Abstract
We report a method for genetically engineering the mitochondria of plant cells. Several peptides selected from sorting signal sequences of plant mitochondrial proteins were tested for cell-penetrating, specific organelle-targeting and nucleic acid-binding properties. Selected sequences were named mitochondrial targeting peptides (mTPs). Five of them were used to deliver a linear dsDNA gene construct into AC Ultima spring triticale (X. Triticosecale Wittmack) protoplasts and microspores. The mitochondrial transient expression of the aadA:gfp reporter gene was qualitatively determined with confocal microscopy and quantitatively measured using qRT-PCR. Significant aadA:gfp transcript abundance was reported in protoplasts from deliveries of all five mTPs, while this transcript abundance remained low in microspores. mTP1 transfected microspores were cultured to produce green plantlets and screened for aadA:gfp by endpoint PCR. Targeted gene integration into the mitochondrial genome of eight regenerated haploid triticale lines was confirmed by sequencing. This represents the first report of a successful in vivo transfection of crop plant somatic and germ cells via mitochondrial peptides and a method that can be used to study transient gene expression and to stably deliver genes into the mitochondria of a plant.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bolender N, Sickmann A, Wagner R, Meisinger C, Pfanner N (2008) Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep 9:42–49. https://doi.org/10.1038/sj.embor.7401126
Bonnefoy N, Remacle C, Fox TD (2007) Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria. Methods Cell Biol 80:525–548. https://doi.org/10.1016/S0091-679X(06)80026-9
Chuah J-A, Yoshizumi T, Kodama Y, Numata K (2015) Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers. Sci Rep 5:7751. https://doi.org/10.1038/srep07751
Chuah J-A, Matsugami A, Hayashi F, Numata K (2016) Self-assembled peptide-based system for mitochondrial-targeted gene delivery: functional and structural insights. BioMolecules 17:3547–3557. https://doi.org/10.1021/acs.biomac.6b01056
Chugh A, Amundsen E, Eudes F (2009) Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores. Plant Cell Rep 28:801–810. https://doi.org/10.1007/s00299-009-0692-4
Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241:779–786
Cornelissen M, Vandewiele M (1989) Nuclear transcriptional activity of the tobacco plastid psbA promoter. Nucl Acids Res 17(1):19–29
Cotlet M, Goodwin PM, Waldo GS, Werner J (2006) A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation. ChemPhysChem 7:250–260. https://doi.org/10.1002/cphc.200500247
Del Gaizo V, Payne RM (2003) A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta. Mol Ther 7:720–730
Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016. https://doi.org/10.1006/jmbi.2000.3903
Eudes F, Amundsen E (2005) Isolated microspore culture of Canadian 6x triticale cultivars. Plant Cell Tissue Org Cult 82:233–241
Farré J-C, Araya A (2001) Gene expression in isolated plant mitochondria: high fidelity of transcription, splicing and editing of a transgene product in electroporated organelles. Nucl Acids Res 29:2484–2491
Flierl A, Jackson C, Cottrell B, Murdock D, Seibel P, Wallace DC (2003) Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid. Mol Ther 7:550–557
Frearson EM, Power JB, Cocking EC (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Develop Biol 33:130–137
Frei U, Peiretti EG, Wenzel G (2010) Significance of cytoplasmic DNA in plant breeding. In: Janick J (ed) Plant breeding reviews, vol 23. Wiley, Hoboken, New Jersey, pp 175–210
Guda C, Guda P, Fahy E, Subramaniam S (2004) MITOPRED: a web server for the prediction of mitochondrial proteins. Nucl Acids Res 32:W372–W374. https://doi.org/10.1093/nar/gkh374
Herce HD, Garcia (2007) Cell penetrating peptides: how do they do it? J Biol Phys 33:345–356. https://doi.org/10.1007/s10867-008-9074-3
Horton KL, Stewart KM, Fonseca SB, Guo Q, Kelley SO (2008) Mitochondria-penetrating peptides. Chem Biol 15:375–382. https://doi.org/10.1016/j.chembiol.2008.03.015
Huang Y, Niu B, Gao Y, Fu L, Li W (2010) CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics 26:680–682. https://doi.org/10.1093/bioinformatics/btq003
Ibrahim N, Handa H, Cosset A, Koulintchenko M, Konstantinov Y, Lightowlers RN, Dietrich A, Weber-Lotfi F (2011) DNA delivery to mitochondria: sequence specificity and energy enhancement. Pharm Res 28:2871–2882. https://doi.org/10.1007/s11095-011-0516-4
José MS-S, Fernando N (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol Plant 134:1–12
Kanki T, Nakayama H, Sasaki N, Takio K, Alam TI, Hamasaki N, Kang D (2004) Mitochondrial nucleoid and transcription factor A. Ann N Y Acad Sci 1011:61–68
