Targeted Gene Delivery into Various Plastids Mediated by Clustered Cell-Penetrating and Chloroplast-Targeting Peptides

The plastid is an organelle that functions as a cell factory to supply food and oxygen to the plant cell and is therefore a potential target for genetic engineering to acquire plants with novel photosynthetic traits or the ability to produce valuable biomolecules. Conventional plastid genome engineering technologies are laborious for the preparation of plant material, require expensive experimental instruments, and are time consuming for obtaining a transplastomic plant line that produces significant levels of the biomolecule of interest. Herein, a transient plastid transformation technique is presented using a peptide-based gene carrier. By formulating peptide/plasmid DNA complexes that combine the functions of both a cell-penetrating peptide and a chloroplast-targeting peptide, DNA molecules are translocated across the plant cell membrane and delivered to the plastid efficiently via vesicle formation and intracellular vesicle trafficking. A simple infiltration method enables the introduction of a complex solution into intact plants, and plastid-localized transgene expression is expeditiously observed in various types of plastids in differentiated cell types of several plants. The gene delivery technology thus provides a useful tool to rapidly engineer plastids in crop species.