Mapping molecular interactions in plants with protein fragments complementation assays

Abstract

The present invention describes a method of expressing PCA interacting partners in plant material comprising: (A) transforming said material with: (1) a first construct coding for a first fusion product comprising (a) a first fragment of a first molecule whose fragments can exhibit a detectable activity when associated and (b) a first protein-protein interacting domain; and (2) a second construct coding for a second fusion product comprising (a) a second fragment of said first molecule and (b) a second protein-protein interacting domain that can bind (1)(b); (B) culturing said material under conditions allowing expression of said PCA interacting partners; and (C) detecting said activity.

1 - 6 . (canceled)  
     
     
         7 . A system for use as a standard or control in a PCA assay or for use in validating a PCA assay comprising: 
 (a) a first fusion product comprising a fragment of a first molecule whose fragments can exhibit a detectable activity when associated and a first protein-protein interacting domain; and    (b) a second fusion product compriging a second fragment of said first molecule and a second protein-protein interaction domain that interacts with said first protein-protein interaction domain.    
     
     
         8 . A system according to  claim 7  where said first and second protein-protein interaction domains are selected from the group consisting of: 
 1) NPR1+TGA2,    2) FKBP+FRB,    3) leucine zippers.    
     
     
         9 - 10 . (canceled)  
     
     
         12 . A plant transgenic for one or more genes, each independently selected from the group consisting of: 
 (A) 1 or more genes coding for 1 or more interacting partners able to participate in a PCA assay, and    (B) 1 more more genes which result, either directly or indirectly, in the presence of 1 or more interacting partners able to participate in a PCA assay.    
     
     
         13 . A plant according to  claim 12  where the plant is of the genus  Arabidopsis.    
     
     
         14 . A plant according to  claim 13  where the plant is  Arabidopsis thaliana.    
     
     
         15 . A plant according to  claim 12  where the interacting partners comprise one or more of a leucine zipper/reporter molecule fusion, a NPR1/reporter molecule fusion, a TGA2/reporter molecule fusion, a FKBP/reporter molecule fusion or, a FRB/reporter molecule fusion.  
     
     
         16 . A method of determining whether a mutated gene acts upstream in a pathway affecting an inducible interaction comprising performing a PCA assay in a mutated plant and correlating a change in PCA activity, relative to that measured in a non-mutated control plant, with the presence of one or more genes acting upstream in said pathway.  
     
     
         17 - 27 . (canceled)