Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide–DNA Complexes

Abstract

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

A centrifugation-assisted peptide-mediated gene transfer method for high-throughput analyses

“…CPPs can serve as a cargo carrier for macromolecules across cellular membranes into cells (Numata 2015;Rádis-Baptista et al 2017). Additionally, we succeeded in the selective delivery of plasmid DNA into chloroplasts or mitochondria using polycationic peptides with chloroplast or mitochondrial targeting signals (Chuah et al 2015;Yoshizumi et al 2018). Yoshizumi et al developed a syringe-based vacuum system for efficient delivery of peptide-DNA complexes into plant cells (Yoshizumi et al 2018).…”

“…Additionally, we succeeded in the selective delivery of plasmid DNA into chloroplasts or mitochondria using polycationic peptides with chloroplast or mitochondrial targeting signals (Chuah et al 2015;Yoshizumi et al 2018). Yoshizumi et al developed a syringe-based vacuum system for efficient delivery of peptide-DNA complexes into plant cells (Yoshizumi et al 2018). However, the drawbacks of this system include its low throughput and the dependence of gene transfer efficiency on the experimenter's ability.…”

“…Sterilized seeds of tobacco were placed on germination (GEM) medium composed of onehalf Murashige-Skoog medium (Murashige and Skoog 1962) with 10 g L −1 sucrose and 2.5 g L −1 Phytagel. For gene transfer efficiency, a (KH) 9 -BP100 fusion peptide (Amino acid sequence: KHKHKHKHKHKHKHKHKHKKLFKKILKYL) (Lakshmanan et al 2013(Lakshmanan et al , 2015) and a CaMV 35S promoter-NanoLuc Luciferase construct (pGWB-Nluc) (England et al 2016) were mixed by the method described previously (Yoshizumi et al 2018). The detached cotyledons of 7-day-old seedlings were added to the peptide-DNA complex solutions and were centrifuged at various g-forces and times by a Tomy MX-307 centrifuge (Tomy Seiko Co., Tokyo, Japan).…”

“…The luciferase activities were evaluated in the relative light units (RLU) per mg of proteins. In a syringe-based vacuum system, the detached cotyledons of 7-day-old tobacco seedlings were transformed using the method described previously (Yoshizumi et al 2018). For shoot induction efficiency, the detached cotyledons of 7-day-old tobacco seedlings were placed on revised medium for organogenesis (shoot regeneration) of Nicotiana plumbaginifolia (RMOP) medium and transferred to fresh RMOP medium every 2 weeks (Svab and Maliga 1993).…”

“…CAPT increases the efficiency of gene transfer over the syringe-based vacuum system in tobacco. For CAPT, the cotyledon segments of 7-day-old seedlings were transformed with a DNA-peptide complex harboring a luciferase construct by centrifugation at 10,000×g for 60 s. The syringe-based vacuum system was performed as described previously (Yoshizumi et al 2018). The bars represent the means±SE (n=23).…”