Selective Gene Delivery for Integrating Exogenous DNA into Plastid

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Selective gene delivery for integrating exogenous DNA into plastid and mitochondrial genomes using peptide-DNA complexes Takeshi Yoshizumi, Kazusato Oikawa, Jo-Ann Chuah, Yutaka Kodama, and Keiji Numata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00323 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on April 1, 2018

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Selective gene delivery for integrating exogenous DNA into plastid and mitochondrial genomes using peptide-DNA complexes Takeshi Yoshizumi,*,† Kazusato Oikawa,† Jo-Ann Chuah,† Yutaka Kodama,‡ and Keiji Numata*,† †

Enzyme Research Team, Biomass Engineering Research Division, RIKEN Center for

Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan. ‡

Center for Bioscience Research and Education, Utsunomiya University, 350 Mine-machi,

Utsunomiya, Tochigi 321-8505, Japan.

*Correspondence

to:

Keiji

Numata

([email protected]),

Takeshi

Yoshizumi

([email protected])

ACS Paragon Plus Environment

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ABSTRACT

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms, including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.


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INTRODUCTION Since plant cells contain multiple organelles with highly polyploid genomes,1 transgenes inserted into plastid2 or mitochondrial genomes3 are expected to exhibit higher expression levels than those inserted into the nuclear genome. Plastids contain various secondary metabolites that could be used as starting materials for drug, chemical, or fuel production.4 Thus, integrating exogenous genes into plastid genomes may allow the efficient biosynthesis of value-added products. Plastid transformation via particle bombardment was first reported in the green alga Chlamydomonas reinhardtii5 and was subsequently achieved in model plants such as Nicotiana tabacum (Nt)6, tomato (Solanum lycopersicum),7 and several other flowering plants.8 Recently, a plastid transformation system in the Arabidopsis thaliana (At) Slavice (Sav-0) accession, which is spectinomycin hypersensitive, was reported and established.9 Although plastid transformation has been reported in the model plant At Columbia (Col-0) accession,10 such work is not routinely performed. Another essential organelle, the mitochondrion, supplies the cell with energy in the form of adenosine triphosphate (ATP), which is necessary to boost the activity of metabolic pathways for metabolic engineering and biological production. To modify mitochondria, gene delivery to mitochondria was achieved by conjugation of mitochondrial targeting signal to nucleic acids.11, 12 The viral vector fused with mitochondrial target signal was also developed to deliver DNA to mammal mitochondria.13 We previously developed a DNA delivery system consisting of a peptide-based DNA carrier fused to a mitochondria-targeting signal sequence, which we successfully used to transiently express exogenous reporter genes in At mitochondria14 and human embryonic kidney cells 293.15 The delivered genes were transiently expressed but not integrated into the organellar genomes. To exploit organelles such as chloroplasts and


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Figure 1. Organellar genome-targeting DNA delivery system using peptide-DNA complexes. (a) The amino acid sequence of KH-AtOEP34 is highlighted in gray and green, to indicate KH and AtOEP34, respectively. DNA and KH-AtOEP34 form peptide-DNA complexes, which deliver DNA to the chloroplasts. (b) The amino acid sequence of Cytcox-KH is highlighted in gray and orange, to indicate KH and Cytcox, respectively. DNA and Cytcox-KH peptide form peptideDNA complexes, which deliver DNA to the mitochondria.

EXPERIMENTAL METHODS Peptide

preparation.

Cytcox-KH

(amino

acid

sequence:

MLSLRQSIRFFKKHKHKHKHKHKHKHKHKH) and KH-AtOEP34 (amino acid sequence: KHKHKHKHKHKHKHKHKHMFAFQYLLVM)

were

synthesized

via

standard

9-

fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis.20 The peptides were purified using high-performance liquid chromatography (HPLC). The purities of these peptides were characterized by HPLC with an InertSustain C18 column (GL Sciences, Tokyo, Japan) for KHAtOEP34 and a YMC-Pack PROTEIN-PR column (YMC co., Ltd., Kyoto, Japan) for CytcoxKH at 25°C (Figure S1). The mobile phase comprised 5–35% CH3CN containing 0.1% TFA. The flow rate was 1.0 mL/min. Their molecular weights were confirmed through matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry. The average molecular weights of KH-ATOEP34 and Cytcox-KH were 3649.4 and 3913.0 Da, respectively. Plant materials and growth conditions. Arabidopsis thaliana (At) (accession Col-0) or Nicotiana tabacum (Nt) (cv SR-1) plants were used for all experiments. Seeds were grown on 0.5 ! Murashige and Skoog (MS) plates without sucrose under continuous light conditions at 22°C for At and at 30 °C for Nt.


