Plastids are semi-autonomous organelles with a relatively small (120-180 kb), highly polyploid genome present in 1,000 to 10,000 copies per cell. The best-known plastids, chloroplasts, convert sunlight into chemical energy. Plastid engineering, in contrast to nuclear engineering, offers higher protein yields, the opportunity to express several genes controlling complex traits, and natural containment that prevents transgene flow via pollen. During the past twenty years we developed protocols for transformation of the tobacco (Nicotiana tabacum) plastid genome, efficient post-transformation excision of the marker genes, and high-level expression of recombinant proteins. Patents based on research from our laboratory cover all aspects of the genetic manipulation of plastid genomes.
Currently, we pursue research in the following areas:
Most nuclear-encoded pentatricopeptide repeat (PPR) proteins are targeted to plastids and mitochondria, and bind specific RNA sequences via a modular recognition mechanism. Study of plant mutants suggests that the P-type PPR proteins are involved in regulating mRNA translation and stability. Our objective is to explore the feasibility of regulating plastid transgene expression by incorporating PPR binding sites in plastid transgenes and regulating the expression of cognate PPR proteins from nuclear transgenes.
Arabidopsis thaliana, an important model plant species, is recalcitrant to plastid transformation. To enable early identification of transplastomic events, we developed a novel marker system that is selectively expressed in chloroplasts. Plastid transformation in Arabidopsis in the appropriate genetic background is now as efficient as in tobacco. We now conduct systematic experimentation to tie shoot regeneration into a streamlined seed production pipeline as part of the refinement of the Arabidopsis plastid transformation protocol. Plastid genome engineering will open up the unique genomic resources of Arabidopsis for studies of plastid-nucleus interactions and for improving crop productivity by engineering the photosynthetic machinery.
To detect organelle movement between cells, we graft two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. We initiate tissue culture from sliced graft junctions and select for clonal lines in which the nuclear gentamycin resistance marker of one line is combined with the plastid-encoded spectinomycin resistance marker of the second. We obtained evidence for cell-to-cell movement of the entire 161-kb plastid genome in the absence of the movement of chromosomes or mitochondrial DNA. In some of the clones, mitochondrial DNA movement was also detected by restoration of pollen fertility in the cytoplasmic male sterile (CMS) graft partner. Homologous recombination yielded fertile and sterile mitochondrial genomes due to recombination at alternative sites, linking CMS to a unique open reading frame in CMS mitochondria. We are now interested in stripping plastid genomes from species-specific features so that the same engineered plastid genome can be utilized in multiple hosts.