Multiple pathways for Cre/lox-mediated recombination in plastids

Plastid transformation technology involves the insertion by homologous reco mbination and subsequent amplification of plastid transgenes to approximate to 10 000 genome copies per leaf cell. Selection of transformed genomes is achieved using a selectable antibiotic resistance marker that has no subse quent role in the transformed line. We report here a feasibility study in t he model plant tobacco, to test the heterologous Cre/lox recombination syst em for antibiotic marker gene removal from plastids. To study its efficienc y, a green fluorescent protein reporter gene activation assay was utilized that allowed visual observation of marker excision after delivery of Cre to plastids. Using a combination of in vivo fluorescence activation and molec ular assays, we show that transgene excision occurs completely from all pla stid genomes early in plant development. Selectable marker-free transplasto mic plants are obtained in the first seed generation, indicating a potentia l application of the Cre/lox system in plastid transformation technology. I n addition to the predicted transgene excision event, two alternative pathw ays of Cre-mediated recombination were also observed. In one alternative pa thway, the presence of Cre in plastids stimulated homologous recombination between a 117 bp transgene expression element and its cognate sequence in t he plastid genome. The other alternative pathway uncovered a plastid genome 'hot spot' of recombination composed of multiple direct repeats of a 5 bp sequence motif, which recombined with lox independent of sequence homology. Both recombination pathways result in plastid genome deletions. However, t he resultant plastid mutations are silent, and their study provides the fir st insights into tRNA accumulation and trans-splicing events in higher plan t plastids.