Abstract
Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for gene delivery systems into animal and plant cells. A non-viral gene carrier containing a cell-penetrating peptide and a cationic sequence was previously developed for use in intact plants and plant cells; however, the permeation pathway of the gene carrier into plant cells is yet to be elucidated, which would facilitate the improvement of the gene delivery efficiency. Here, we identified the vacuum/compression infiltration-mediated permeation pathway of a non-viral gene carrier into plant tissues and cells using a complex of plasmid DNA and a peptide-based gene carrier. This complex was taken up via the hydathodes in Arabidopsis thaliana, and from root hairs in Nicotiana benthamiana. Remarkably, these structurally weak tissues are also routes of bacterial invasion in nature, suggesting that peptide-pDNA complexes invade intact plants through similar pathways as bacterial pathogens.
Highlights
Non-viral carriers, which can be used to transform any plant species without specialized equipment, are an effective alternative to overcome species-specific transformation problems
For the evaluation of the vacuum/compression infiltration-mediated permeation of the peptide-plasmid DNA (pDNA) complex into different tissues of plant seedlings, we prepared an ionic complex of pDNA encoding NanoLuc luciferase (Nluc) with Bp100(KH)[9] as a cell-penetrating peptides (CPPs)-containing fusion peptide
The peptide-pDNA complex was infiltrated into N. benthamiana and A. thaliana seedlings at seven days after germination (DAG) by degassing and pressurizing seedlings immersed in the complex solution
Summary
Non-viral carriers, which can be used to transform any plant species without specialized equipment, are an effective alternative to overcome species-specific transformation problems. Studies using cell-penetrating peptides (CPPs) as a non-viral carrier have been reported[10]. In our previous study, a fusion peptide consisting of a CPP and a cationic sequence interacting with a nucleic acid was used as a non-viral carrier to introduce nucleic acids into plant cells[16]. Confocal laser scanning microscopy (CLSM) revealed that the uptake of the complexes in A. thaliana mainly occurred via the hydathodes in the leaves, whereas the uptake in N. benthamiana occurred via the root hairs. These plant tissues are structurally weak regions that are targeted by bacterial invasion pathways. This research provides a new insight into the relationship between bacterial infection and non-viral gene delivery systems in plant cells, and could facilitate the enhancement of the transformation efficiency of various plant species
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