Rapid and Efficient Gene Delivery into Plant Cells Using Designed

Dec 6, 2012 - To develop a new easy and quick gene delivery system for any types of plants, we prepared ionic complexes of plasmid DNA with designed p...
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Rapid and Efficient Gene Delivery into Plant Cells Using Designed Peptide Carriers Manoj Lakshmanan,†,‡ Yutaka Kodama,§ Takeshi Yoshizumi,∥ Kumar Sudesh,‡ and Keiji Numata*,† †

Enzyme Research Team, RIKEN Biomass Engineering Program, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Ecobiomaterial Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia § Center for Bioscience Research and Education, Utsunomiya University, 350 mine-machi, Utsunomiya, Tochigi 321-8505, Japan ∥ Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017, Japan ‡

S Supporting Information *

ABSTRACT: To develop a new easy and quick gene delivery system for any types of plants, we prepared ionic complexes of plasmid DNA with designed peptide carriers, each of which combined a cell-penetrating peptide (Bp100 or Tat2) with a polycation (nona-arginine or a copolymer of histidine and lysine). The present system via the designed peptides demonstrated rapid and efficient transient transfections into intact leaf cells of Nicotiana benthamiana and Arabidopsis thaliana without protoplast preparations. The designed peptides demonstrated significantly higher transfection efficiency in comparison to the nonfusion peptides (Bp100, Tat2, nona-arginine, and copolymer of histidine and lysine), indicating that the combination of functional peptides was a key to develop an efficient peptide-based gene delivery system. On the basis of the results, we exhibited the versatility of the designed peptide-based gene delivery system, which will explore the application of plant biotechnology.

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INTRODUCTION Plant gene delivery, which is the introduction of exogenous genes into plant cells, has proven to be invaluable in a wide variety of applications for basic plant science and plant biotechnology.1 Although the Agrobacterium-mediated method2 and particle bombardment3 are currently practical plant transformations, they still have several critical limitations, including the requirement of expensive equipment, risk of gene damage, low transformation efficiency, limitation of transgene sizes, and the limitation of applicable plant types (species and/or tissues).4 Thus, new gene delivery systems that are facile and widely applicable to all types of plants and genes are needed to develop industrial and basic plant biotechnology. Peptide-based gene delivery systems have been studied and have received much attention as potential new gene carriers for animal cells.5,6 For plant cells, however, this new technology is still in its early stage, with several studies reporting the use of cell-penetrating peptides (CPPs) to deliver plasmid DNA (pDNA) into permeabilized wheat embryo,7 mung bean and soy bean roots,8 and others using double-stranded RNA to induce post-transcriptional gene silencing in tobacco suspension cells.9 The potential advantages of the peptide-based gene delivery system are that, unlike Agrobacterium-mediated delivery, it would not be limited with respect to the plant types to be transformed, or with respect to transgene sizes. On the other hand, the permeability and transfection efficiency of the peptide-based gene carrier through the cell walls have not been adequately quantified. © 2012 American Chemical Society

We have previously demonstrated that fusion peptides combining silk and a CPP are effective in animal cells both in vivo and in vitro.10,11 Also, greater presence of functional peptides such as tumor-homing peptide at the surface of the pDNA−peptide complexes was reported to increase transfection efficiency, indicating that the amount of functional peptide at the surface of the complexes is critical for transfection efficiency of gene delivery systems.12 Therefore, we designed the fusion peptides of a CPP and polycation to enhance the presence of CPP at the surface of the complexes, which leads to higher transfection efficiency. In this design, pDNA preferentially interacts with polycation and is condensed via ionic interactions, while CPP, which interacts less pDNA, functions as CPP. On the basis of the success in animal cells, in this study, three fusion peptides that combined a polycationic peptide, i.e., nona-arginine (R9)13 or a copolymer of histidine and lysine, (KH) 9 (18 a.a.), 14 with a CPP, Bp100 (KKLFKKILKYL)15 or Tat2 (RKKRRQRRRRKKRRQRRR),16 were designed for plant gene delivery. The CPP, Bp100, was originally designed and optimized as an antimicrobial peptide against plant pathogens.15 Tat2, on the other hand, is a dimer peptide of the HIV-1 Tat basic domain (RKKRRQRRR)16 and was reported to internalize triticale mesophyll protoplasts and deliver pDNA into permeabilized wheat immature embryos. A Received: June 14, 2012 Revised: November 8, 2012 Published: December 6, 2012 10