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Plasmid construction. For At plastid transfection, the plastid genome between nucleotides 11,502 and 15,877 was amplified using the AtCp1 primer set (AtCp1FKpn and AtCp1RKpn). All the primers used in this study are listed in Table S1. The PCR product was cloned into the pUC19 vector at the KpnI site. The GFP and spectinomycin resistance (SPECr) genes were amplified using the following primer sets: GFP (GFPF and GFPR) and SPECr (SPECr-Fw-GFP3’ and SPECr-Rev). The annealed PCR products were used as a template for PCR with GFPF and SPECr-Rev, resulting in the GFPSPECr fragment. The psbA promoter (psbAp) and psbA terminator (psbAt) were amplified with the following primer sets: psbAp (psbApFBglII and psbApRGFP), psbAt (psbAtFSPECr and psbAtRBglII). The construct psbAp:GFP:psbAt (psbApt:GFP) was generated by combining each PCR fragment, and the PCR amplification procedure was the same as for GFPSPECr. The psbApt:GFP fragment was cloned into the BglII site of the cloned AtCp1. The size of the plasmid was approximately 9.3 kb. The construct for At plastid genome is shown in Figure 2a. Mitochondrial genomic fragments from At (nucleotides 73,056 to 77,283) and for Nt (nucleotides 62,401 to 66,760) were amplified using the following primer sets: AtMt1 (AtMtF and AtMtR), NtMtB (NtmtBFkpn and NtmtBRkpn) (see the sequence in Table S1). TOPO cloning (Life Technologies, Carlsbad, CA) was used to clone the AtMt1 fragment into the pUC19 vector at the EcoRI site and to clone NtMtB into the pCR2.1 vector. The yeast cox2 promoter (cox2p) and cox2 terminator (cox2t) fragments were amplified using the following primer sets: cox2p (cox2pFBgl and cox2pR-GFP), cox2t (cox2tFSPECr and cox2tRBgl). Employing the cox2p, cox2t, and GFPSPECr fragments, cox2p:GFP:cox2t (cox2pt:GFP) was constructed via the same method used for psbApt:GFP. The cox2pt:GFP product was inserted into the cloned AtMt1 fragment at the BglII site. NdeI sites were added to cox2pt:GFP using the


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Preparation of DNA-peptide complexes. DNA-peptide complexes were prepared using a previously reported method.16 Briefly, 3 "L of 1 mg/mL DNA was added to 1 "l of 1 mg/mL peptide solution at an N/P ratio (number of amine groups in the peptide/number of phosphate groups in the DNA) of 0.5. The mixture solution of DNA and peptide was diluted with autoclaved Milli-Q water to obtain a final volume of 100 "L. The solution was thoroughly mixed by repeated pipetting and was allowed to stabilize for 30 minutes at 25 °C. Characterization of DNA-peptide complexes. The complexes were characterized with a zeta potentiometer as previously reported.21 The solution containing the DNA-peptide complexes was diluted to a final volume of 800 "L using Milli-Q water for zeta potential and size measurements. The zeta potential and zeta deviation of the samples were measured three times with a zeta potentiometer (Zetasizer Nano-ZS; Malvern Instruments, Ltd., Worcestershire, UK), and the averaged data were obtained using Zetasizer software version 6.20 (Malvern Instruments, Ltd). Dynamic light scattering (DLS) was performed on the same instrument to determine the hydrodynamic diameter using a 633 nm He-Ne laser at 25 °C with a backscatter detection angle of 173 °. Then, the polydispersity index (PDI) was determined with Zetasizer software (Malvern Instruments, Ltd). Treatment of plants with the complexes. For each infiltration, 100 "L of a DNA-peptide complex solution containing 1.0 µg of DNA was infiltrated directly into 2-day-old seedlings using a syringe-based vacuum system (Figure 3). The vacuum conditions consisted of compression at 2 bar for 1 minute after applying vacuum at 0.1 bar for 1 minute. The infiltrated seedlings were incubated in a plant incubator for 1 day for plastid transfection and for 2 days for mitochondrial transfection. The leaves of the infiltrated plants were observed using confocal laser scanning microscopy (CLSM) and were excised to isolate genomic DNA. Genomic DNA


