Gene silencing materials and methods

Abstract

Disclosed are methods for silencing a target nucleotide sequence (preferably representing one or more endogenous genes, preferably in a systemic fashion) which is present in a first part of the plant, which method comprises transiently introducing into the cytoplasm of a cell in a second part of the plant, which cell comprises a nucleic acid encoding the target sequence and which is remote from said first part of the plant, a nucleic acid construct.

1 . A method for silencing a target nucleotide sequence present in a first part of a plant, which method comprises transiently introducing into the cytoplasm of a cell in a second part of the plant, which cell comprises a nucleic acid encoding the target sequence and which is remote from said first part of the plant, a nucleic acid construct, wherein said construct: 
 (i) encodes a sequence which shares sequence identity with the target nucleotide sequence or the complement thereof, and    (ii) does not encode proteins which are capable of blocking systemic movement of a gene silencing signal, such that a silencing signal not comprising the construct is initiated in the first part of the plant and propagated to the second part of the plant such as to cause the silencing of said target nucleotide sequence.    
     
     
         2 . A method as claimed in  claim 1  wherein the proteins which are capable of blocking systemic movement of a gene silencing signal are those which are capable of mediating intercellular viral movement.  
     
     
         3 . A method as claimed in  claim 1  or  claim 2  wherein the part of the plant into which the nucleic acid is introduced corresponds to a region in which photosynthetic products are concentrated and the target nucleotide sequence is present in a remote region in which such products are used.  
     
     
         4 . A method as claimed in any one of the preceding claims wherein the target nucleotide sequence, or a nucleotide sequence sharing homology with the target nucleotide sequence, is transcribed in the cells of the tissues connecting the first and second parts of the plant through which the gene silencing signal is propagated.  
     
     
         5 . A method as claimed in any one of the preceding claims wherein the target nucleotide sequence is silenced systemically in the plant.  
     
     
         6 . A method as claimed in any one of the preceding claims wherein the construct is not capable of autonomous replication.  
     
     
         7 . A method as claimed in any one of the preceding claims wherein the construct introduced into the plant cell does not encode a viral coat protein  
     
     
         8 . A method as claimed in any one of the preceding claims wherein the sequence sharing sequence identity with the target gene does not include translation-recognition signals such that said sequence is not translated to a protein product.  
     
     
         9 . A method as claimed in any one of the preceding claims wherein the nucleic acid construct is DNA.  
     
     
         10 . A method as claimed in any one of the preceding claims wherein the construct comprises a promoter operably linked to a nucleotide sequence, wherein said nucleotide sequence: 
 (i) encodes a viral replicase,    (ii) encodes a replicable sequence which shares sequence identity with the target nucleotide sequence or its complement, and which is operably linked to one or more cis acting elements recognised by said replicase, such that the replicable sequence is replicated in the cytoplasm of the cell into which it is introduced,    (iii) does not encode proteins which are capable of mediating intercellular viral movement.    
     
     
         11 . A method as claimed in  claim 11  wherein the viral replicase is a PVX replicase.  
     
     
         12 . A method as claimed in  claim 10  or  11  wherein the promoter is an inducible promoter.  
     
     
         13 . A method as claimed in any one of  claims 10  to  12  wherein the construct comprises Ti-derived sequences which permit integration of the construct into the plant genome.  
     
     
         14 . A method as claimed in any one of  claims 1  to  9  wherein the construct does not comprise any of the following: 
 (i) promoter or terminator sequences,  
 (ii) Ti-derived sequences which permit integration of the construct into the plant genome.  
 
     
     
         15 . A method as claimed in  claim 13  wherein the construct is introduced into the plant using  Agrobacteriun tumafaciens.    
     
     
         16 . A method as claimed in any one of  claims 1  to  14  wherein the construct is introduced into the plant cell by microprojectile bombardment.  
     
     
         17 . A method as claimed in any one of the preceding claims wherein the target nucleotide sequence encodes a heterologous gene.  
     
     
         18 . A method as claimed in any one of  claims 1  to  16  wherein the target nucleotide sequence encodes a gene which is endogenous to the plant.  
     
     
         19 . A method as claimed in  claim 18  wherein the plant is not a transgenic plant.  
     
     
         20 . A method as claimed in any one of the preceding claims wherein the target nucleotide sequence encodes all or part of a viral genome of a virus in the plant.  
     
     
         21 . A method as claimed in any one of the preceding claims wherein two or more target genes which share sequence identity are silenced.  
     
     
         22 . A method of assessing a phenotypic characteristic associated with a target nucleotide sequence in a plant, the method comprising: 
 (a) silencing the nucleotide sequence in a plant in accordance with a method as claimed in any one of the preceding claims,    (b) observing the phenotype of the plant, and optionally    (c) comparing the result of the observation with the phenotype of a control plant.    
     
     
         23 . A method for regulating the expression of a target nucleotide sequence in a plant comprising use of a method as claimed in any one of  claims 1  to  21 .  
     
     
         24 . A method of systemically altering the phenotype of a plant comprising use of a method as claimed in any one of  claims 1  to  21 .  
     
     
         25 . A nucleic acid construct comprising a promoter operably linked to a nucleotide sequence, wherein said nucleotide sequence: 
 (i) encodes a viral replicase,    (ii) encodes a replicable sequence which shares sequence identity with the target nucleotide sequence or its complement, and which is operably linked to one or more cis acting elements recognised by said replicase, such that the replicable sequence is replicated in the cytoplasm of the cell into which it is introduced,    (iii) does not encode proteins which are capable of mediating intercellular viral movement.    
     
     
         26 . A construct as claimed in  claim 25  which is a DNA plasmid.  
     
     
         27 . A construct as claimed in  claim 26  which is a Ti plasmid vector.  
     
     
         28 . A method for producing a systemic gene silencing signal in a plant, said method comprising the steps of introducing a construct as claimed in any one of  claims 25  to  27  into a cell of that plant.  
     
     
         29 . A method as claimed in  claim 28  wherein the signal produced by the construct is subsequently stably maintained in the absence of the construct.  
     
     
         30 . A plant cell comprising a construct as claimed in any one of  claims 25  to  27 .  
     
     
         31 . A plant comprising a plant cell as claimed in  claim 30 .  
     
     
         32 . A plant comprising a target nucleotide sequence which has been silenced in accordance with the method of any one of  claims 1  to  21 .