Methods and means for obtaining modified phenotypes

Abstract

Methods and means are provided for reducing the phenotypic expression of a nucleic acid of interest in eucaryotic cells, particularly in plant cells, by introducing chimeric genes encoding sense and antisense RNA molecules directed towards the target nucleic acid, which are capable of forming a double stranded RNA region by base-pairing between the regions with sense and antisense nucleotide sequence or by introducing the RNA molecules themselves. Preferably, the RNA molecules comprises simultaneously both sense and antisense nucleotide sequence.

1 . A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a plant cell, comprising the step of introducing a chimeric DNA comprising the following operably linked parts: 
 a) a promoter, operative in said plant cell;    b) a DNA region, which when transcribed, yields an RNA molecule comprising an RNA region capable of forming an artificial hairpin RNA structure comprising two annealing RNA sequences, wherein one of the annealing RNA sequences of the stem-loop structure comprises a sense sequence, essentially similar to at least part of the nucleotide sequence of said nucleic acid of interest, and wherein the second of said annealing RNA sequences comprises an antisense sequence essentially similar to at least part of the complement of at least part of said nucleotide sequence of said nucleic acid of interest; and    c) a DNA region involved in transcription termination and polyadenylation.    
     
     
         2 . A method for reducing the phenotypic expression of a nucleic acid of interest, which is normally capable of being expressed in a eukaryotic cell, comprising the step of introducing a chimeric DNA comprising the following operably linked parts; 
 a) a promoter, operative in said plant cell;    b) a DNA region, which when transcribed, yields an RNA molecule with a nucleotide sequence comprising 
 i. a sense nucleotide sequence of at least 10 consecutive nucleotides having between 95% and 100% sequence identity with at least 10 consecutive nucleotides of the nucleotide sequence of said nucleic acid of interest; and  
 ii. an antisense nucleotide sequence including at least 10 consecutive nucleotides, having between about 95% to about 100% sequence identity with the complement of said at least 10 consecutive nucleotides of said sense nucleotide sequence;  
 wherein the RNA is capable of forming an artificial hairpin RNA structure with a double stranded RNA stem by base-pairing between the regions with sense and antisense nucleotide sequence such that at least said 10 consecutive nucleotides of the sense sequence basepair with said 10 consecutive nucleotides of the antisense sequence; and  
   c) a DNA region involved in transcription termination and polyadenylation.    
     
     
         3 . The method of  claim 2 , wherein said RNA molecule further comprises a spacer nucleotide sequence located between said sense and said antisense nucleotide sequence.  
     
     
         4 . The method of  claim 2 , wherein said sense nucleotide sequence comprises at least 550 consecutive nucleotides having between 75% and 100% sequence identity with at least part of the nucleotide sequence of said nucleic acid.  
     
     
         5 . The method of  claim 2 , wherein said nucleic acid of interest is a gene integrated in the genome of said plant cell.  
     
     
         6 . The method of  claim 5 , wherein said gene is an endogenous gene.  
     
     
         7 . The method of  claim 5 , wherein said gene is a foreign transgene.  
     
     
         8 . The method of  claim 2 , wherein said chimeric DNA is stably integrated in the genome of the DNA.  
     
     
         9 . The method of  claim 2 , wherein said nucleic acid of interest is comprised in the genome of an infecting virus.  
     
     
         10 . The method of  claim 9 , wherein said infecting virus is an RNA virus.  
     
     
         11 . (canceled)  
     
     
         12 . The method of  claim 2 , wherein said plant cell is comprised within a plant.  
     
     
         13 . (canceled)  
     
     
         14 . A method for reducing the gene expression of a gene of interest in plant cells, said method comprising the step of introducing a first and second chimeric DNA, linked on one recombinant DNA such that both chimeric DNAs are integrated together in the nuclear genome of the transgenic plant cells; wherein said first chimeric DNA comprises the following operably linked parts: 
 a) a plant-expressible promoter;    b) a first DNA region capable of being transcribed into a sense RNA molecule with a nucleotide sequence comprising a sense nucleotide sequence of at least 10 consecutive nucleotides having between 95% and 100% sequence identity with at least part of the nucleotide sequence of said gene of interest; and    c) a DNA region involved in transcription termination and polyadenylation functioning in plant cells;    and wherein said second chimeric DNA comprises the following operably linked parts:    a) a plant-expressible promoter;    b) a second DNA region capable of being transcribed into an antisense RNA molecule with a nucleotide sequence comprising an antisense nucleotide sequence including at least 10 consecutive nucleotides, having between about 95% to about 100% sequence identity with the complement of said at least 10 consecutive nucleotides of said sense nucleotide sequence; and;    c) a DNA region involved in transcription termination and polyadenylation functioning in plant cells;    wherein said sense and antisense RNA molecules are capable of forming a double stranded RNA by base-pairing between the regions which are complementary.    
     