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Confocal laser scanning microscopy analysis. The intracellular localization of chloroplasts and mitochondria was detected using chlorophyll autofluorescence and MitoTracker staining (MitoTracker Red CMXRos; Molecular Probes, the Netherlands), respectively. For chloroplast imaging, time gating of chloroplast autofluorescence was used according to a previous literature.23 For mitochondrial imaging, leaves were incubated for at least 30 minutes in 200 nM MitoTracker dissolved in water. GFP, chlorophyll and MitoTracker fluorescence were observed via CLSM (TCS SP8 X, Leica Microsystems, Wetzlar, Germany). Images of the Cy3-labeled peptide-DNA complexes associated with chloroplasts and the process of entering a chloroplast were obtained through CLSM using a Zeiss LSM880 with Airyscan (Carl Zeiss, Jena, Germany). A transgenic plant expressing the N-terminal hydrophobic region of CHUP1 fused with GFP was used to examine the outer membrane of chloroplasts, as described in a previous study24. Chloroplast-binding

assay.

AtOEP34-FLAG

fusion

peptide

(sequence:

MAMQAMFAFQYLLVMGGGGSDYKDDDDK) was synthesized as previously described. Chloroplasts were isolated from deveined spinach leaves (30-35 g fresh weight) using a method described by Nakatani and Barber.25 The resultant chloroplast preparation (0.9 mL) was 89% intact, with a chlorophyll concentration of 0.922 mg/mL. Intact chloroplasts were pelleted via centrifugation (5000 !g, 2 minutes, 4 °C) and resuspended in assay buffer containing 50 mM Tris-acetate, 0.1% (w/v) Tween 20, 30 mM MgCl2, and 10 mM NaCl. For the binding assay, different ratios of chloroplasts to AtOEP34-FLAG (stock concentration: 3 mM) were tested; each tube contained 100 "L of one of the following chloroplast/peptide/assay buffer mixtures: a) 50 "L/no peptide/50 "L (control); b) 5 "L/10 "L/85 "L (1:2 ratio); and c) 1 "L/10 "L/89 "L (1:10 ratio). The components were incubated at 4 °C for 1 hour with agitation at 80 rpm and were


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washed 3 times with the assay buffer to remove unbound peptides. For the detection of chloroplast-bound peptides, a monoclonal anti-FLAG Cy3 antibody (Sigma-Aldrich, St. Louis, MO, USA, Product No. A9594) was added to each tube at a final concentration of 10 "g/mL, and the mixture was then incubated at 25 °C for 1 hour. Unbound antibodies were removed via 3 washes with 1! Tris-buffered saline, and 10 "L of each solution was subjected to CLSM analysis. Genomic PCR analysis. Recombinant genomic fragments were amplified using the following primer sets: AtCp1insF and SPECrRev for At plastids, AtMt1insR and cox2tFSPECr for At mitochondria, and NtMtBinsF and cox2tFSPECr for Nt mitochondria (see the sequence in Table S1). The sequences of the primers are listed in Table S1. The amplified recombinant genomic fragments were cloned into the pCR2.1 vector using TOPO cloning. Sequence analysis was performed using the following primer sets: Atplastid1 and Atplastid2 for At plastids, Atmito1 and Atmito2 for At mitochondria, and Ntmito1 and Ntmito2 for Nt mitochondria (see the sequence in Table S1). For control experiments, wild-type genomic DNA; the plasmid alone; a mixture of wild-type genomic DNA and the plasmid; or transformed genomic DNA was employed as the PCR template. The transfection ratios were determined based on the results obtained for 70 seedlings, and the average value was calculated from 4 repeats (4 repeats ! 70 seedlings). Southern blot analysis. Total cellular DNA was isolated using the DNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany). The DNA was then digested with XhoI, and the digested DNA was electrophoretically separated in a 0.8% agarose gel with 1! TAE buffer. Subsequently, separated DNA was blotted onto Hybond N+ (GE Healthcare UK Ltd., Buckinghamshire, UK). As a probe, a fragment of the SPECr was amplified with the primers SPECr-Fw and SPECrR-


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Rev (see the sequence in Table S1). The primers were designed as shown in Figure S2. The primer sequences are listed in Table S1. The AlkPhos Direct Kit (GE Healthcare UK Ltd., Buckinghamshire, UK) was used for probe labeling, hybridization, washing, and signal generation, according to the manufacturer’s instructions. Hybridization was performed at 55 °C overnight, and the membrane was washed twice at 55 °C for 10 minutes. Chemiluminescent signals were detected using a luminescent imaging analyzer, LAS-3000 (Fujifilm, Tokyo, Japan).

