ORIGINAL ABSTRACT

A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50-fold less active. A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development. However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate. The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis. As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal-specific promoter and treating the mature plants with glyceryl glyphosate. A chromogenic phosphonate ester substrate, 5-bromo-4-chloro-indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene. The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed.