Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco

Plastid transformation (transplastomic) technology has several potential ad vantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid nu mber and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and repr oductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate syntha se (EPSPS). Immunological, enzymatic and whole-plant assays were used to pr ove the efficacy of three different prokaryotic (Achromobacter, Agrobacteri um and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10 000-f old range of protein accumulation (to > 10% total soluble protein in leaves ). Plastid-expressed EPSPS could provide very high levels of glyphosate res istance, although levels of resistance in vegetative and reproductive tissu es differed depending on EPSPS accumulation levels, and correlated to the p lastid abundance in these tissues. Paradoxically, higher levels of plastid- expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration o f high-level glyphosate tolerance in vegetative and reproductive organs usi ng transplastomic technology provides a necessary step for transfer of this technology to other crop species.