METHOD AND CONSTRUCTS FOR DELIVERING DOUBLE STRANDED RNA tO PEST ORGANISMS

Abstract

The present invention relates in generally to RNAi and its use in gene silencing. In particular, the present invention relates to methods and constructs for delivering double stranded RNA (dsRNA) to pest organisms.

1 . An RNA delivery molecule consisting of a polypeptide sequence comprising
 i) at least one RNA-binding domain,   ii) at least one targeting polypeptide able to bind to a cellular endocytosis and/or transcytosis receptor molecule, and   iii) optionally at least one peptide linker for linking the RNA binding domain (i) to the targeting polypeptide (ii).   
     
     
         2 . An RNA delivery molecule according to  claim 1 , wherein said at least one RNA-binding domain (i) is chosen from:
 a polypeptide comprising the coliphage HK022 Nun protein (for instance as represented in SEQ ID NO 2), a homologue thereof, or a fragment thereof comprising the RNA-binding domain, said fragment preferably comprising amino acids 1 to 47 (SEQ ID NO 4) of the aminoterminal sequence, more preferably comprising amino acids 13 to 47 (SEQ ID NO 6) of the aminoterminal sequence, most preferably comprising amino acids 22 to 47 (SEQ ID NO 8) of the aminoterminal sequence,   a polypeptide comprising the  Bacillus subtilis  LicT protein (for instance as represented in SEQ ID NO 10), a homologue thereof, or a fragment thereof comprising the RNA-binding domain, said fragment preferably comprising amino acids 1 to 56 (SEQ ID NO 12) of the aminoterminal sequence, and   a polypeptide comprising the bacteriophage MS2 coat protein (for instance as represented in SEQ ID NO 14), a homologue thereof, or a fragment thereof comprising the RNA-binding domain.   
     
     
         3 . An RNA delivery molecule according to  claim 1 , wherein said at least one targeting polypeptide (ii) binds to a gut cell endocytosis or transcytosis receptor molecule. 
     
     
         4 . An RNA delivery molecule according to  claim 1 , wherein said at least one targeting polypeptide (ii) binds to a tissue cell endocytosis or transcytosis receptor molecule. 
     
     
         5 . An RNA delivery molecule according to  claim 1 , further comprising a purification tag. 
     
     
         6 . A method for delivering a double-stranded RNA molecule to a pest species, comprising
 co-expressing in a plant cell of:   a) an RNA delivery molecule according to  claim 1 , and   b) an RNA molecule which comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene of the pest species, and which further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule of (a).   feeding said plant cell to said pest species.   
     
     
         7 . A method for delivering double-stranded RNA to a pest species, comprising feeding the pest species with a mixture of an RNA molecule and an RNA delivery molecule according to  claim 1 ,
 wherein the RNA molecule comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene of the pest species, and which further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule,   whereby the mixture is taken up into the gut of the pest species, and   whereby the complex of the double-stranded RNA bound to the RNA delivery molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell.   
     
     
         8 . A method for down-regulating expression of a target gene in a pest species, comprising feeding the pest species with a mixture of an RNA molecule and an RNA delivery molecule according to  claim 1 ,
 wherein the RNA molecule comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, and further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule,   whereby the mixture is taken up into the gut of the pest species, and   whereby the complex of the double-stranded RNA bound to the RNA delivery molecule is transcytosed and/or endocytosed by a gut cell and/or a tissue cell and thereby down-regulates expression of the target gene in a pest cell.   
     
     
         9 . A method for down-regulating expression of a target gene in a pest species, comprising:
 co-expressing in a plant cell of:   a) an RNA delivery molecule according to  claim 1 ,   b) an RNA molecule which comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene to be down-regulated, and which further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule of (a), and   feeding said plant cell to said pest species.   
     
     
         10 . A method for producing a transgenic plant that is resistant to a pest species, comprising:
 co-expressing in a plant cell of:   a) an RNA delivery molecule according to  claim 1 , and   b) an RNA molecule which comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of the target gene of said pest species, and which further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule of (a), and   regenerating a plant from said plant cell.   
     
     
         11 . A method according to  claim 6 , wherein in (a) the RNA-binding domain comprises the coliphage HK022 Nun protein or a fragment thereof, said fragment comprising the RNA-binding domain of the HK022 Nun protein, and wherein the double stranded RNA molecule (b) comprises a nucleotide sequence corresponding to the binding motif for binding to the coliphage HK022 Nun protein or a fragment thereof. 
     
     
         12 . A method according to  claim 11 , wherein in (a) the RNA-binding domain is a polypeptide comprising the sequence represented in any of SEQ ID NOs 2, 4, 6 or 8, preferably comprising the sequence represented in SEQ ID NO 8, and wherein (b) comprises the binding motif represented in SEQ ID NO 15 or 16. 
     
     
         13 . A method according to  claim 6 , wherein in (a) the RNA binding domain is the  Bacillus subtilis  LicT protein or a fragment thereof, said fragment comprising the RNA binding domain and wherein the double stranded RNA molecule (b) comprises a nucleotide sequence corresponding to the binding motif for binding to the  Bacillus subtilis  LicT protein or a fragment thereof. 
     
     
         14 . A method according to  claim 13 , wherein in (a) the RNA binding domain is a polypeptide comprising the sequence represented in SEQ ID NO 10 or 12, preferably a polypeptide comprising the sequence represented in SEQ ID NO 12, and wherein the double stranded RNA molecule (b) comprises the binding motif represented in  FIG. 41 , or as represented in any of SEQ ID NOs 17 to 34. 
     
     
         15 . A method according to  claim 6 , wherein in (a) the RNA binding domain is MS2 or a fragment thereof, said fragment comprising the RNA binding domain and wherein the double stranded RNA molecule (b) comprises a nucleotide sequence corresponding to the binding motif for binding to MS2 or a fragment thereof. 
     
