Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

Abstract

Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide–DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.

Will epigenetics be a key player in crop breeding?

“…Alternatively, transient expression or external application of epigenome modifying transgene or proteins may be delivered to the plant for epigenome modification without transgene integration into the genome. Cell penetrating peptide is one such method that allows DNA/RNA/proteins bound to a designed peptide to transfer across the cell wall/membrane via endocytosis (Numata et al, 2018;Oikawa et al, 2021). Transfection of preassembled Cas9/sgRNA complex has been demonstrated (Woo et al, 2015;Murovec et al, 2018), and cell penetrating peptide has efficiently delivered proteins into plant cells (Bilichak et al, 2015;Terada et al, 2020).…”

Organelle-targeted gene delivery in plants by nanomaterials

“…The former mechanism has been proposed for highly charged NMs, while the latter has been frequently observed for peptide-based complexes. 52,54,[101][102][103][104] For gene delivery into intact plants, the Numata group has developed the use of peptide-based NMs to specifically deliver nucleic acid cargo into the nuclei of intact plants. 52,70,73 To facilitate access into the plant cells, most peptides contain a CPP or CPP-like domain and are either coincubated or fused with another peptide to facilitate cargo complexation (Fig.…”

“…The former mechanism has been proposed for highly charged NMs, while the latter has been frequently observed for peptide-based complexes. 52,54,101–104…”

Cell-Penetrating Peptides for Use in Development of Transgenic Plants

“…Further, they used the above complexes as nanocarriers, transfecting the plasmid DNA into chloroplasts after spraying on leaf surfaces [101]. The CTP/DNA complexes were transported from the extracellular space to the chloroplast stroma in Arabidopsis leaves [79]. These studies on the CPP(CTP)/DNA complex system dramatically enhanced the transgenesis without protoplast preparations nor callus formations, and provided a useful tool for rapid and effective plastids engineering in plants.…”