Fig. 1.

Recombination-induced knockout of plastid translation (RIKT). (A) Region of the tobacco plastid genome from which the chloroplast transformation vector pRB115 was derived. (B) Physical map of the plastid targeting region in transformation vector pRB115 (cloned as SacI/KpnI fragment into pBluescript). Note that the selectable marker gene aadA is flanked by only one region of homology with the plastid genome. Restriction sites lost because of ligation of heterologous ends are shown in parentheses. Sites from the pBluescript (pBS) polylinker are underlined. Prrn, promoter derived from the plastid rRNA operon; TpsbA, 3′ UTR derived from the psbA gene. (C) Cointegrate produced by plastid transformation with pRB115. A single homologous recombination event (indicated by the dotted arrow between the two maps in A and B) results in incorporation of the entire plasmid vector into the plastid genome. The cointegrate is unstable, and homologous recombination in the directly repeated psaA/psaB/rps14/trnfM region will recreate the wild-type genome and eliminate the aadA-resistance gene. In the presence of the translational inhibitor spectinomycin, cell lines that have completely lost the aadA as the result of such recombination events will no longer produce plastid-encoded proteins. pBS, pBluescript backbone (not drawn to scale).