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

Abstract

). ² These authors contributed equally to this work. SummaryPlastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodi®ed prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the ef®cacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the ef®cacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identi®ed translational control sequences that direct a 10 000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for ef®cacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

“…For example, integrating the cry genes into the chloroplast genome generated plants that were insecticidal to Bacillus thuringiensis (Bt)-sensitive [15] and highly resistant insects [16]. Chloroplasts have also been engineered recently to generate plants tolerant to bacterial and fungal diseases [17], drought [8] or herbicides [2,[18][19][20]. Transgenes recently engineered via the chloroplast genome are listed in Table 2.…”

“…Recently, the Agrobacterium EPSPS gene (C4) was expressed in tobacco plastids and resulted in 250-fold higher levels of the glyphosate-resistant C4 protein than were achieved via nuclear transformation [19]. Even though C4 expression in plastids was enhanced more than nuclear expression levels, field tolerance to glyphosate remained the same, showing that higher levels of expression do not always proportionately increase herbicide tolerance.…”

“…For example, the bacteriophage T7 gene10 leader sequence (G10L), which enhances translation of foreign genes in bacteria, has the same effect in chloroplasts [7,19,21,45]. Accumulation of a herbicide-tolerant EPSPS protein was increased 200-fold when its chloroplast transgene included the G10L sequence [19].…”

“…Accumulation of a herbicide-tolerant EPSPS protein was increased 200-fold when its chloroplast transgene included the G10L sequence [19]. This translational enhancement was attributed to complementarity between the G10L ribosome-binding site (rbs) and the anti-rbs on chloroplast 16S rRNA [7,19]. However, complementarity of a foreign transcript to 16S rRNA is not always beneficial.…”

“…Furthermore, silent mutations in the fused N-terminal coding sequences were found to decrease reporter protein accumulation without influencing RNA level [46]. In a separate study, fusion of sequences encoding the 14 N-terminal amino acids of GFP to a synthetic EPSPS gene increased EPSPS protein accumulation 33-fold without influencing transcript levels [19]. Therefore, when designing foreign genes for maximal expression in chloroplasts, one must optimize not only 5′UTR elements but also sequences downstream of the initiation codon.…”