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Figure 1. The peptide-based gene delivery system. (a) The negatively charged pDNA and designed peptides: R9-Bp100, (KH)9-Bp100 and R9-Tat2. (b) Formation of ionic complexes between pDNA and the designed peptide. (c) Infiltration of the pDNA complex into plant cells of leaves. The pDNA complex penetrates through the cell wall and the cell membrane, and then the pDNA is expressed throughout the cell, including in the nucleus. force microscope (AFM, Seiko Instruments, Japan). The solution containing the complexes was diluted to a final volume of 800 μL using ultrapure water (Milli-Q) and used for zeta potential and size measurements. Zeta potential and zeta deviation of samples were measured three times by a zeta potentiometer, and the average data were obtained using Zetasizer software ver. 6.20 (Malvern Instruments, Ltd.). Dynamic light scattering (DLS) was performed to determine the hydrodynamic diameter, and the polydispersity index (PDI) was determined with a zeta nanosizer (Zetasizer software ver 6.20) using a 633 nm He−Ne laser at 25 °C with a backscatter detection angle of 173°. The pDNA complex solution was cast on cleaved mica and observed in air at room temperature using a silicon cantilever with a spring constant of 1.3 N/m in tapping mode AFM. The calibration of the cantilever tip-convolution effect was performed to obtain the true dimensions of the pDNA complexes using the methods reported previously.19,20 For gel retardation assay, 40 μL of each sample containing 1.0 μg of pDNA was mixed with loading buffer and analyzed on 1% agarose gel (TAE buffer, 100 V, 30 min) and stained with ethidium bromide. Treatment of Leaves with pDNA Complexes. Approximately 100 μL of complexes solution containing pDNA encoding GFP and RLuc were infiltrated directly into fully expanded leaves of N. benthamiana and A. thaliana using a syringe without a needle (see Figure S1). The treated N. benthamiana and A. thaliana were incubated at 29 and 21 °C, respectively, under 16 h of constant light per day in a plant incubator for up to 6 days. To evaluate the RLuc gene expression quantitatively, Renilla luciferase assay (Promega, Madison, WI) was performed (n = 4) according to the manufacturer’s protocol. Briefly, the infiltrated leaves were periodically sampled from 12 to 144 h by cutting out 1 cm2 around the infiltrated section and lysed with Renilla Luciferase Assay Lysis Buffer (Promega). The lysate was briefly centrifuged, and the supernatant was mixed with Renilla Luciferase Assay Substrate and Renilla Luciferase Assay Buffer (Promega). The gene expression was evaluated based on the intensity of photoluminescence (relative light units) using a multimode microplate reader (Spectra MAX M3; Molecular Devices Corporation, Sunnyvale, CA). The amount of protein in the supernatant was determined using a BCA protein assay (Pierce Biotechnology, Rockford, IL), and the ratio of relative light units/weight of protein (RLU/mg) was obtained. Particle bombardment was performed as a positive control using PDS1000/He system (Bio-Rad Laboratories, Hercules, CA). pDNA of 1.0 μg was precipitated on gold particles of an average size of 0.6 μm, along the manufacture protocol provided by Bio-Rad Laboratories. For each bombardment, pDNA of 1.0 μg with the gold particles was spread on the surface of the microcarrier. The pressure employed in this experiment was 1100 psi. The leaves after the bombardment were incubated under a wet condition at 26 °C for 12 h. The treated leaves were cut out 1 cm2 and lysed with Renilla Luciferase Assay Lysis Buffer for Renilla Luciferase Assay as mentioned above. Qualitative evaluation of GFP gene expression in the leaves was performed by fluorescence microscopy (Axio Observer Z1; Carl Zeiss, Oberkochen, Germany) and confocal laser scanning microscopy (CLSM, Leica Microsystems, Wetzlar, Germany). The GFP expressed