     
         15 . A method for obtaining a virus resistant plant, said method comprising the steps of: 
 1) providing the cells of said plant with a first and second chimeric DNA wherein said first chimeric DNA comprises the following operably linked parts: 
 a) a promoter operative in said cells;  
 b) a first DNA region capable of being transcribed into a sense RNA molecule with a nucleotide sequence comprising a sense nucleotide sequence of at least 10 consecutive nucleotides having between 95% and 100% sequence identity with at least at least 10 consecutive nucleotides of the nucleotide sequence of the genome of a virus, capable of infecting said plant; and  
 c) a DNA region involved in transcription termination and polyadenylation functioning in said cells; and  
 wherein said second chimeric DNA comprises the following operably linked parts:  
 a) a promoter operative in said plant;  
 b) a second DNA region capable of being transcribed into an antisense RNA molecule with a nucleotide sequence comprising an antisense nucleotide sequence including at least 10 consecutive nucleotides, having between about 95% to 100% sequence identity with the complement of said at least 10 consecutive nucleotides of said sense nucleotide sequence; and  
 c) a DNA region involved in transcription termination and polyadenylation functioning in said cells; and  
 wherein said sense and antisense RNA are capable of forming a double stranded RNA by base-pairing between the regions which are complementary.  
   
     
     
         16 . (canceled)  
     
     
         17 . The method of  claim 15 , wherein said cells of said plants are provided with said first and second chimeric DNA by crossing parent plants comprising either said first or said second chimeric DNA.  
     
     
         18 . The method of  claim 15 , wherein said cells of said plants are provided with said first and second chimeric DNA by transforming a plant cell with said first and second chimeric DNA, and regenerating a plant from said transformed plant cell.  
     
     
         19 . The method of  claim 15 , wherein said first and second chimeric DNA are integrated separately in said nuclear genome of said plant cell.  
     
     
         20 . The method of  claim 15 , wherein said first and second chimeric DNA are linked on one recombinant DNA such that both chimeric DNAs are integrated together in the nuclear genome of the transgenic plant cells.  
     
     
         21 - 38 . (canceled)  
     
     
         39 . A method to inhibit expression of a target gene in a cell in a plant comprising contacting the cell with a construct encoding at least one ribonucleic acid (RNA) in an amount sufficient to inhibit the expression of a target gene, wherein the RNA comprises or forms a double-stranded structure containing a first strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide sequences are complementary sequences that hybridize to each other to form said double-stranded structure, and the RNA comprising the double-stranded structure inhibits expression of the target gene.  
     
     
         40 . The method of  claim 39 , wherein said plant is selected from the group consisting of monocots, dicots and gymnosperms.  
     
     
         41 . The method of  claim 39 , wherein said plant is selected from the group consisting of  Arabidopsis , field crops, vegetable crops, fruit crops, nut crops and ornamentals.  
     
     
         42 . The method of  claim 39 , wherein said cell is transfected with said construct.  
     
     
         43 . The method of  claim 39 , wherein said cell is transformed with said construct.  
     
     
         44 . The method of  claim 39 , wherein said construct is an expression construct.  
     
     
         45 . The method of  claim 39  wherein said double-stranded structure is formed by a single self-complementary RNA strand containing said first and second ribonucleotide sequences.  
     
     
         46 . The method of  claim 39  wherein the RNA is a double-stranded molecule containing two separate complementary RNA strands.  
     
     
         47 . The method of  claim 42  wherein said construct is introduced into said target cell by a method selected from the group consisting of particle bombardment, electroporation, lipid-mediated carrier transport and chemical-mediated transport.  
     
     
         48 . The method of  claim 39  wherein said target cell is at risk for infection by a pathogen.  
     
     
         49 . The method of  claim 40  wherein inhibition of expression of said target gene provides a plant with altered fruit ripening characteristics.  
     
     
         50 . The method of  claim 43  wherein said RNA is synthesized in said cell in response to a condition selected from the group consisting of infection, stress, temperature and chemical induction.  
     
     
         51 . The method of  claim 48 , wherein said infection is caused by a pathogen selected from the group consisting of arachnids, insects, nematodes, protozoans, bacteria and fungi.  
     