RESULTS AND DISCUSSION Chloroplast-binding of chloroplast transit peptide. For chloroplast localization, we selected a signal peptide of Arabidopsis origin (AtOEP34), which was previously demonstrated to be capable of both chloroplastic membrane targeting and insertion,17 with a peptide chain length (10 residues) that is amenable to chemical synthesis. Detailed analyses of this signal peptide have revealed that the import of AtOEP34 does not occur spontaneously but, rather, through a proteinaceous receptor. As a preliminary experiment, we performed an in vitro chloroplast-binding assay and confirmed the high affinity of AtOEP34 for the chloroplasts through microscopic visualization (Figure S3). Thus, we chose the transit peptide of AtOEP34 as the signal for the DNA carrier. AtOEP34 has been shown to function in chloroplast targeting and is sufficiently short for peptide synthesis.

Selective DNA delivery to At chloroplasts. For integration into the plastid genome, the plasmid DNA contained At plastid genome sequences of approximately 2.4 kb and 2.0 kb for homologous recombination (Figure S1a). As a reporter system, the green fluorescent protein


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(GFP) and spectinomycin resistance (SPECr) genes were inserted between the homologous recombination sites under the control of the Nt psbA promoter and terminator (Figure 2a). The peptide-DNA complex was prepared by mixing DNA and KH-AtOEP34 at an N/P ratio of 0.5. This is because an N/P ratio of 0.5 was best for DNA delivery into plant organelles according to a previous study.14 The hydrodynamic diameter and zeta potential of the complex were approximately 359 nm and -26 mV (Figure S4), respectively. Ten 2-day-old At (Col-0) seedlings were infiltrated with this complex via the application of vacuum at 0.1 bar for 1 minute and compression at 2 bar for 1 minute. To confirm the delivery behavior, the complexes of Cy3labeled DNA and KH-AtOEP34 were observed on transgenic At chloroplasts 6 hours after infiltration using confocal laser scanning microscopy (CLSM) (Figure 4). To visualize chloroplast outer membranes, transgenic Arabidopsis expressing CHUP-GFP, which was localized on chloroplast outer membranes, was used. The complexes were observed at the surface (outer membrane) of chloroplasts (Figure 4a), indicating that the complexes were covered with the chloroplast membrane. This might be because the cationic complexes interacted with the components of chloroplast membranes, namely, lipids. The uptake process of the complex into chloroplasts was also observed, which showed that uptake occurs relatively quickly—on the scale of several minutes (Figure 4b).


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The integration of the transgene into the plastid genome was confirmed via PCR using specific primers to amplify only the recombined sequence containing the plastid genomic region and the exogenous DNA (Figures 5a and S5a). The recombined sequence was detected in the plastid genomes of all ten samples 1 day after infiltration (Figure 5a; DNA + Pep). The bands the arrow indicates were positive bands to indicate the homologous recombination. The recombined sequence was maintained even after the treated seedlings were cultured for 10 days (Figure 5b). In addition, we sequenced the junctions between the exogenous DNA and the plastid genomes (Figure 5c). It was observed that the endogenous genomic sequence had been completely replaced with the identical region of exogenous DNA, with no insertions or deletions, indicating that the exogenous DNA had been integrated into the plastid genome via homologous recombination. Furthermore, we confirmed exogenous DNA integration at the appropriate genomic position through Southern blot analysis using gene-specific probes designed with a SPECr fragment as a template. The negative controls did not show any bands (Figure 5d; lanes 1 and 2), whereas the band in lane 4 of Figure 5d corresponded free plasmid DNA, which was not integrated into the plastid genome. As expected based on the sequence and restriction sites (Figure S6a), a fragment of approximately 8 kb was detected (Figure 5d; lane 3). Therefore, this result indicated that the exogenous DNA was internalized into chloroplasts and was integrated into the plastid genomes of the At seedlings after infiltration of the peptide-DNA complexes.