positively charged polycationic peptide has the ability to condense the negatively charged pDNA via ionic interactions.17 The polycationic peptide interacts with pDNA to form complexes, while the CPP transports the complexes into plant cells by penetrating the cell walls and plasma membranes (Figure 1). The fusion peptides, R9-Bp100, (KH)9-Bp100, and R9-Tat2, were chemically synthesized and mixed with pDNA to form complexes. The transfection behaviors using the peptide− pDNA complexes into Nicotiana benthamiana and Arabidopsis thaliana were investigated, and the potential of the peptidebased plant gene delivery system was assessed.

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EXPERIMENTAL SECTION

Peptide Synthesis. R9-Bp100 (RRRRRRRRRKKLFKKILKYLNH 2 , theoretical pI/M w : 12.55/2827.56 Da), (KH) 9 -Bp100 (KHKHKHKHKHKHKHKHKHKKLFKKILKYL-NH2, theoretical 10.81/3809.71 Da), and R9-Tat2 pI/Mw: (RRRRRRRRRKKRRQRRRRKKRRQRRR-NH2, theoretical pI/Mw: 13.28/3910.72 Da) were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis.18 The polypeptides were purified using high-performance liquid chromatography (HPLC) and the molecular weights were confirmed by matrix assisted laser desorption/ionization−time-of-flight (MALDI-TOF) mass spectrometry. (KH) 9 (KHKHKHKHKHKHKHKHKH-NH 2 ), R9 (RRRRRRRRR-NH2), Bp100 (KKLFKKILKYL-NH2), and Tat2 (KKRRQRRRRKKRRQRRR-NH2) were used as controls. Preparation of Plants. Seeds of N. benthamiana and A. thaliana were germinated in pots with planting medium containing a mixture of soil (Pro-Mix, Canada) and vermiculite in a ratio of 2:1. Both plants were grown and incubated under the optimal conditions as follows: Nicotiana plants were grown in a plant incubator (Biotron NK System, Japan) under 24 h of constant light and at a temperature of 29 °C for up to 3 weeks. The light intensity was approximately 80 μmol photons m−2·s−1. Arabidopsis plants were grown under daylength conditions of 16 h of light/8 h of dark and at 22 °C, unless noted otherwise. Preparation and Characterization of Peptide−pDNA Complexes at Various N/P Ratios. Two types of pDNA, i.e., P35SGFP(S65T)-TNOS, which encodes a green fluorescent protein (GFP), and P35S-RLuc-TNOS, which encodes Renilla luciferase (RLuc), were used as reporter genes. All pDNAs were amplified in competent DH5α Escherichia coli (Takara, Japan) and purified using an Endofree Plasmid Giga Kit (Qiagen, Hilden, Germany). To prepare the peptide−pDNA complexes, 0.5 g/L of peptides were mixed with pDNA solution (approximately 1.0 mg/mL) at various N/P ratios (0.5, 1, 2, 5, 10 and 20) at 25 °C. Here, N/P ratio refers to the number of amine groups from the peptide/the number of phosphate groups from pDNA. The final concentration of pDNA was 25 μg/mL. The final concentrations of R9-Bp100, (KH)9-Bp100, and R9-Tat2 are summarized in Table S1. The complexes were characterized immediately after mixing by a zeta potentiometer (Zetasizer NanoZS; Malvern Instruments, Ltd., Worcestershire, UK) and an atomic 11

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in the leaves was directly observed. The pDNA encoding GFP was labeled with Cy3 using a NimbleGen One-Color DNA Labeling Kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacture procedure. Complexes of the labeled pDNA (1.0 μg) with (KH)9-Bp100 (N/P 0.5) were infiltrated into A. thaliana leaves. After 12 h incubation, the leaves were collected and washed with phosphate buffer saline (D-PBS(-), Wako Pure Chemical Industries, Ltd., Osaka, Japan) twice and incubated with 300 nM 4′,6-diamidino2-phenylindole (DAPI, Lonza Walkersville, Inc., Walkersville, MD) PBS solution for 10 min under reduced pressure (approximately 0.06 MPa). The intracellular distributions of the pDNA complex labeled by Cy3 and the nuclei stained with DAPI were observed by CLSM at excitation wavelengths of 405, 488, and 555 nm (diode). Statistical Analysis. Statistical differences in transfection efficiency were determined by an unpaired t-test with a two-tailed distribution, and differences were considered statistically significant at p < 0.05. The data in the cell viability experiments are expressed as means ± standard deviation (n = 4).

