Engineering a root‐specific, repressor‐operator gene complex

Summary Strong, tissue‐specific and genetically regulated expression systems are essential tools in plant biotechnology. An expression system tool called a ‘repressor‐operator gene complex’ (ROC) has diverse applications in plant biotechnology fields including phytoremediation, disease resistance, plant nutrition, food safety, and hybrid seed production. To test this concept, we assembled a root‐specific ROC using a strategy that could be used to construct almost any gene expression pattern. When a modified E. coli lac repressor with a nuclear localization signal was expressed from a rubisco small subunit expression vector, S1pt::lacIn , LacIn protein was localized to the nuclei of leaf and stem cells, but not to root cells. A LacIn repressible Arabidopsis actin expression vector A2pot was assembled containing upstream bacterial lacO operator sequences, and it was tested for organ and tissue specificity using β‐glucuronidase ( GUS ) and mercuric ion reductase ( merA ) gene reporters. Strong GUS enzyme expression was restricted to root tissues of A2pot::GUS/S1pt::lacIn ROC plants, while GUS activity was high in all vegetative tissues of plants lacking the repressor. Repression of shoot GUS expression exceeded 99.9% with no evidence of root repression, among a large percentage of doubly transformed plants. Similarly, MerA was strongly expressed in the roots, but not the shoots of A2pot::merA/S1pt::lacIn plants, while MerA levels remained high in both shoots and roots of plants lacking repressor. Plants with MerA expression restricted to roots were approximately as tolerant to ionic mercury as plants constitutively expressing MerA in roots and shoots. The superiority of this ROC over the previously described root‐specific tobacco RB7 promoter is demonstrated.