Mechanisms for introducing 250 kDa fluorescent molecules and Cas9/sgRNA into plant cells by plasma treatment

Plant cell walls prevent molecules with high molecular weights from reaching the cell membrane, challenging genome editing in plants. To overcome this challenge, the microplasma method, established as a gene and molecule transfection technology in animal cells, was investigated in tobacco plants. We found that plasma treatment of tobacco leaves and calluses introduced fluorescent molecules into epidermal and callus cells. Scanning electron microscopy revealed that plasma treatment decomposed the cuticula layer on the surface of tobacco leaves and that plasma treatment decomposed the extracellular matrix and caused cracks on the cell wall surface of tobacco callus. These results suggest that when external molecules are introduced into plant cells by plasma treatment, the external molecules’ transport pathway reaches the cell membrane by degradation of the cuticula layer and extracellular matrix. Additionally, the introduction of molecules by plasma treatment was inhibited by an endocytosis inhibitor, indicating that plasma stimulation induces endocytosis. In summary, plasma treatment decomposes the cuticula layer and cellular interstitium, allowing molecules to reach the cell membrane, after which they are introduced into the cell via endocytosis.


Introduction
Plant diseases have become an increasingly pressing issue in recent years, with wheat diseases such as black rust causing extensive damage in parts of Africa and the Middle East and epidemics of yellow rust and rice blast also occurring. Developing disease-resistant varieties is therefore crucial from the perspective of crop disease control and food security. However, conventional breeding methods such as selective breeding of naturally occurring varieties and crossbreeding of different varieties are time-consuming and unsuitable for combating acute plant diseases. Transgenic breeding, a new method in which the target gene is introduced from another plant, has become popular owing to the establishment of the Agrobacterium method 1) for introducing genes into plant cells. In 1996, the global area of genetically modified crops (GMOs) was 1.7 million hectares, and herbicide-tolerant soybeans, pest-resistant corn, and potatoes had already been commercially cultivated. 2,3) In 2019, GMO cultivation had increased to 190.4 million hectares. However, consumer concerns about GMOs' safety and stringent approval screening requirements have become challenges to their widespread use. Therefore, genome-editing technology is being explored as a promising alternative to transgenic breeding.
Genome-editing, unlike genetic modification, changes the genes that plants naturally possess, making it a safer breeding method. It involves deleting, replacing, or inserting any part of the genome sequence by cutting double-stranded DNA [4][5][6] using a genome-editing tool such as ribonucleoprotein (RNP), a complex of sgRNA, which binds to the target genomic region, and Cas9 protein, which causes doublestrand breaks in DNA. However, this technology is still at the basic research stage in agriculture, 7) mainly because plant cells have a cell wall that prevents molecules with molecular weights of several hundred kilodaltons or more, such as RNPs, from reaching the cell membrane. The current introduction method, particle bombardment, [8][9][10] is invasive and has low efficacy, necessitating the development of more efficient and minimally invasive techniques for molecular introduction into plant cells.
Research on atmospheric pressure non-thermal equilibrium plasma in the agricultural field has attracted much attention recently. In particular, many studies have been reported on the plasma treatment of seeds to promote germination. Early studies suggested that changes in seed water absorption due to plasma treatment affected germination. [11][12][13] However, gene expression and phytohormone content analysis recently revealed that plasma affects germination-related factors. [14][15][16][17][18] On the other hand, due to the growing expectation for plant breeding by genome editing, molecular transfer into plant cells by plasma treatment has also been reported. Our group has established the microplasma method as a gene and molecule transfection technology in animal cells, [19][20][21][22][23][24][25][26] and its application in plant cells is under investigation. Yanagawa et al. reported successfully introducing molecules into plant cells by treating them with a plasma jet. 27) They also report the successful introduction of RNP into plant cells for genome editing. 28) However, the introduction mechanism has not been clarified in those reports. In addition, genome editing is only the result of expression analysis by PCR, and there is no fluorescent image of the plants with actual GFP expression, which poses problems in the efficiency of introduction and the success rate of genome editing. In this study, we utilized microplasma treatment to introduce RNPs into tobacco (Nicotiana benthamiana, Nicotiana tabacum) leaves and calluses, using a fluorescent molecule of almost the same molecular size as RNP. We also tried to introduce RNPs into tobacco leaves. As a result, we observed GFP fluorescence, which is thought to be caused by genome editing by the introduced RNPs. Furthermore, we observed the surface morphology of plasma-treated calluses using a field-emission scanning electron microscope and found that the cell wall surface structure had changed to allow proteins to reach the cell membrane. We also confirmed that the inhibition of endocytosis, [22][23][24] a cellular mechanism responsible for spontaneous membrane trafficking, prevented the introduction of molecules. Figure 1 schematically illustrates the plasma system comprising a high-voltage amplifier (Trek: PD05034) for highvoltage sine wave generation and a plasma generator. The input to the high-voltage amplifier is a signal generated by a function generator (Agilent: 33220 A). The plasma generator has a Fe/Ni-plated needle electrode with a length of 26 mm and an outer diameter of 0.73 mm for high-voltage application. To ensure uniform contact between the tobacco leaf and the ground (GND) electrode, a conductive gel pad (Nippon Medical Next: Thermoguard 51-7810) was attached to the tobacco leaf as a counter-electrode during plasma treatment. For treating the tobacco callus, a copper plate served as the GND electrode, and the callus was placed on this plate at the center of a 3.5 cm dish. The distance between the needle electrode and the treated sample was 1 mm.