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RESULTS AND DISCUSSION Characterization of Peptide−DNA Complexes. Two types of CPP, Bp100 and Tat2, and two types of polycation, R9 and (KH)9, were selected for component of fusion peptides. To clear which peptide is a better component of fusion peptide for plant gene delivery, we designed and synthesized three types of fusion peptides, namely, R9-Bp100, (KH)9-Bp100, and R9Tat2. Ionic complexes of peptides (R9-Bp100, (KH)9-Bp100, R9-Tat2, Figure 1a) with pDNA encoding reporter genes at different N/P ratios (0.1−20) were prepared and characterized using DLS, AFM, zeta-potential measurement, and agarose gel electrophoresis. Based on the DLS results (Table 1 and Figure

Figure 2. Morphologies of the pDNA complexes of the fusion peptides. AFM amplitude images of the pDNA complexes. The pDNA complexes of R9-Bp100 at an N/P ratio of 0.5 (a) and N/P ratio of 20 (b). The pDNA complexes of (KH)9-Bp100 at an N/P ratio of 0.5 (c) and N/P ratio of 20 (d). The pDNA complexes of R9-Tat2 at an N/P ratio of 0.5 (e) and N/P ratio of 20 (f). Each scale bar denotes 500 nm.

complexes, the dimensions determined by DLS were larger than those determined by AFM, since the pDNA complexes on mica decreased in size by approximately 80% due to drying in air.21 The DLS and AFM results confirmed that complexes were formed for both peptides, and their sizes clearly decreased with increases in the N/P ratio. The zeta potential for pDNA complexes of R9-Bp100, (KH)9-Bp100 and R9-Tat2 showed negative values at N/P ratios of 0.5 and 1 but increased to positive values at N/P ratios ranging from 2 to 20 (Figure 3). This saturation of the zeta

Table 1. Size and PDI of pDNA Complexes of R9-Bp100, (KH)9-Bp100 and R9-Tat2 Prepared at Various N/P Ratios R9-Bp100

a

(KH)9-Bp100

R9-Tat2

N/P ratio

size, nm

PDI

size, nm

PDI

size, nm

PDI

0.1 0.5 1 2 5 10 20

508, 4720a 319 321 151 127 114 116

a

348, 4240a 291 322 139 106 96 114

a

308, 2256a 472 428 172 115 122 126

a

0.34 0.38 0.21 0.21 0.23 0.25

0.31 0.30 0.23 0.23 0.23 0.24

0.47 0.58 0.27 0.26 0.26 0.22

No useful PDI due to bimodal distribution.

S2), the average hydrodynamic diameters of complexes of R9Bp100, (KH)9-Bp100 and R9-Tat2 decreased with increasing N/P ratios from 0.1 to 20. The pDNA complexes prepared at N/P ratios of more than 0.5 were successfully formed based on the average hydrodynamic diameters. On the other hand, the pDNA complexes at N/P 0.1 exhibited bimodal distributions, suggesting heterogeneous formation of the pDNA complexes. The diameters of the complexes prepared at N/P ratios of 0.5 and 1 were approximately 300 nm, whereas the complexes at N/P ratios of 5, 10, and 20 had diameters of around 120 nm. The morphologies of the pDNA complexes on mica were imaged by AFM (Figure 2). All of the complexes formed homogeneous globular complexes, and the pDNA complexes of R9-Bp100 at N/P 0.5 and 20 were 190 ± 21 nm wide and 12.8 ± 5.3 nm high and 123 ± 13 nm wide and 9.1 ± 2.2 high, respectively (n = 10). The pDNA complexes of (KH)9-Bp100 and R9-Tat2 also demonstrated similar sizes for the respective N/P ratios. On the basis of the diameters of the pDNA