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To further confirm the integration of the GFP gene into the plastid genomes, we used CLSM to visualize GFP fluorescence in the chloroplasts from At cotyledon cells (Figure 6). GFP fluorescence was observed in the stroma of chloroplasts and was not overlapped with the autofluorescence of chloroplasts. Thus, the GFP gene was successfully introduced and expressed via peptide-DNA complexes. To confirm the integration of the SPECr gene into the At plastid genome, the spectinomycin resistance of At cotyledons treated with the peptide-DNA complex was evaluated (Figure 7). The At seedlings treated with the complex showed significant resistance to 50 mg/L spectinomycin compared with the control (Figure 7a), even though the differences between the control (peptide only) and the seedlings treated with the peptide-DNA complexes at 0 and 100 mg/L spectinomycin (Figure 7a and S6). The transplastomic seedlings were significantly larger (Figure 7b and S7) and exhibited a greater dry weight than the seedlings treated with peptides without DNA in 50 mg/L spectinomycin (Figure 7c). Thus, our “plastid genome-targeting” gene delivery system can integrate exogenous DNA into target positions of the plastid genome.


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mitochondrial genome in multicellular plants has not yet been reported because of a lack of a selective delivery system. However, C. reinhardtii mitochondria have been successfully transformed using particle bombardment,26 and electroporation has been employed in vitro to introduce genes into isolated Nt mitochondria (in organello).27 To develop a gene delivery system for the mitochondrial genome, we used the fusion peptide Cytcox-KH (Figure 1b). The DNA complex of Cytcox-KH at N/P 0.5 used in this study was previously reported to show 356 nm of hydrodynamic diameter and -25 mV of zeta potential.14 The size, zeta potential, stability of the DNA complexes of Cytcox-KH at various N/P ratios were studied for mitochondrial transfection, resulting in the highest transfection efficiency at an N/P ratio of 0.5.14, 15 The stability of the peptide-DNA complex was analyzed by DNA migration assay. The DNA binding stability and condensation capacity of the peptide were characterized at various N/P ratios up to 20.15 The complex prepared at an N/P ratio of 0.5, which is the complex used in the present study, demonstrated a medium stability enough for the delivery system. These complexes are effective for gene delivery into At mitochondria14 and human embryonic kidney 293 cells.15 In the current study, plasmid DNA containing GFP, the Sc mitochondrial cox2 promoter, and a terminator was used to transform the At mitochondrial genome (Figure 2b). The cox2 promoter activates the transcription of reporter genes only in mitochondria and not in the nucleus,28 excluding the possibility that the fluorescent signals resulted from integration of GFP into the nuclear genome. The reporter cassette worked in plant mitochondria without codon optimization for organellar genomes, according to a previous report.14 We expected the exogenous gene to be integrated into the mitochondrial genome via homologous recombination, which occurs in both plant mitochondria and plastids.27 The reporter gene cassette was inserted into the BglII site of an approximately 4.3 kb At mitochondrial


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genome fragment (Figure S2b). We treated At seedlings with the peptide-DNA complex for gene delivery into their mitochondrial genomes. PCR analysis of genomic DNA extracted 2 days after treatment confirmed that the exogenous sequence had been integrated into the mitochondrial genome (Figure 8a and S5b). The positive bands indicated by an arrow confirmed that exogenous DNA was integrated into the mitochondrial genome in nine of the ten samples examined (Figure 8a; DNA + Pep). However, no integration events were detected in the samples treated with DNA or carrier peptide alone (Figure 8a; DNA, Pep). The integrated sequence was not only detected immediately after the gene delivery but was maintained within the genome even after the treated seedlings were cultured for 10 days (Figure 8b). Sequencing of the junctions between the exogenous DNA and the mitochondrial genome revealed that the exogenous DNA underwent precise homologous recombination in At mitochondria (Figure 8c). Furthermore, we confirmed through Southern blot analysis that the exogenous DNA had integrated into the expected position of the mitochondrial genome (Figure 8d and S6b). Lanes 1 and 2 show the results of negative controls treated with either peptide or DNA, while lanes 4 and 5 show bands corresponding to DNA only before and after the restriction enzyme digestion. The positive band in lane 3 confirmed the exogenous DNA had integrated into the mitochondrial genome (Figure 8d). Thus, the exogenous DNA was successfully internalized into the mitochondria using Cytcox-KH, resulting in the integration of the DNA into the mitochondrial genome. Considering the DNA delivery efficiency into mitochondria of human embryonic kidney 293 cell line,15 the delivery efficiency into plant mitochondria was relatively lower. This might be because the peptide-DNA complex used in this study did not contain functions to penetrate cell membrane and cell walls.


