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

Chonprakun Thagun,Jo-Ann Chuah,Keiji Numata

doi:10.1002/advs.201902064

Abstract

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.

Highlights

Introduction of Clustered Complexes into DifferentPlant Tissues: For leaf infiltration, ≈100 μL of complex solution containing 1.0 μg of plasmid DNA was introduced into the fully expanded leaves of A. thaliana and N. benthamiana from the abaxial side of the leaf using a 1 mL syringe without a needle
The analyses of free peptides in the Plasmid DNA (pDNA)/chloroplast-targeting peptide (CTP) complex solutions formed at N/P ratios = 0.5–5.0 revealed that up to 64% of CTP gradually bound to the pDNA molecules
This binding between pDNA and CTP is in pDNA concentration-dependent manner as indicated by the excessive amount of free CTP in the complex solutions formed at higher N/P ratios (Figure S1d,e, Supporting Information)

Summary

Introduction

Plant Tissues: For leaf infiltration, ≈100 μL of complex solution containing 1.0 μg of plasmid DNA was introduced into the fully expanded leaves of A. thaliana and N. benthamiana from the abaxial side of the leaf using a 1 mL syringe without a needle. Root segments were placed into 500 μL of complex solution containing 1.0 μg plasmid DNA 100 μL−1, and the solution was vacuum infiltrated into the plant tissue at 600 mmHg for 5 min. The root segments were incubated in the complex solution for 30 min before three washes with 1⁄2 Gamborg’s B5 media supplemented with 2% sucrose without agar. Transformed root segments were incubated in 1⁄2 Gamborg’s B5 media supplemented with 2% sucrose with 0.7% agar in the dark at 22 °C for 24 h

Results

Discussion

Conclusion