Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle‐Complex‐Mediated DNA Delivery to Specific Plant Organelles

Abstract Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle‐targeted DNA delivery in plants. However, nanocarrier‐mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall‐loosening zwitterionic liquid (ZIL) with a peptide‐displaying micelle complex for organelle‐specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier‐mediated cargo delivery to specific organelles in living plants.


Fig. S1
Effects of ZIL and ILs on plant growth. p. S8

Fig. S2
AFM height images of cellulose microcrystals pretreated with various concentrations of ZIL.

Fig. S3
Effects of ZIL pretreatment on A. thaliana cotyledon cell walls examined by electron microscopy.

Fig. S5
Comparison of peptide-displaying micelle complexes (MCs) in terms of the transfection efficiency.

Fig. S6
Effects of ZIL on hydrodynamic diameters of CPP-MC determined by DLS measurements.

Fig. S8
Time course evaluation of CPP-MC-mediated nuclear GFP expression in A. thaliana cotyledons.

Fig. S9
CLSM images showing GFP expression in roots 24 h after CPP-MC-mediated transfection with or without ZIL.

Fig. S10
Validation of CPP-MC-mediated GFP expression in A. thaliana seedlings.

Fig. S11
Time course evaluation of Nluc-based transfection efficiency of CPP-MC in A. thaliana cotyledons.

Fig. S12
Comparison of transfection efficiency of CPP-MC between shoots and roots from A. thaliana seedlings with or without ZIL pretreatment.

Fig. S14
Effects of ZIL on CPP-MC-mediated transfection in mature plant leaves.

Fig. S16
Effects of ZIL on hydrodynamic diameters of CTP/CPP-MC determined by DLS measurements.

Fig. S19
Validation of chloroplast-specific GFP expression in the transfected A. thaliana seedlings.
Mature plants at 10 weeks after germination were also used for transfection experiments.

Synthesis and characterization of zwitterionic liquid (ZIL)
ZIL was synthesized by a modified protocol according to the previous report. [  medium plate, the viability of the seedlings was determined by the previously reported Evans blue assay. [ 1 ] Data were obtained from four biologically independent samples (each sample consisted of five infiltrated seedlings). The seedlings were photographed at 1, 10, and 20 days after treatment.

Evaluation of the ability of ZIL to dissolve cellulose microcrystals by wide-angle X-ray diffraction (WAXD) and atomic force microscopy (AFM) analyses
The synchrotron WAXD measurements of cellulose nanocrystal pellets were performed similarly as previously reported. in an epoxy resin block as previously described. [ 5 ] The serial ultrathin sections (70 nm and 100 nm per section for TEM and FE-SEM, respectively) were obtained from the resin blocks and stained with an uranyl acetate solution for 10 min and a lead citrate solution for 1 min. The samples were observed on a JEM 1400 Flash transmission electron microscope (JEOL) at 80 kV or a SU8220 scanning electron microscope (Hitachi) at acceleration voltage of 5.0 kV.

Quantitative evaluation of cell wall permeability by quenching assay
To quantitatively evaluate cell wall permeability, we performed a quenching assay reported by Liu et al. [ 6 ] The quenching assay was based on CLSM imaging and image analysis.

Preparation and characterization of peptide-displaying micelle complexes
The CPP-modified micelle complex (CPP-MC) was prepared as previously reported. analysis as previously described. [2] Briefly, the GFP band on the membrane was detected using a combination of a rabbit anti-GFP polyclonal antibody (NB600-308, Novus Biologicals) with a horseradish peroxidase (HRP)-conjugated goat antirabbit IgG polyclonal antibody (ab6721, Abcam), and imaged with a Fusion Solo

Statistical analysis
Statistical tests and graphs were generated with GraphPad Prism 9. Figure S1. Effects of ZIL and ILs on plant growth. Pictures of A. thaliana seedlings at 1, 10, and 20 days after pretreatment with ZIL, IL-1, IL-2, and water for 3 h. Scale bar, 10 mm.