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Figure 10. Gene integration into the Nicotiana tabacum (Nt) mitochondrial genome. (a) PCR analysis of exogenous DNA integration into the mitochondrial genome. DNA and Pep indicate treatment of Nt seedlings with DNA or the peptide alone, respectively, and (DNA + Pep) indicates seedlings treated with the DNA-peptide complex. The arrowhead indicates the size of the expected PCR products. (b) Sequence analysis of the junctions between the inserted DNA and the mitochondrial genomes. Black arrows indicate the sequencing primers. The sequencing results and the predicted sequences of the junctions are shown in the left and right panels, respectively. “Homologous” and “Endogenous” indicate the portions of the mitochondrial genome sequence cloned into the vector and the genomic sequence flanking the integration site, respectively. cox2t indicates the sequence of the cox2 terminator. (c) CLSM observations of transformed Nt mitochondria. Left, GFP fluorescence; center, MitoTracker Red florescence; right, merged image. Magnified images of several mitochondria expressing GFP are shown in the far right panels.

Selectivity of the fusion peptides. One advantage of using peptides as DNA carriers is that cells or organelles can be selectively targeted employing transit/signal peptides29. To evaluate the selectivity of the peptides used in this study, we investigated the integration efficiency of other combinations of peptides and DNA for At mitochondrial transfection. When we used a combination of chloroplast transit peptide-bearing KH-AtOEP34 and DNA containing the At mitochondrial homologous recombination site (Figure 11a), PCR products were weakly amplified from a few samples (Figure 11b; lanes 1 and 6), indicating that the selectivity of KHAtOEP34 for mitochondria is not perfect but is sufficient for a gene delivery system. On the other hand, no DNA was integrated into the plastid genome using the combination of Cytcox-KH and DNA targeting the At plastid genome (Figure 11c), while the same DNA was successfully integrated into the plastid genome by KH-AtOEP34 (Figure 11d). These control experiments indicated that exogenous DNA was delivered to the target organelle genome due to the


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selectivity of the localization signals. However, transit/signal peptides with lower selectivity for their respective organelles might deliver genes into multiple types of organelles via off-target localization. Considering the off-target localization into nucleus, we previously investigated the target selectivity by using the complex of Cytcox-KH and DNA encoding a reporter gene driven by 35S promoter (for nucleus).14 As a result, the reporter gene was not detected, indicating that Cytcox-KH did not delivery DNA to nucleus instead of mitochondria. Taken together, the selectivity of the fusion peptide-mediated system is high enough for the gene delivery system targeting to specific organelles.


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This study is the first to demonstrate a selective system for the delivery of exogenous DNA into the genomes of plastids and mitochondria. Although particle bombardment is commonly used to integrate exogenous DNA into both plastid30 and nuclear genomes31, this integration occurs without selectivity for target genomes, as the particles with attached exogenous DNA can hit either the nucleus or plastids, sometimes leading to unexpected patterns of genomic integration. In contrast, our peptide-mediated gene delivery system is organelleselective and is sufficiently efficient to achieve expression of reporter genes in the target organelle. Remarkably, our system can control the integration position of the target genome by incorporating homologous recombination sites into the DNA integration vector. Thus, our peptide-mediated gene delivery system may represent a breakthrough platform technology in plant science, due to its selectivity for organellar genomes and their sequences.

CONCLUSIONS This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants but also multicellular organisms in general. Since the peptide containing the mitochondrial targeting signal Cytcox, which originated in yeast, was successfully used to integrate exogenous DNA into both At and Nt mitochondrial genomes in vivo, this peptide may also be applicable to crop and non-model plants. To establish T1 plants harboring transformed mitochondrial genomes, suitable selectable markers need to be developed in addition to our gene delivery system for the mitochondrial genome3, 28. This report should motivate and provide the basis for the development of an organellar selection system using marker genes. As a target phenotype for mitochondrial modification, cytoplasmic male sterility (CMS) is an important


