Engineering of the rpl23 gene cluster to replace the plastid RNA polymerase a subunit with the Escherichia coli homologue

The Escherichia coli RNA polymerase (RNAP) alpha, beta, and beta' core subu nits are evolutionarily conserved among bacteria and plastids, and the plas tid specificity factors form a functional holoenzyme with the E. coli core. To investigate whether the E. coli core subunits may form a functional hyb rid enzyme with the plastid core subunits, we replaced the tobacco plastid RNAP a subunit gene (rpoA) with the E. coli a subunit gene by targeted gene insertion. The transplastomic tobacco plants look similar to tobacco rpoA deletion mutants in that they are chlorophyll-deficient and non-photoautotr ophic. In addition, they lack transcripts from promoters recognized by the E. coli-like plastid RNA polymerase. These results indicate that evolutiona ry conservation between the E. coli and plastid RNA polymerase a subunits i s insufficient to allow substitution of the tobacco a subunit with its bact erial counterpart. Interestingly, the cyanobacterial cc subunits are as dif ferent as the E. coli a subunits: and therefore it is unlikely that replace ment of the tobacco cc subunit with cyanobacterial alpha subunits would yie ld a functional enzyme. Replacement of plastid rpoA With the E. coli RNA po lymerase alpha subunit gene represents the first engineering of a plastid o peron in higher plants.