Receptor/histidine kinase fusion constructs and uses thereof

Abstract

The present disclosure provides compositions and methods for sensing a target substance of interest in the environment and inducing gene expression in response thereto, useful for detection of biological and chemical agents and environmental pollutants.

What is claimed is: 
     
         1 . A fusion protein comprising a chemotactic receptor protein, a receptor involved in quorum sensing, or a receptor from a receptor histidine kinase operably linked at the A/D position to a histidine kinase protein, wherein the fusion protein comprises a kinase activation region. 
     
     
         2 . The fusion protein of  claim 1 , wherein the chemotactic receptor protein is Trg, Tar, Tap or Tsr. 
     
     
         3 . The fusion protein of  claim 1 , wherein the receptor involved in quorum sensing is the  Xylella  DSF receptor RpfC or the LuxPQ receptor LuxP. 
     
     
         4 . The fusion protein of  claim 1 , wherein the histidine kinase protein is PhoR or EnvZ. 
     
     
         5 . The fusion protein of  claim 1 , wherein the histidine kinase protein is a EnvZ/PhoR chimera. 
     
     
         6 . The fusion protein of  claim 1 , wherein the kinase activation region of the fusion protein has been engineered to restore inducible kinase activity or engineered to allow the interaction of maltose-bound maltose binding protein with the receptor to functionally activate kinase activity. 
     
     
         7 . The fusion protein of  claim 1 , wherein the histidine kinase protein is activated when the chemotactic receptor protein or the receptor involved in quorum sensing binds to a sensor protein bound to a target substance. 
     
     
         8 . The fusion protein of  claim 7 , wherein the target substance is a chemical agent, a heavy metal, a poison, a pollutant, a toxin, an herbicide, a polycyclic aromatic hydrocarbon, a benzene, a toluene, a xylene, a halogenated hydrocarbon, a steroid or other hormone, an explosive, or a degradation product of one of the foregoing compounds. 
     
     
         9 . The fusion protein of  claim 1 , further comprising a plasma membrane targeting signal sequence operably linked to an N-terminus of the chemotactic receptor protein or receptor involved in quorum sensing. 
     
     
         10 . A DNA construct comprising a nucleic acid segment that encodes the fusion protein of  claim 1 . 
     
     
         11 . The DNA construct of  claim 10 , wherein the nucleic acid segment is operably linked to a promoter. 
     
     
         12 . A transgenic plant comprising:
 a) a first DNA construct comprising a first plant operable promoter operably linked to a nucleic acid segment encoding a sensor protein, said protein comprising a secretory sequence for directing the protein to the extracellular space of a plant cell and a binding region specific for a target substance of interest, wherein said protein undergoes a conformational change when the target substance is bound;   b) a second DNA construct comprising a second plant operable promoter operably linked to a nucleic acid segment encoding a protein that comprises the following domains: a plasma membrane targeting signal sequence, an extracellular domain for binding the sensor protein, a transmembrane domain and a histidine kinase domain for phosphorylating a protein with nuclear shuttling or transcriptional activating functions, wherein the histidine kinase is activated when the sensor protein binds to the extracellular domain; and   c) a third DNA construct comprising a third plant operable promoter operably linked to a nucleic acid segment encoding a detectable marker or a response gene, wherein the third plant operative promoter is responsive to the transcriptional activator protein, and wherein the detectable marker is expressed when the external target substance of interest is bound to the sensor protein.   
     
     
         13 . The transgenic plant of  claim 12 , wherein the transmembrane domain and the histidine kinase domain of the second DNA construct are derived from one or more bacterial genes, and the membrane targeting signal sequence of the second DNA construct is derived from a plant gene. 
     
     
         14 . The transgenic plant of  claim 12 , wherein the detectable marker of the third DNA construct is a chlorophyll degradation enzyme or a functional fragment thereof. 
     
     
         15 . A method for detecting an external substance of interest, the method comprising:
 a) exposing the transgenic plant of  claim 12  to an external substance of interest; and   b) detecting a change resulting from expression of the detectable marker.