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agronomic trait in plants32. Furthermore, mutations in human mitochondrial genomes are associated with severe diseases, which could be treated genetically with our mitochondriontargeted delivery system33. Thus, our organelle-selective gene delivery system represents a breakthrough platform for the development of artificial CMS and high-yield crops in plants and gene therapy for mitochondrial diseases in animals, in addition to stimulating basic science and the field of mitochondrial genetics.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Figures S1-S7 and Table S1 (PDF) AUTHOR INFORMATION Corresponding Author * Keiji Numata ([email protected]), Takeshi Yoshizumi ([email protected]) Author Contributions T.K., Y.K. and K.N designed the research. K.O. and Y.K. performed CLSM observations. J.C. performed preparations and biochemical assays of peptides. T.K. performed the other experiments. T.K., Y.K. and K.N analyzed the whole data. All the authors prepared the manuscript. ACKNOWLEDGMENT


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This work was supported by JST ERATO Grant Number JPMJER1602, Japan (Y.K and K.N.) and the New Energy and Industrial Technology Development Organization, Japan (T.Y., Y.K., and K.N.). REFERENCES (1) Li, W.; Ruf, S.; Bock, R. Mol. Genet. Genomics. 2006, 275, 185-192. (2) Bock, R. Methods. Mol. Biol. 2014, 1132, 93-106. (3) Larosa, V.; Remacle, C. Int. J. Dev. Biol. 2013, 57, 659-665. (4) Maliga, P.; Bock, R. Plant Physiol. 2011, 155, 1501-1510. (5) Boynton, J. E.; Gillham, N. W.; Harris, E. H.; Hosler, J. P.; Johnson, A. M.; Jones, A. R.; Randolph-Anderson, B. L.; Robertson, D.; Klein, T. M.; Shark, K. B.; Et Al. Science 1988, 240, 1534-1538. (6) Svab, Z.; Hajdukiewicz, P.; Maliga, P. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8526-8530. (7) Ruf, S.; Hermann, M.; Berger, I. J.; Carrer, H.; Bock, R. Nat. Biotechnol. 2001, 19, 870875. (8) Bock, R. Annu. Rev. Plant Biol. 2015, 66, 211-241. (9) Yu, Q.; Lutz, K. A.; Maliga, P. Plant Physiol. 2017, 175, 186-193. (10) Sikdar, S. R. ; Serino, G. ; Chaudhuri, S. ; Maliga, P. Plant Cell Rep. 1998, 18, 4. (11) Chinnery, P. F.; Taylor, R. W.; Diekert, K.; Lill, R.; Turnbull, D. M.; Lightowlers, R. N. Gene Ther. 1999, 6, 1919-1928. (12) Flierl, A.; Jackson, C.; Cottrell, B.; Murdock, D.; Seibel, P.; Wallace, D. C. Mol. Ther. 2003, 7, 550-557. (13) Yu, H.; Koilkonda, R. D.; Chou, T. H.; Porciatti, V.; Ozdemir, S. S.; Chiodo, V.; Boye, S. L.; Boye, S. E.; Hauswirth, W. W.; Lewin, A. S.; Guy, J. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, E1238-1247. (14) Chuah, J. A.; Yoshizumi, T.; Kodama, Y.; Numata, K. Sci. Rep. 2015, 5, 7751. (15) Chuah, J. A.; Matsugami, A.; Hayashi, F.; Numata, K. Biomacromolecules 2016, 17, 3547-3557. (16) Lakshmanan, M.; Kodama, Y.; Yoshizumi, T.; Sudesh, K.; Numata, K. Biomacromolecules 2013, 14, 10-16. (17) Li, Hm; Chen, L. J. J. Biol. Chem. 1997, 272, 10968-10974. (18) Li, H. M.; Teng, Y. S. Trends Plant Sci. 2013, 18, 360-366. (19) Hurt, E. C.; Pesold-Hurt, B.; Schatz, G. EMBO J. 1984, 3, 3149-3156. (20) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (21) Numata, K.; Ohtani, M.; Yoshizumi, T.; Demura, T.; Kodama, Y. Plant Biotechnol. J. 2014, 12, 1027-1034. (22) Parker, N.; Wang, Y.; Meinke, D. Plant Physiol. 2014, 166, 2013-2027. (23) Kodama, Y. PLoS One 2016, 11, e0152484. (24) Oikawa, K.; Yamasato, A.; Kong, S. G.; Kasahara, M.; Nakai, M.; Takahashi, F.; Ogura, Y.; Kagawa, T.; Wada, M. Plant Physiol. 2008, 148, 829-842. (25) Nakatani, H. Y.; Barber, J. Biochim. Biophys. Acta 1977, 461, 500-512.


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Selective Gene Delivery for Integrating Exogenous DNA into Plastid

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