Figure 3. Surface charges of the pDNA complexes with the fusion peptides. The zeta-potentials of the pDNA complexes prepared at N/P ratios ranging from 0.5 to 20 are shown.

potentials indicated that the peptides covered the surface of the pDNA complexes at an N/P ratio of 5. Agarose gel electrophoresis was also conducted to characterize the ionic interaction and electrolytic stabilities of the complexes (Figure 4). Larger migration of pDNA in the agarose gel denotes that the ionic complexes are less stable, which also means that the interaction between the peptides and pDNA are weaker. The complexes of R9-Bp100 prepared at N/P ratios from 0.5 to 2 12

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Figure 4. Stabilities of the pDNA complexes with the fusion peptides. The electrical stability of the pDNA complexes was characterized by agarose gel electrophoresis. (a) The stability of the pDNA complexes of R9-Bp100 prepared at different N/P ratios. (b) The stability of the pDNA complexes of (KH)9-Bp100 prepared at different N/P ratios. (c) The stability of the pDNA complexes of R9-Tat2 prepared at different N/P ratios.

Figure 5. Transfection efficiency of the fusion peptide-based gene delivery system with different N/P ratios of the pDNA complexes based on the RLuc assay results. The efficiencies for the pDNA complexes of R9-Bp100, (KH)9-Bp100, and R9-Tat2 at various N/P ratios ranging from 0.1 to 20 at 12 h after the injection of the complexes into N. benthamiana leaves are shown. Error bars represent the standard deviation of samples (n = 4). *Significant difference between two groups at p < 0.05.

showed migration of pDNA across the gel similar to that seen for the free pDNA (Figure 4a). The amount of migrated pDNA decreased with an increase in N/P ratio, indicating that more stable complexes of pDNA and fusion peptides formed at higher N/P ratios. Also, all bands of the complexes in Figure 4 were shown at different positions from free pDNA. Therefore, based on the agarose electrophoresis of the complexes, almost all pDNA molecules were involved in the formation of the complexes with the peptides. The negative or less positive zeta potentials of the complexes of R9-Bp100 showed the migration of pDNA at N/P 0.5 to 2. On the other hand, the pDNA complexes of (KH)9-Bp100 and R9-Tat2 demonstrated the migration of pDNA at N/P ratios from 0.5 to 10 and from 0.5 to 1, respectively (Figure 4b,c). This different migration of the pDNA indicates that the fusion peptides with higher pI showed stronger ionic interactions and stabilities with pDNA, because the pI and molecular weight of R9-Bp100 were 12.55 and 2827.56 Da and differed slightly from those of (KH)9-Bp100 (10.81 and 3809.71 Da) and R9-Tat2 (13.28/3910.72 Da). The present results confirmed the formation of the complexes of pDNA and fusion peptides, which suggests that positively charged fusion peptides, mainly polycation sequences, condensed pDNA as illustrated in Figure 1. Transfection into N. benthamiana Leaves. To elucidate the transfection function of the designed peptides, the complexes of R9-Bp100, (KH)9-Bp100 and R9-Tat2 with pDNA encoding Renilla luciferase (RLuc) as a reporter gene were directly infiltrated into N. benthamiana leaves. To determine the most efficient N/P ratio of the pDNA complexes between 0.1 and 20, the transfection efficiency was characterized quantitatively using an RLuc assay (Figure 5). The pDNA complexes of R9-Bp100, (KH)9-Bp100 and R9-Tat2 prepared at an N/P ratio of 0.5 showed a higher transfection efficiency than those prepared at other N/P ratios, whereas the efficiency of the complexes prepared at over N/P 0.5 decreased with an increase in the N/P ratio, implying that excess polycation induced cytotoxicity to plant cells and reduced the transfection efficiency, just as observed in the case of animal cells.21 The observation of the leaves after the infiltration of the complexes was also performed to confirm that higher cytotoxicity to cells was induced by a higher N/P ratio (Figure S3). The complexes prepared at N/P ratios of 0.5 and 1 induced no significant cell death (Figure S3B). However, the