Experimental methods
Tobacco leaves were grown for approximately 4 weeks in an artificial weather chamber with a 16/8 h day/night cycle after germination. FITC-dextran (Sigma Aldrich: FD2000S-250MG), a fluorescent substance that emits green fluorescence at 518 nm upon exposure to blue light excitation at 492 nm, was used as an introductory substance. Specifically, 4 μl of a solution containing 10 μM FITC-dextran with 0.1% surfactant (Momentive: SILWET L-77) was dropped onto the plasma-treated tobacco leaves. After 60 min of incubation, the leaves were washed for 40 min in running water to remove FITC-dextran remaining on the leaf surface. After washing, the leaves were cut into approximately 10 × 10 mm sections centered on the plasma-treated area and observed under a macro fluorescence microscope (Olympus: MVX10).

Introduction of molecules into tobacco leaf cells
The tobacco leaf surface was treated with plasma. As experimental conditions, the power supply voltage was set to 11.0 kV, and the sample was treated for 25 ms. The treatment time was set to the minimum as far as the introduction was confirmed. Since the discharge voltage has a probability distribution and conditions such as the shape are different for each sample, the actual discharge voltage fluctuates by about ±0.5 kV with respect to the set voltage. FITC-dextran was used as the introductory substance with a molecular weight of 2 MDa. Figures 2(a) and 2(b) show dark-field fluorescence images of tobacco leaf epidermal cells treated with drops of FITC-dextran solution with and without (control) plasma treatment, respectively. The plasma-treated sample exhibited puzzle piece-shaped areas emitting green fluorescence. In contrast, the untreated control sample fluoresced green only at the edges of the epidermal cells, suggesting that FITC-dextran had entered the epidermal cells of the plasma-treated leaf but was only surface-attached in the control leaf.

Introduction of molecules into the tobacco callus
The tobacco callus was treated with plasma. As experimental conditions, the power supply voltage was set to 11.0 kV and the sample was treated for 12 ms. The treatment time was set to the minimum as far as the introduction was confirmed. Due to the same reason as the case of tobacco leaf, the discharge voltage fluctuates by about ±0.5 kV with respect to the set voltage. FITC-dextran was used as the introductory substance with a molecular weight of 250 kDa. Figures 3(a) and 3(b) show darkfield fluorescence images of tobacco calluses treated with drops of FITC-dextran solution with and without (control) plasma treatment, respectively. The plasma-treated sample emitted green fluorescence throughout the imaging area, with particularly strong fluorescence in the cell-like particle regions. In contrast, the control sample showed almost no green fluorescence. These findings suggest that FITC-dextran was present inside the callus cells of plasma-treated samples, whereas control samples only exhibited surface attachment.
Next, we attempted genome editing in the tobacco callus by introducing RNPs (a complex of NLS-SpCas9 and sgRNA). Transgenic tobacco callus harboring the GFP gene was used in this experiment. This transgenic tobacco callus does not usually express GFP protein, however, when a mutation is introduced by genome editing, it becomes possible to express GFP protein. Therefore, transgenic tobacco callus with RNPs introduced by plasma treatment will show GFP fluorescence upon successful genome editing. After plasma treatment at an applied voltage of 11 kVpp for 12 ms in the treatment group, the RNP solution was dropped onto the callus and incubated for one day before fluorescence was observed. Figures 4(a) and 4(b) show dark-field fluorescence images of calluses treated with drops of RNP solution with and without (control) plasma treatment, respectively. The plasma-treated sample showed strong green fluorescence in the cell-like particle region, suggesting

Inhibition of endocytosis
The confirm the contribution of clathrin-dependent endocytosis (CME) as a spontaneous membrane trafficking mechanism of cells, ES9-17 (Sigma Aldrich: SML2712) was used as a CME inhibitor. 29) Before plasma treatment, the tobacco callus was immersed in a 30 μM ES9-17 solution for 30 min, and FITCdextran fluorescent molecules (Sigma Aldrich: FD250S) were used as introductory substances. Figures 5(a) and 5(b) show fluorescence images without and with endocytosis inhibition treatment, respectively. Without endocytosis inhibition treatment, the green fluorescence of FITC-dextran was observed, indicating the introduction of FITC-dextran into the cell [ Fig. 5(a)]. However, with endocytosis inhibition treatment, no green fluorescence was observed [ Fig. 5(b)]. These findings indicate that endocytosis is the route of plasma treatmentmediated molecule introduction into the tobacco callus.

