Method of determining the presence of a trait in a plant by transfecting a nucleic acid sequence of a donor plant into a different host plant in an anti-sense orientation

Abstract

The present invention provides a method of compiling a plant functional gene profile, a method of changing the phenotype or biochemistry of a plant, a method of determining a change in phenotype or biochemistry of a plant, and a method of determining the presence of a trait in plant. The methods comprise expressing transiently a nucleic acid sequence of a plant into a host plant to affect phenotypic or biochemical changes in the host plant. A viral vector functional genomic screen has been developed to identify nucleotide sequences in transfected plants by systemically knocking out endogenous gene expression in an antisense mechanism. Once the presence of a trait in a plant is identified by phenotypic or biochemical changes in the host plant, the nucleic acid insert in the cDNA clone or in the vector that results in the changes is then sequenced. The present invention exemplifies that genes encoding GTP binding proteins in one plant can silence endogenous gene expression in a different plant.

What is claimed is:  
     
         1 . A method of changing the phenotype or biochemistry of a plant, comprising: 
 a) preparing a library of DNA or RNA sequences from a donor plant, and constructing recombinant viral nucleic acids obtained from a tobamo virus comprising an unidentified nucleic acid insert obtained from said library in an antisense orientation relative to said DNA or RNA sequence of said donor plant;    b) infecting a host plant with one or more said recombinant viral nucleic acids;    c) transiently expressing said unidentified nucleic acid in the host plant; and    d) determining one or more phenotypic or biochemical changes in the host plant.    
     
     
         2 . A method of changing the phenotype or biochemistry of a plant, comprising: 
 a) expressing transiently a nucleic acid sequence from a donor plant in an antisense orientation in a host plant, wherein said donor plant and said host plant belong to a different family, order, class, subdivision, or division; and    b) determining one or more phenotypic or biochemical changes in said host plant.    
     
     
         3 . A method of determining a change in phenotype or biochemistry in a plant due to a transient expression of a nucleic acid in an antisense orientation, comprising: 
 a) preparing a library of nucleic acid sequences from a donor plant, and constructing recombinant viral nucleic acids obtained from a tobamo virus comprising an unidentified nucleic acid insert obtained from said library in an antisense orientation relative to said DNA or RNA sequence of said donor plant;    b) infecting a host plant with one or more said recombinant viral nucleic acids;    c) transiently expressing said unidentified nucleic acid in the host plant;    d) determining one or more phenotypic or biochemical changes in the host plant; and    e) correlating said one or more biochemical or phenotypic changes to a host plant that is uninfected.    
     
     
         4 . A method of determining a change in phenotype or biochemistry in a plant due to a transient expression of a nucleic acid in an antisense orientation, comprising: 
 a) expressing transiently a nucleic acid sequence from a donor plant in an antisense orientation in a host plant, wherein said donor plant and said host plant belong to a different family, order, class, subdivision, or division;    b) determining one or more biochemical or phenotypic changes in said host plant; and    c) correlating said one or more biochemical or phenotypic changes to a host plant that is uninfected.    
     
     
         5 . A method of determining the presence of a trait in a plant, comprising: 
 a) preparing a library of nucleic acid sequences from a donor plant, and constructing recombinant viral nucleic acids obtained from a tobamo virus comprising an unidentified nucleic acid insert obtained from said library in an antisense orientation relative to said DNA or RNA sequence of said donor plant;    b) infecting a host plant with one or more said recombinant viral nucleic acids;    c) transiently expressing said unidentified nucleic acid in the host plant;    d) determining one or more phenotypic or biochemical changes in the host plant,    e) correlating said one or more biochemical or phenotypic changes to a host plant that is uninfected; and    f) identifying a trait present in said infected or uninfected host plant.    
     
     
         6 . A method of determining the presence of a trait in a plant, comprising: 
 a) expressing transiently a nucleic acid sequence from a donor plant in an antisense orientation in a host plant, wherein said donor plant and said host plant belong to a different family, order, class, subdivision, or division;    b) determining one or more biochemical or phenotypic changes in said host plant;    c) correlating said one or more biochemical or phenotypic changes to a host plant that is uninfected; and    d) identifying a trait present in said infected or uninfected host plant.    
     
     
         7 . The method according to  claim 3 , further comprises the step of correlating said one or more biochemical or phenotypic changes to a host plant that is infected with a viral vector that contains a known nucleic acid sequence but in an antisense orientation, wherein said known nucleic acid sequence has similar size but is different in sequence from said nucleic acid sequence in (a).  
     
     
         8 . The method according to  claim 3  or  5 , wherein said donor plant is selected from the group consisting of food crops, seed crops, oil crops, ornamental crops and forestry crops.  
     
     
         9 . The method according to  claim 3  or  5 , wherein said host plant is selected from the group consisting of food crops, seed crops, oil crops, ornamental crops and forestry.  
     
     
         10 . The method according to  claim 9 , wherein said host plant is Nicotiana.  
     
     
         11 . The method according to  claim 10 , wherein said host plant is  Nicotiana benthamina  or  Nicotiana cleavlandii.    
     
     
         12 . The method according to  claim 3  or  5 , wherein said nucleic acid sequence is derived from a library of cDNAs, genomic DNAs, or a pool of RNAs, which represents all or part of the donor plant genome.  
     
     
         13 . The method according to  claim 3  or  5 , wherein the recombinant viral nucleic acids are capped or uncapped.  
     
     
         14 . The method according to  claim 3  or  5 , wherein an antisence RNA is produced in the cytoplasm of said host plant, and said antisense RNAs results in a reduced expression of an endogenous gene in said host plant.  
     
     
         15 . The method according to  claim 3  or  5 , wherein said recombinant viral nucleic acid further comprises a native plant viral subgenomic promoter and a plant viral coat protein coding sequence.  
     
     
         16 . The method according to  claim 15 , wherein said recombinant viral nucleic acid further comprises a non-native plant viral subgenomic promoter, said native plant viral subgenomic promoter initiates transcription of said plant viral coat protein sequence and said non-native plant viral subgenomic promoter initiates transcription of said nucleic acid sequence.  
     
     
         17 . The method according to  claim 3  or  5 , wherein said phenotypic changes are changes in growth rates, morphology, or color.  
     
     
         18 . A method of determining the presence of a trait in a plant, comprising: 
 a) expressing transiently a nucleic acid sequence of a donor plant in an antisense orientation in a host plant;    b) determining phenotypic or biochemical changes in said host plant; and    c) correlating said expression with said phenotypic or biochemical changes, wherein said nucleic acid sequence comprising a GTP binding protein open reading frame having an antisense orientation.    
     
     
         19 . The method according to  claim 18 , wherein said GTP binding protein is selected from the group consisting of rab family, and ADP-ribosylation factor family.  
     
     
         20 . A method of changing the phenotype or biochemistry of a plant, comprising: 
 a) expressing transiently a nucleic acid sequence encoding ribosomal protein S18 from a donor plant in an antisense orientation in a host plant, which inhibits an endogenous gene expression in the meristem of said host plant; and    b) determining one or more phenotypic or biochemical changes in said host plant.