Isolated dsRNA Molecules And Methods Of Using Same For Silencing Target Molecules Of Interest

Abstract

An isolated dsRNA molecule comprising an antisense RNA sequence for regulating a target gene of interest in a plant or a phytopathogen of the plant, wherein the dsRNA sequence is flanked by two complementary sites to an smRNA or smRNAs expressed in the plant and wherein the dsRNA molecule further comprises a helicase binding site positioned so as to allow unwinding of the strands of the isolated dsRNA molecule to single stranded RNA (ssRNA) and recruitment of an RNA-dependent RNA polymerase so as to amplify the dsRNA molecule in the plant cell and generate secondary siRNA products of the dsRNA sequence.

1 . An isolated double-stranded RNA (dsRNA) molecule comprising
 (a) a first RNA strand having
 at least one antisense RNA sequence for suppressing expression of a target gene of interest in a plant or a phytopathogen of a plant, 
 a first heterologous smRNA-binding sequence for binding to a first small RNA (smRNA) expressed in said plant or phytopathogen, and 
 a helicase-binding sequence comprising the helicase binding site of SEQ ID NO: 14; and 
   (b) a second RNA strand that is a reverse complement of said first RNA strand.   
     
     
         2 . The isolated dsRNA molecule of  claim 1 , wherein said first smRNA comprises a nucleic acid sequence wherein said nucleic acid sequence shares between 100% and 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1 to 288, and complements thereof. 
     
     
         3 .- 5 . (canceled) 
     
     
         6 . The isolated dsRNA molecule of  claim 1 , wherein said first RNA strand further comprises a second heterologous smRNA-binding sequence for binding a second smRNA expressed in said plant or phytopathogen, and said first heterologous smRNA-binding sequence and said second heterologous smRNA-binding sequence flank said at least one antisense RNA sequence. 
     
     
         7 . The isolated dsRNA molecule of  claim 6 , wherein said first smRNA and said second smRNA comprise a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1 to 288, and complements thereof. 
     
     
         8 . The isolated dsRNA molecule of  claim 6 , wherein said second heterologous smRNA-binding sequence is the complement of said second smRNA. 
     
     
         9 . The isolated dsRNA molecule of  claim 6 , wherein said second smRNA is identical to said first smRNA. 
     
     
         10 . The isolated dsRNA molecule of  claim 6 , wherein said second smRNA is non-identical to said first smRNA. 
     
     
         11 . The isolated dsRNA molecule of  claim 6 , wherein said first heterologous smRNA-binding sequence and said second heterologous smRNA-binding sequence comprise a nucleotide sequence selected from the group consisting of:
 a direct sequence of said first smRNA and a direct sequence of said second smRNA;   a reverse complement of said first smRNA and a direct sequence of said second smRNA;   a reverse complement of said first smRNA and a reverse complement of said second smRNA;   a direct sequence of said first smRNA and a reverse complement of said second smRNA;   a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA;   a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA;   a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA;   a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA;   a direct sequence of said first smRNA and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;   a reverse complement of said first smRNA and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;   a reverse complement of said first smRNA and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage;   a direct sequence of said first smRNA and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage;   a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;   a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a direct sequence of said second smRNA and further comprising a mutation rendering it resistant to cleavage;   a reverse complement of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage; and   a direct sequence of said first smRNA and further comprising a mutation rendering it resistant to cleavage and a reverse complement of said second smRNA and further comprising a mutation rendering it resistant to cleavage.   
     
     
         12 .- 14 . (canceled) 
     
     
         15 . The isolated dsRNA molecule of  claim 1 , wherein said first smRNA has a nucleotide sequence selected from the group consisting of an RNA sequence of a microRNA (miRNA) and an RNA sequence of an siRNA. 
     
     
         16 . The isolated dsRNA molecule of  claim 6 , wherein said first smRNA has a nucleotide sequence selected from the group consisting of an RNA sequence of a miRNA and an RNA sequence of an siRNA, and said second smRNA has a nucleotide sequence selected from the group consisting of an RNA sequence of a miRNA and an RNA sequence of an siRNA. 
     
     
         17 . (canceled) 
     
     
         18 . The isolated dsRNA molecule of  claim 1 , wherein said first smRNA is a miRNA. 
     
     
         19 . The isolated dsRNA molecule of  claim 6 , wherein said first smRNA is a miRNA and said second smRNA is a miRNA. 
     
     
         20 . (canceled) 
     
     
         21 . The isolated dsRNA molecule of  claim 1 , wherein said first smRNA is a miRNA selected from the group consisting of miR390, miR161.1, miR168, miR393, miR828, and miR173. 
     
     
         22 . The isolated dsRNA molecule of  claim 6 , wherein said first smRNA is a miRNA selected from the group consisting of miR390, miR161.1, miR168, miR393, miR828, and miR173. 
     
     
         23 .- 48 . (canceled) 
     
     
         49 . A method of suppressing gene expression in a plant or a phytopathogen of the plant comprising:
 a. contacting a seed with an isolated double-stranded RNA (dsRNA) molecule of  claim 1  under conditions which allow penetration of said dsRNA molecule into said seed,
 thereby introducing said dsRNA molecule into said seed; and optionally 
 b. generating a plant of said seed. 
   
     
     
         50 . The method of  claim 49 , wherein said dsRNA molecule penetrates a cell of said seed selected from the group consisting of an endosperm cell, an embryo cell, and combinations thereof. 
     
     
         51 .- 69 . (canceled) 
     
     
         70 . An isolated double-stranded RNA (dsRNA) molecule comprising:
 a first RNA strand having a nucleic acid sequence comprising in a sequential order from 5′ to 3′;   an endovirus 5′ UTR sequence;   an endovirus RNA Dependent RNA Polymerase (RDRP) coding sequence;   a multiple cloning site;   an endovirus 3′ UTR sequence; and   a second RNA strand that is a reverse complement of said first RNA strand.   
     
     
         71 . An isolated dsRNA molecule comprising:
 a first RNA strand having a nucleic acid sequence comprising in a sequential order from 5′ to 3′;   an endovirus 5′ untranslated region (UTR) sequence;   an endovirus RNA Dependent RNA Polymerase (RDRP) coding sequence;   an antisense nucleic acid sequence for regulating a target gene;   an endovirus 3′ UTR sequence; and   a second RNA strand that is a reverse complement of said first RNA strand.   
     
     
         72 . The isolated dsRNA molecule of  claim 70 , wherein said endovirus 5′ UTR sequence, said endovirus RNA Dependent RNA Polymerase (RDRP) coding sequence and said endovirus 3′ UTR sequence are capable of autonomous replication when introduced into a plant cell. 
     
     
         73 . The isolated dsRNA molecule of  claim 71 , wherein said endovirus 5′ UTR sequence, said endovirus RNA Dependent RNA Polymerase (RDRP) coding sequence and said endovirus 3′ UTR sequence are capable of autonomous replication when introduced into a plant cell. 
     
     
         74 .- 113 . (canceled)