Site-specific recombinases to manipulate the plastid genome

The plastid genome (ptDNA) of higher plants is highly polyploid and the 1,000 to 10,000 copies are compartmentalized in up to ~100 plastids per cell. I report here two different site-specific recombinases to manipulate the tobacco plastid genome, the P1 phage Cre recombinase and the phiC31 phage Integrase (Int). Thus far plastid transformation in higher plants was based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. I describe here an alternative approach that relies on integration of foreign DNA by Int mediating recombination between bacterial attB and phage attP sequences. During the plastid transformation protocol marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable once homoplastomic plants are obtained. I describe plastid marker gene excision with both the Cre-loxP and the Int-att site-specific recombinases. The Cre-loxP approach uses a transiently expressed Cre, a novel protocol that enables rapid removal of marker genes from the ~10,000 plastid genome copies without transformation of the plant nucleus. The Int-att system uses a nuclear-encoded plastid-targeted Int to efficiently excise plastid marker genes flanked by directly oriented attB and attP sites. Lastly, I describe use of the Int-att system to study plastid sorting with the conclusion that a newly arising minor plastid DNA type can get established in a developing tobacco shoot and transmitted to the seed progeny.Ph.D.Includes bibliographical references