Expression of bar in the plastid genome confers herbicide resistance

Phosphinothricin (PPT) is the active component of a family of environmental ly safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinot hricin acetyltransferase, a detoxifying enzyme. We report here expression o f a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Hava na) plastids that confers field-level tolerance to Liberty, an herbicide co ntaining PPT. We also describe a second bacterial bar gene (b-bar2) and a c odon-optimized synthetic bar (s-bar) gene with significantly elevated level s of expression in plastids (>7% of total soluble cellular protein). Althou gh these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct select ion of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in cod on use. We propose to use codon usage differences as a precautionary measur e to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in t he plastid genome is an attractive alternative to incorporation in the nucl ear genome since there is no transmission of plastid-encoded genes via poll en.