complexes prepared at N/P ratios of 10 and 20 induced cell death around the infiltrated area (Figure S3D). Based on the observations (Figure S3A-D), the complexes prepared at a higher N/P ratio were confirmed to induce cytotoxicity to plant cells. On the other hand, the cytotoxicity of pDNA was also characterized (Figure S3E-G), and the present amount of pDNA (5.0 μg) showed no significant cytotoxicity. According to previous studies,10,11 the most suitable pDNA complexes for transfection into animal cells were smaller complexes with a slight positive charge. In the present study, however, the peptide−pDNA complex prepared at N/P 0.5, which was negatively charged and approximately 300 nm in diameter, demonstrated the best transfection efficiency into N. benthamiana leaves. Generally, plant cell walls are composed of fibrous structures, which are cross-links of cellulose, pectin, hemicellulose, lignin, and some charged proteins. Plant cell walls are known to contain cell wall modifications, such as pit, perforation plate, spiral thickening, and wart.22 One possible mechanism for the present pDNA delivery system was that the pDNA complexes went through the cell wall via pits of the cell walls, because some pits are more than 1 μm in diameter. CPP functions to penetrate cell membrane after going through the cell wall, similar to a previous report on transfection into animal cells using CPP.10 On the other hand, the complexes with relatively small sizes (less than approximately 120 nm) and relatively high positive charges (more than approximately 40 mV) did not show obvious transfection efficiency. This might be because plant cell walls with cross-link structure trapped the complexes, due to their too small sizes (size exclusion effect) and/or positive charges (ionic interaction), while the complexes went through the cell walls. Also, the N/P 0.5 was lower than usual complexes. Herein, CPP sequences seemed to play a role of polycation in addition to cell-penetrating function, because CPP contains several cations and helps to condense pDNA. Therefore, the pDNA complex of the fusion peptides could be formed even at N/P 0.5. The transfection efficiency via R9-Bp100, (KH)9-Bp100, and R9-Tat2 for N. benthamiana leaves was also measured to quantitatively characterize the time-course effects of the present gene delivery system (Figure 6a,b). The pDNA complexes of all 13

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Figure 6. Transfection efficiency and behavior based on RLuc expression. Transfection efficiency using the designed peptides (R9-Bp100, (KH)9Bp100 and R9-Tat2) into N. benthamiana leaves (a) and A. thaliana leaves (b). Transfection efficiency using the CPP Bp100 and Tat2 as well as polycation (KH)9 and R9 into N. benthamiana leaves (c) and A. thaliana leaves (d) (controls). Error bars represent the standard deviation of samples (n = 4). *Significant difference between two groups at p < 0.05.

Figure 7. Transfection behavior with the peptide-based gene delivery method based on microscopic characterization. (a) A N. benthamiana leaf infiltrated with pDNA complexes. White circles denote the infiltrated portions. (b) A transfected epidermis cell of the N. benthamiana leaf via the peptide-based delivery. The nucleus (a white arrow) of the transfected epidermis cell demonstrated the expression of GFP. (c) An A. thaliana leaf infiltrated with the pDNA complexes. A white circle denotes the infiltrated part. (d) Spongy mesophyll cells of A. thaliana leaves infiltrated with pDNA complexes showed GFP expression (green) in the cytosol, which was obviously different from the autofluorescence of chloroplasts (red).