Cell surface observation
In the treatment group, tobacco leaves were treated with plasma at an applied voltage of 15 kVpp for 5 ms, followed by t-butanol lyophilization for scanning electron microscopy (SEM) observation 30) with a field-emission SEM (JEOL: JSM-7500F). Figures 6(a) and 6(b) show SEM images of samples with and without (control) plasma treatment, respectively. The sedimentary layer, i.e. the cuticular layer, on the cell surface was thinner in the plasma-treated sample than in the control sample, with exposed veins observed in the former. The cuticular layer is hydrophobic and prevents the penetration of molecules from the outside, and the reduction in its thickness suggests that FITC-dextran may have passed through it and reached the epidermal cells.
In the treatment group, the tobacco callus was treated with plasma at an applied voltage of 11 kVpp for 12 ms, followed by t-butanol lyophilization for SEM observation 30) with a field-emission SEM (Hitachi: S-4800). Figures 7(a) and 7(b) show SEM images of samples with and without (control) plasma treatment. The large grains in the 1000× images are single cells in the callus. In the plasma-treated samples, particles were agglomerated and bonded, creating gaps where no particles were present, some of which were tens to hundreds of nanometers in size. These gaps may have allowed FITC-dextran and RNPs to pass through the cell wall and reach the vicinity of the plasma membrane.

Discussion
Plasma treatment of tobacco leaves and calluses resulted in reduced cuticular layer thickness and the formation of gaps in the cell wall surface, which were considered the effects of plasma treatment because they were not observed in untreated control samples. In this section, we discuss the mechanism of molecular introduction by plasma treatment.
First, we focus on the decomposition mechanism of cuticula layer and cell wall. SEM observations of the tobacco leaf surface show the reduction of the cuticula layer by plasma treatment. Cutin, which constitutes the cuticula layer, forms polymers through ester bonds. The main chains of polymers, The Japan Society of Applied Physics by IOP Publishing Ltd such as cutin, which constitute the cuticula layer, can be decomposed by the radicals generated by a plasma. This breaking of the chains may lead to the decomposition reaction of the cuticula layer. Plant cell walls consist of a thin primary cell wall and a thick secondary cell wall, and the cell wall of the callus is mainly composed of the primary cell wall, which consists of cellulose, hemicellulose, and pectin. Cellulose has a crystallized microfiber structure attached to hemicellulose, forming cross-links between the cellulose. Pectin, a complex polysaccharide, fills the gaps between microfibers and hemicellulose. 31) In the present study, no microfiber structure was observed using SEM, so the particle layer in which gaps were produced was likely a pectin layer. Homogalacturonan, the major domain of pectin, maintains its cell wall structure through ion binding of non-methyl esterified carboxyl groups to divalent ions such as Ca 2+ ions (Fig. 8). Plasma treatment produces various chemically active species, including H 2 O 2 , which contributes to the introduction of molecules. The presence of OH radicals, precursors of H 2 O 2 , suggests that plasma also produces OH radicals that bind to pectin and cause a decomposition reaction that cleaves the sugar chain, forming cavities on the cell wall surface. 32) This suggests that in our system, the pectin is also decomposed by OH radicals. On the other hand, the joule heating by the electric current can be another candidate for pectin decomposition. To reveal which one is the main mechanism is our future task. Next, we focus on the mechanism of cell membrane permeation by plasma treatment. The mechanism by which plasma treatment introduced molecules into plant cells is shown schematically in Fig. 9. First, OH radicals generated primarily by the microplasma partially decompose the cell wall. As a result, gaps of tens to hundreds of nanometers are formed in the cell wall (Fig. 7), allowing molecules to pass through the cell wall and approach the cell membrane. Molecules that reach the cell membrane are internalized by clathrin-dependent endocytosis, a spontaneous membrane trafficking mechanism of the cell. We have previously reported that a combination of electrical factors, such as plasma-generated current and charge, and chemical factors, such as H 2 O 2 and OH radicals, induces endocytosis in animal cells. [20][21][22][23][24] In this study, using the same plasma treatment system as in these animal cell studies, we found that plasma-derived electrical and chemical factors also stimulate plant cells and that clathrin-dependent endocytosis is induced in plant cells as in animal cells. In animal cells, endocytosis, including caveolae-dependent endocytosis, lipid raft endocytosis, and clathrin-dependent endocytosis, accounts  for approximately 80% of molecular introduction. 19) Endocytosis inhibition experiments revealed that ES9-17 inhibited almost 100% of molecular introduction into tobacco calluses. Therefore, the pathway of molecular introduction into tobacco calluses via plasma treatment is almost exclusively through clathrin-dependent endocytosis.

Conclusions
In this study, tobacco leaves and callus were treated with plasma, and the results indicate successful FITC-dextran introduction into tobacco cells and suggest that genome editing could be done in tobacco. SEM observations confirmed that the plasma treatment reduced the cuticular layers and the generated gaps in the cell wall surface. The introduced molecules could pass through the gaps in the cell wall and reach the cell membrane, where they were internalized via clathrin-dependent endocytosis. Inhibition experiments showed that almost 100% of plasma treatment-mediated molecular introduction into the tobacco callus occurred through clathrin-dependent endocytosis.