaja viroid, the genome nucleotide sequence of Tomato apical stunt viroid, the genome nucleotide sequence of Coconut cadang-cadang viroid, the genome nucleotide sequence of Citrus exocortis viroid, the genome nucleotide sequence of  Columnea  latent viroid, the genome nucleotide sequence of Hop stunt viroid and the genome nucleotide sequence of Citrus bent leaf viroid. 
     
     
         131 . The chimeric RNA molecule according to  claim 130 , wherein the viroid genome nucleotide sequence is selected from group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. 
     
     
         132 . The chimeric RNA molecule according to  claim 124 , wherein the largely double-stranded RNA region comprises a genomic nucleotide sequence of Potato spindle tuber viroid. 
     
     
         133 . The chimeric RNA molecule according to  claim 132 , wherein the viroid genome nucleotide sequence is the genome nucleotide sequence of Potato spindle tuber viroid strain RG1. 
     
     
         134 . The chimeric RNA molecule of  claim 133 , wherein the genome nucleotide sequence has the nucleotide sequence of SEQ ID NO: 3. 
     
     
         135 . The chimeric RNA molecule according to  claim 123 , wherein the largely double-stranded RNA region comprises at least about 35 repeats of the trinucleotide CUG. 
     
     
         136 . The chimeric RNA molecule according to  claim 135 , wherein the largely double stranded RNA region comprises between about 44 and about 2000 repeats of the trinucleotide CUG. 
     
     
         137 . The chimeric RNA molecule according to  claim 123 , wherein the RNA molecule comprises multiple target gene-specific regions. 
     
     
         138 . The chimeric RNA molecule according to  claim 123 , wherein the RNA molecule comprises an intron sequence. 
     
     
         139 . The chimeric RNA molecule according to  claim 138 , wherein the intron sequence is selected from the group consisting of the pdk2 intron, the catalase intron from Castor bean, the Delta12 desaturase intron from cotton, the Delta 12 desaturase intron from  Arabidopsis , the Ubiquitin intron from maize, the Actin intron from rice, the triose phosphate isomerase intron from  Aspergillus  and the intron from SV40. 
     
     
         140 . A chimeric DNA molecule for reduction of the expression of a target gene in a cell of a eukaryotic organism, comprising
 a promoter or promoter region capable of being recognized by RNA polymerases in the cells of the eukaryotic organism; operably linked to   a DNA region that, when transcribed, yields a chimeric sense RNA molecule as described in  claim 123 .