three fusion peptides showed the highest transfection efficiency at 12 h, and their transfection efficiencies gradually decreased up to 144 h. This time-course study revealed that the present peptide-based gene delivery system is an outstanding system to perform transfection over a relatively short period of time. R9Bp100 and R9-Tat2 showed faster transfection behavior than (KH)9-Bp100 (Figure 6a). This difference in transfection behavior may have been due to the slower degradation of the (KH)9 sequence by proteases inside cells as compared to the R9 peptide. It is noteworthy that the transfection behavior can be changed and controlled by the selection of peptide-based gene carriers with appropriate amino acid sequences. As control experiments, transfection into N. benthamiana leaves via one of the CPPs (Bp100 and Tat2) plus one of the polycations ((KH)9 and R9) was performed using an N/P ratio of 0.5 from 3 to 144 h. The transfection efficiency of the nonfusion peptides was significantly lower than that of the fusion peptide-based gene delivery (Figure 6b). On the basis of the control results, the combination of a CPP and polycation as a fusion peptide clearly enhanced the transfection efficiency by approximately 4-fold compared with the CPP alone. This may be because pDNA interacts with the polycationic sequence

rather than with CPPs, and hence the CPP preferentially exists at the surface of the ionic complex and functions to penetrate cell membranes. The particle bombardment, which is one of the most common methods for plant transformation, was also performed as a positive control. N. benthamiana leaves are large enough to obtain the transfection efficiency of one bombardment, contrary to A. thaliana leaves. pDNA of 1.0 μg was used for each bombardment, and the incubation time after the bombardment was 12 h. On the basis of the RLuc assay, the transfection efficiency by the particle bombardment method into N. benthamiana leaves was 209 ± 60 RLU/mg, indicating that the efficiency of the peptide-based system was significantly higher than that of the established method, namely, particle bombardment. To further investigate the transfection behavior within the cells via the fusion peptides as gene carriers, the complexes of (KH)9-Bp100 and pDNA encoding GFP was also infiltrated into N. benthamiana leaves (Figure 7a), and the complexes looked noncytotoxic to the leaves. The epidermis cells around the infiltrated region were partially transfected using the fusion peptides (Figure 7b), and the GFP expression was observed in 14

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Figure 8. Intracellular distribution of pDNA complexes with the designed peptide ((KH)9-Bp100) in N. benthamiana leaves. (a,f) Cy3, (b,g) the autofluorescence of chloroplasts, (c,h) DAPI, (d,i) differential interference contrast, and (e,j) overlay of the four images (a−d and f−i). Images f−j are enlargements of the area indicated by white arrows in images a−e. pDNA was labeled with Cy3 (orange), chloroplasts have the autofluorescence (red), and the nuclei were stained with DAPI (blue). Each scale bar in a−e and f−j represents 20 and 5 μm, respectively.

Intracellular Distribution of the Complexes in A. thaliana Leaves. The intracellular distribution of the complexes of (KH)9-Bp100 with Cy3-labeled pDNA and the nuclei stained with DAPI were investigated by CLSM. Figure 8 shows typical CLSM images of the epidermis cells around the region infiltrated with the complexes of (KH)9-Bp100 with Cy3-labeled pDNA. The Cy3-labeled pDNA (orange) was distributed around the nuclei (blue), indicating that the pDNA was transferred near the nucleus via the (KH)9-Bp100 fusion peptide. This result also directly supported that the fusion peptides function as gene carriers into plant cells.

the nucleus and cytosol (Figure 7b, white arrow). GFP is known to localize at the edge of cytsol and often diffuse into the nucleus in plant cells due to the large vacuoles in plant cells.23,24 Therefore, the observation confirmed general subcellular localization of GFP in plant cells. The result suggests that the present peptide-based gene delivery system is available for study on subcellular localization of GFP-tagged proteins. Transfection into A. thaliana Leaves. In vivo transfection experiments with A. thaliana leaves were performed to investigate the difference in transfection between plant types. The pDNA complexes of R9-Bp100, (KH)9-Bp100, and R9Tat2 prepared at an N/P ratio of 0.5 were directly infiltrated into A. thaliana leaves. The transfection efficiencies via R9Bp100, (KH)9-Bp100, and R9-Tat2 for A. thaliana leaves were much higher due to its smaller leaves and showed a tendency similar to that for N. benthamiana leaves (Figure 6c); namely, the pDNA complexes of the fusion peptides, especially (KH)9Bp100, showed rapid transfection within 12 h. The control experiments using the nonfusion peptides demonstrated that the fusion peptides enable efficient transfection into A. thaliana leaves in addition to N. benthamiana leaves (Figure 6d). The transfection efficiencies by the bombardment method into A. thaliana leaves was 480 ± 122 RLU/mg. Therefore, the efficiency of the peptide-based system into A. thaliana leaves was significantly higher than that of the particle bombardment (Figure 6c), which was in a manner similar to that of N. benthamiana leaves. The complexes of the peptide and pDNA seemed noncytotoxic against the infiltrated leaves (Figure 7c). Also, the spongy mesophyll cells of A. thaliana leaves after the infiltration of the pDNA complexes of the fusion peptides showed GFP expression in the cytosol (green in Figure 7d), which was obviously different from the autofluorescence of chloroplasts (red in Figure 7d, also see Figures S4 and S5). The expression of GFP was observed in the epidermis cells of the A. thaliana leaves (Figure S6). The fusion peptide was capable of delivering pDNA into not only the epidermis cells but also the mesophyll cells inside A. thaliana leaves. On the basis of the transfection results related to A. thaliana leaves, the transfection via the fusion peptides was successfully performed and showed no significant difference between N. benthamiana and A. thaliana.