     
         52 . The method of  claim 43  wherein said RNA is synthesized in said cell at a specific development stage or age.  
     
     
         53 . The method of  claim 43  wherein said RNA is synthesized in said plant in a tissue-specific manner.  
     
     
         54 . The method of  claim 53 , wherein said tissue is selected from the group consisting of fruit seed, anther, flower, leaf and root.  
     
     
         55 . A method to reduce the phenotypic expression of a nucleic acid of interest in a plant cell comprising introducing into the plant cell a construct encoding at least one ribonucleic acid (RNA), wherein the RNA comprises or forms a double-stranded structure containing a first strand comprising a ribonucleotide sequence which is essentially similar to a nucleotide sequence of the target gene and a second strand comprising a ribonucleotide sequence which is essentially similar to a nucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide sequences are complementary sequences that base-pair to each other to form said double-stranded structure, and the RNA comprising the double-stranded structure reduces the phenotypic expression of the target gene.  
     
     
         56 . The method of  claim 55 , wherein said plant is selected from the group consisting of monocotyledonous and dicotyledonous plants.  
     
     
         57 . The method of  claim 55 , wherein said plant is selected from the group consisting of  Arabidopsis , crop plants including corn, rice, wheat, barley, sugarcane, cotton, oilseed rape, soybean vegetables (including chicory,  brassica  vegetables, lettuce, tomato), tobacco, potato, sugarbeet, plants used in horticulture, floriculture or forestry, oilseed rape ( Brassica juncea, napus, rapa, oleracea, campestris ), corn, cotton, groundnut, sunflower, castor beans, flax, coconut, linseed, soybean and plants important in viticulture or in fruit production.  
     
     
         58 . The method of  claim 55 , wherein said construct is introduced transiently into said plant cell.  
     
     
         59 . The method of  claim 55 , wherein said cell is transformed with said construct.  
     
     
         60 . The method of  claim 55 , wherein said construct is an expression construct.  
     
     
         61 . The method of  claim 55  wherein said double-stranded structure is formed by a single self-annealing RNA strand containing said first and second ribonucleotide sequences.  
     
     
         62 . The method of  claim 55  wherein the RNA is a double-stranded molecule containing two separate complementary RNA strands.  
     
     
         63 . The method of  claim 58  wherein said construct is introduced into said target cell by a method selected from the group consisting of direct gene transfer, microprojectile bombardment, lipid-mediated carrier transport and chemical-mediated transport.  
     
     
         64 . The method of  claim 55  wherein said plant cell is a plant cell at risk for infection by a pathogen.  
     
     
         65 . The method of  claim 55  wherein inhibition of expression of said target gene provides a plant with shatter resistance, modified flower color patterns, nematode resistance, delayed fruit ripening or male sterility.  
     
     
         66 . The method of  claim 59  wherein the expression of said RNA from said construct in said cell is controlled at will by the application of an appropriate chemical inducer, by operably linking the transcribed DNA region of the chimeric genes of the invention to a promoter whose expression is induced by a chemical compound.  
     
     
         67 . The method of  claim 64 , wherein said infection is caused by a pathogen selected from the group consisting of viruses and nematodes.  
     
     
         68 . The method of  claim 59  wherein the expression of said RNA from said construct in said cell is under control of an organ-specific promoter.  
     
     
         69 . The method of  claim 59  the expression of said RNA from said construct in said cell is under control of a tissue-specific promoter.  
     
     
         70 . The method of  claim 69 , wherein said tissue-specific promoter is selected from the group consisting of a seed-specific promoter, an organ-primordia specific promoter, a leaf specific promoter, a mesophyl-specific promoters, a root specific promoter, a tuber-specific promoter, a vascular tissue specific promoter, a stamen-selective promoter and a dehiscence zone specific promoters.  
     
     
         71 . The method of  claim 2 , wherein said sense nucleotide sequence includes at least 20 consecutive nucleotides having between 95% and 100% sequence identity with at least 20 consecutive nucleotides of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 20 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 20 consecutive nucleotides of said sense nucleotide sequence.  
     
     
         72 . The method of  claim 2 , wherein said sense nucleotide sequence includes at least 50 consecutive nucleotides having between 95% and 100% sequence identity with at least 50 consecutive nucleotides of the nucleotide sequence of said nucleic acid of interest, and said antisense nucleotide sequence includes at least 50 consecutive nucleotides having between 95% and 100% sequence identity with the complement of said at least 50 consecutive nucleotides of said sense nucleotide sequence.