     
         16 . A method according to  claim 15 , wherein in (a) the RNA binding domain is a polypeptide comprising the sequence represented in SEQ ID NO 14, and wherein the double stranded RNA molecule (b) comprises the binding motif represented in  FIG. 42 , or as represented in any of SEQ ID NOs 35 to 40. 
     
     
         17 . A method according to  claim 6 , wherein said pest species is chosen from the group comprising yeast, fungi, insects and nematodes. 
     
     
         18 . A nucleic acid encoding an RNA delivery molecule as defined in  claim 1 . 
     
     
         19 . A nucleic acid according to  claim 18  comprising an in frame signal sequence for directing the encoded polypeptide to a plant organel, such as a chloroplast, a plastide, a mitochondrium. 
     
     
         20 . A vector comprising a nucleic acid of  claim 18 . 
     
     
         21 . A vector according to  claim 20 , wherein the nucleic acid is under the control of regulatory sequences for expression of the nucleic acid in a plant cell. 
     
     
         22 . A vector according to  claim 20  further comprising a tissue-specific or cell-specific promoter. 
     
     
         23 . A vector according to  claim 22 , wherein the promoter is an inducible or a constitutive promoter. 
     
     
         24 . A host cell comprising a nucleic acid according to  claim 18 , or a vector according to  claim 20 . 
     
     
         25 . A host cell according to  claim 24 , wherein said host cell is a bacterial, yeast, fungal or plant cell. 
     
     
         26 . A composition comprising at least one RNA delivery molecule as defined in  claim 1 , and optionally further comprising at least one suitable excipient. 
     
     
         27 . A composition comprising:
 a) at least one RNA delivery molecule according to  claim 1 , and   b) at least one RNA molecule which comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene of a pest species, and which further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule of (a), and   
       optionally further comprising at least one suitable excipient. 
     
     
         28 . A kit comprising at least one RNA delivery molecule as defined in  claim 1 . 
     
     
         29 . A method for producing an RNA delivery molecule according to  claim 1  comprising:
 introducing into a host cell an isolated DNA molecule encoding the RNA delivery molecule according to  claim 1 ; 
 growing the host cell under conditions suitable for expression of the RNA delivery molecule; and 
 isolating the RNA delivery molecule produced by the host cell. 
 
     
     
         30 . A transgenic plant resistant to a pest species, an essential derived variety thereof, plant part, plant cell or protoplast thereof obtainable by a method according to  claim 10 . 
     
     
         31 . A transgenic plant, essentially derived variety thereof, plant part, plant cell or protoplast thereof which comprises a nucleic acid encoding an RNA delivery molecule as defined in  claim 1 , wherein said nucleic acid is heterologous to the genome of said transgenic plant, or an essentially derived variety, plant part, plant cell or plant protoplast thereof. 
     
     
         32 . A plant, essentially derived variety thereof, plant part, plant cell or protoplast thereof wherein the plant, essentially derived variety thereof, plant part, plant cell or protoplast thereof has been transformed with a nucleic acid encoding an RNA delivery molecule as defined in  claim 1 . 
     
     
         33 . A plant, essentially derived variety, plant part, plant cell or protoplast thereof which co-expresses:
 a) an RNA delivery molecule according to  claim 1 , and   b) an RNA molecule which comprises double-stranded RNA comprising annealed complementary strands, one of which has a nucleotide sequence which is complementary to at least part of a target nucleotide sequence of a target gene in a pest species, and which further comprises a nucleotide sequence which binds to the RNA-binding domain of the RNA delivery molecule of (a).   
     
     
         34 . The plant according to  claim 32 , wherein the plant has been stably transformed. 
     
     
         35 . The plant according to  claim 32 , wherein the plant has been transiently transformed. 
     
     
         36 . A transgenic plant which comprises a vector according to  claim 20 . 
     
     
         37 . Progeny of a plant or essentially derived variety thereof as claimed in  claim 30 , obtained in a generative or vegetative manner. 
     
     
         38 . Parts or derivatives of plants obtainable from a plant or essentially derived variety thereof as claimed in  claim 30 . 
     
     
         39 . The parts or derivatives of plants according to  claim 38  comprising leaves, stems, roots, shoots, cuttings or explants and the like, protoplasts, somatic embryos, anthers, petioles, cells in culture, seeds, flowers, fruits and tubers. 
     
     
         40 . A chimeric RNAi molecule comprising
 at least one nucleotide sequence recognized by an RNA-binding protein or RNA-binding domain, for instance as represented in any of SEQ ID NOs 15 to 40, or an RNA secondary structure as represented in  FIG. 41  or  42 , and,   at least one nucleotide sequence corresponding to a target nucleotide sequence of a target gene of a pest species.   
     
     
         41 . A chimeric RNAi molecule according to  claim 40 , as represented in any of SEQ ID NOs 41 to 52 and in any of SEQ ID NOs 54 to 56, and in any of SEQ ID NOs 57 to 71, and in any of SEQ ID NOs 72 to 86. 
     
     
         42 . A complex comprising an RNA delivery molecule according to  claim 1 , and a chimeric RNAi molecule according to  claim 40 . 
     
     
         43 . Use of an RNA delivery molecule according to  claim 1  for delivery of a double stranded RNA molecule to a target sequence in a pest species. 
     
     
         44 . Use of an RNA delivery molecule according to  claim 1  for producing a transgenic plant. 
     
     
         45 . Use of a complex according to  claim 42  for down-regulating expression of a target gene in a pest species. 
     
     
         46 . Use of a complex according to  claim 42  for producing a transgenic plant that is resistant to a pest species.