Viral amplification of recombinant messenger RNA in transgenic plants

Abstract

A novel method of over expressing genes in plants is provided. This method is based on the RNA amplification properties of plus strand RNA viruses of plants. A chimeric multicistronic gene is constructed containing a plant promoter, viral replication origins, a viral movement protein gene, and one or more foreign genes under control of viral subgenomic promoters. Plants containing one or more of these recombinant RNA transcripts are inoculated with helper virus. In the presence of helper virus recombinant transcripts are replicated producing high levels of foreign gene RNA. Sequences are provided for the high level expression of the enzyme chloramphenicol acetyltransferase in tobacco plants by replicon RNA amplification with helper viruses and movement protein genes derived from the tobamovirus group.

1 - 25 . (canceled)  
     
     
         26 . A method of expressing a foreign antisense RNA in a plant cell, the method comprising: 
 providing a chromosomally integrated transgene in a plant cell, wherein the transgene comprises a replicon having a plus sense, RNA viral replication origin and at least one foreign gene for the antisense RNA, which gene is capable of expression in a plant or plant cell, wherein said replicon is transcribed from a promoter, and wherein said replicon is dependent for replication on a helper virus;    providing the helper virus, wherein the helper virus comprises a plus sense RNA viral replicase;    transcribing the transgene to form recombinant mRNA comprising the replicon;    replicating the recombinant mRNAs with the plus sense RNA viral replicase, thereby producing additional replicon components; and,    producing subgenomic mRNA from the additional replicon components, thereby expressing the antisense RNA in the plant cell.    
     
     
         27 . The method of  claim 26 , wherein providing the chromosomally integrated transgene comprises: 
 a) introducing the transgene into the plant cell by transformation; and,    b) growing a transgenic plant to an optimal stage prior to providing the helper virus.    
     
     
         28 . The method of  claim 26 , wherein providing the helper virus comprises infecting the plant cell with the helper virus.  
     
     
         29 . The method of  claim 26 , wherein producing the subgenomic mRNA further comprises self-cleaving the mRNA.  
     
     
         30 . The method of  claim 26 , wherein transcribing the transgene comprises transcription with a host RNA polymerase.  
     
     
         31 . The method of  claim 26 , wherein the replicon codes for at least one sequence upon which the helper virus is dependent.  
     
     
         32 . The method of  claim 31 , wherein the at least one sequence upon which the helper virus is dependent comprises a functional movement protein.  
     
     
         33 . The method of  claim 31 , wherein the at least one sequence upon which the helper virus is dependent enables systemic infection of a whole plant and systemic expression of the foreign antisense RNA in the plant.  
     
     
         34 . The method of  claim 26 , wherein the plant cell comprises a cell susceptible to tobamovirus, and wherein the replicon comprises tobamoviral-derived sequences.  
     
     
         35 . A transgenic plant comprising the plant cell of  claim 26 .  
     
     
         36 . A method of expressing a foreign ribozyme in a plant cell, the method comprising: 
 providing a chromosomally integrated transgene in a plant cell, wherein the transgene comprises a replicon having a plus sense, RNA viral replication origin and at least one foreign gene for the ribozyme RNA, which gene is capable of expression in a plant or plant cell, wherein said replicon is transcribed from a promoter, and wherein said replicon is dependent for replication on a helper virus;    providing the helper virus, wherein the helper virus comprises a plus sense RNA viral replicase;    transcribing the transgene to form recombinant mRNA comprising the replicon;    replicating the recombinant mRNAs with the plus sense RNA viral replicase, thereby producing additional replicon components; and,    producing subgenomic mRNA from the additional replicon components, thereby expressing the ribozyme RNA in the plant cell.    
     
     
         37 . The method of  claim 36 , wherein providing the chromosomally integrated transgene comprises: 
 a) introducing the transgene into the plant cell by transformation; and,    b) growing a transgenic plant to an optimal stage prior to providing the helper virus.    
     
     
         38 . The method of  claim 36 , wherein providing the helper virus comprises infecting the plant cell with the helper virus.  
     
     
         39 . The method of  claim 36 , wherein producing the subgenomic mRNA further comprises self-cleaving the mRNA.  
     
     
         40 . The method of  claim 36 , wherein transcribing the transgene comprises transcription with a host RNA polymerase.  
     
     
         41 . The method of  claim 36 , wherein the replicon codes for at least one sequence upon which the helper virus is dependent.  
     
     
         42 . The method of  claim 41 , wherein the at least one sequence upon which the helper virus is dependent comprises a functional movement protein.  
     
     
         43 . The method of  claim 41 , wherein the at least one sequence upon which the helper virus is dependent enables systemic infection of a whole plant and systemic expression of the foreign ribozyme RNA in the plant.  
     
     
         44 . The method of  claim 36 , wherein the plant cell comprises a cell susceptible to tobamovirus, and wherein the replicon comprises tobamoviral-derived sequences.  
     
     
         45 . A transgenic plant comprising the plant cell of  claim 36 .  
     
     
         46 . A method of expressing a foreign protein in a plant cell, the method comprising: 
 providing a chromosomally integrated transgene in a plant cell, wherein the transgene comprises a replicon having a plus sense, RNA viral replication origin and at least one foreign gene for the foreign protein, which gene is capable of expression in a plant or plant cell, wherein said replicon is transcribed from a promoter, and wherein said replicon is dependent for replication on a helper virus;    providing the helper virus, wherein the helper virus comprises a plus sense RNA viral replicase;    transcribing the transgene to form recombinant mRNA comprising the replicon;    replicating the recombinant mRNAs with the plus sense RNA viral replicase, thereby producing additional replicon components;    producing subgenomic mRNA from the additional replicon components, thereby expressing the foreign protein gene in the plant cell; and,    translating the subgenomic mRNA of the expressed foreign protein gene, thereby producing the foreign protein.    
     
     
         47 . The method of  claim 46 , wherein providing the chromosomally integrated transgene comprises: 
 a) introducing the transgene into the plant cell by transformation; and,    b) growing a transgenic plant to an optimal stage prior to providing the helper virus.    
     
     
         48 . The method of  claim 46 , wherein providing the helper virus comprises infecting the plant cell with the helper virus.  
     
     
         49 . The method of  claim 46 , wherein producing the subgenomic mRNA further comprises self-cleaving the mRNA.  
     
     
         50 . The method of  claim 46 , wherein transcribing the transgene comprises transcription with a host RNA polymerase.  
     
     
         51 . The method of  claim 46 , wherein the replicon codes for at least one sequence upon which the helper virus is dependent.  
     
     
         52 . The method of  claim 51 , wherein the at least one sequence upon which the helper virus is dependent comprises a functional movement protein.  
     
     
         53 . The method of  claim 51 , wherein the at least one sequence upon which the helper virus is dependent enables systemic infection of a whole plant and systemic expression of the foreign protein in the plant.  
     
     
         54 . The method of  claim 53 , wherein the foreign protein comprises one or more of: an enzyme, a regulatory enzyme, a structural protein, or a therapeutic protein.  
     
     
         55 . The method of  claim 54 , wherein the foreign protein comprises an interleukin, a colony stimulating factor, CSF-G, CSF-GM, or CSF-M.  
     
     
         56 . The method of  claim 46 , wherein the plant cell comprises a cell susceptible to tobamovirus, and wherein the replicon comprises tobamoviral-derived sequences.  
     
     
         57 . A transgenic plant comprising the plant cell of  claim 46.