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CONCLUSIONS The fusion peptide-based rapid gene delivery is a novel transient transformation method for plant, which has the potential to be used for any type of plant (particularly leaves) or gene without the need for special equipment or protoplast preparations. The fusion peptides demonstrated significantly higher transfection efficiency in comparison to the nonfusion peptides, indicating that combination of functional peptides was a key to design peptide-based gene delivery system. This is because the sequences of the fusion peptides, which contain polycationic sequences, are more appropriate for formation of the complexes with pDNA. The present gene delivery system induces transient transformation, resulting in no risk of contamination of transformed genes in natural environment. It is also possible to produce exogenous proteins into only selected plant tissues, such as inedible tissues of agricultural plants, by infiltration of the peptide−DNA complexes. Using our system, various endogenous signal transductions could be regulated by local infiltration of signal molecules in intact plants. For example, regulations of flowering, development, and resistance against abiotic and biotic stresses would be useful for agriculture and crop science. Furthermore, we demonstrated the versatility of the designed peptide-based gene delivery system in plant science and biotechnology areas.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S4 and Table S1 are available free of charge via the Internet at http://pubs.acs.org. 15

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(20) Numata, K.; Kikkawa, Y.; Tsuge, T.; Iwata, T.; Doi, Y.; Abe, H. Macromol. Biosci. 2006, 6, 41−50. (21) Numata, K.; Subramanian, B.; Currie, H. A.; Kaplan, D. L. Biomaterials 2009, 30, 5775−5784. (22) Harada, H.; Côté, W. A., Structure of wood. In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic Press: Orlando, FL, 1985; pp 1−42. (23) Tamura, K.; Shimada, T.; Ono, E.; Tanaka, Y.; Nagatani, A.; Higashi, S.; Watanabe, M.; Nishimura, M.; Hara-Nishimura, I. Plant J. 2003, 35, 545−555. (24) von Arnim, A. G.; Deng, X. W.; Stacey, M. G. Gene 1998, 221, 35−43.

AUTHOR INFORMATION

Corresponding Author

*Mailing address: RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. Phone: +81-48-467-9525; Fax: +81-48-4624664; E-mail: [email protected]. Author Contributions

M.L. performed all experiments except for the plasmid constructions and the microscopy experiments under the supervision of K.N.; T.Y. and Y.K. constructed the plasmids; K.N. and Y.K. performed the microscopy experiments; all experiments were designed by K.N., T.Y., and Y.K.; the manuscript was prepared by all authors with discussion and improvements from all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.N. acknowledges funding from the New Energy and Industrial Technology Development Organization, Japan. ABBREVIATIONS CPP, cell-penetrating peptides; pDNA, plasmid DNA; R9, nona-arginine; Bp100, KKLFKKILKYL; Tat2, RKKRRQRRRRKKRRQRRR; DLS, dynamic light scattering; RLuc, Renilla luciferase; GFP, green fluorescent protein

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dx.doi.org/10.1021/bm301275g | Biomacromolecules 2013, 14, 10−16