Keeney PM, Quigley CK, Dunham LD, Papageorge CM, Iyer S, Thomas RR, Schwarz KM, Trimmer PA, Khan SM, Portell FR, Bergquist KE, Bennett JP Jr (2009) Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson’s disease cell model. Hum Gene Ther 20:897–907. https://doi.org/10.1089/hum.2009.023
Khan MS, Maliga P (1999) Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat Biotechnol 17:910–915. https://doi.org/10.1038/12907
Koulintchenko M, Konstantinov Y, Dietrich A (2003) Plant mitochondria actively import DNA via the permeability transition pore complex. EMBO J 22:1245–1254. https://doi.org/10.1093/emboj/cdg128
Leon P, Walbot V, Bedinger P (1989) Molecular analysis of the linear 2.3 kb plasmid of maize mitochondria: apparent capture of tRNA genes. Nucl Acids Res 17:4089–4099
Lonsdale DM, Brears T, Hodge TP, Melville SE, Rottmann WH (1988) The plant mitochondrial genome - homologous recombination as a mechanism for generating heterogeneity. Philos Trans R Soc Lond B 319:149–163
Madani F, Linberg S, Langel U, Futaki S, Graslund A (2011) Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys. https://doi.org/10.1155/2011/414729
Maréchal A, Brisson N (2010) Recombination and the maintenance of plant organelle genome stability. New Phytol 186:299–317. https://doi.org/10.1111/j.1469-8137.2010.03195.x
Matsushima R, Hamamura Y, Higashiyama T, Arimura S, Tsutsumi N, Sakamoto W (2008) Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging. Plant Cell Physiol 49:1074–1083. https://doi.org/10.1093/pcp/pcn084
Mileshina D, Koulintchenko M, Konstantinov Y, Dietrich A (2011) Transfection of plant mitochondria and in organello gene integration. Nucleic Acids Res 39(17):e115. https://doi.org/10.1093/nar/gkr517
Millar AH, Whelan J, Soole KL, Day DA (2011) Organization and regulation of mitochondrial respiration in plants. Ann Rev Plant Biol 62:79–104. https://doi.org/10.1146/annurev-arplant-042110-103857
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–35
Neupert W, Herrmann JM (2007) Translocation of proteins into mitochondria. Ann Rev Biochem 76:723–749. https://doi.org/10.1146/annurev.biochem.76.052705.163409
Ogihara Y, Yamazaki Y, Murai K, Kanno A, Terachi T, Shiina T, Miyashita N, Nasuda S, Nakamura C, Mori N, Takumi S, Murata M, Futo S, Tsunewaki K (2005) Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome. Nucleic Acids Res 33:6235–6250. https://doi.org/10.1093/nar/gki925
Placido A, Gagliardi D, Gallerani R, Grienenberger J-M, Maréchal-Drouard L (2005) Fate of a larch unedited tRNA precursor expressed in potato mitochondria. J Biol Chem 280:33573–33579. https://doi.org/10.1074/jbc.M505269200
Remacle C, Cardol P, Coosemans N, Gaisne M, Bonnefoy N (2006) High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes. Proc Natl Acad Sci USA 103:4771–4776. https://doi.org/10.1073/pnas.0509501103
Scheller A, Oehlke J, Wiesner B, Dathe M, Krause E, Beyermann M, Melzig M, Bienert M (1999) Structural requirements for cellular uptake of α-helical amphipathic peptides. J Pep Sci 5:185–194. https://doi.org/10.1002/(SICI)1099-1387(199904)5:4%3C185::AID-PSC184%3E3.0.CO;2-9
Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569–1572
Seibel P, Trappe J, Villani G, Klopstock T, Papa S, Reichmann H (1995) Transfection of mitochondria—strategy towards a gene-therapy of mitochondrial-DNA diseases. Nucleic Acids Res 23:10–17
Shen Y, Maupetit J. Derreumaux P, Tufféry P (2014) Improved PEP-FOLD approach for peptide and miniprotein structure prediction. J Chem Theor Comput 10(10):4745–4758. https://doi.org/10.1021/ct500592m
Sieber F, Placido A, El Farouk-Ameqrane S, Duchêne A-M, Maréchal-Drouard L (2011) A protein shuttle system to target RNA into mitochondria. Nucl Acids Res 39:e96. https://doi.org/10.1093/nar/gkr380
Small I, Peeters N, Legeai F, Lurin C (2004) Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4:1581–1590. https://doi.org/10.1002/pmic.200300776
Vassarotti A, Stroud R, Douglas M (1987) Independent mutations at the amino terminus of a protein act as surrogate signals for mitochondrial import. EMBO J 6:705–711
Vestweber D, Schatz G (1989) DNA-protein conjugates can enter mitochondria via the protein import pathway. Nature 338:170–172. https://doi.org/10.1038/338170a0
Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272:16010–16017
von Heijne G (1986) Mitochondrial targeting sequences may form amphiphilic helices. EMBO J 5:1335–1342
Yoshizumi T, Oikawa K, Chuah J-A, Kodama Y, Numata K (2018) Selective gene delivery for integrating exogenous DNA into plastid and mitochondrial genomes using peptide-DNA complexes. Biomacromolecules 19:1582–1591. https://doi.org/10.1021/acs.biomac.8b00323
Yu H, Koilkonda RD, Chou TH, Porciatti V, Ozdemir SS, Chiodo V, Boye SL, Boye SE, Hauswirth WW, Lewin AS, Guy J (2012) Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model. Proc Natl Acad Sci USA 109(20):E1238–E1247. https://doi.org/10.1073/pnas.1119577109
Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 279:34682–34690. https://doi.org/10.1074/jbc.M402999200
Zhou J, Fan J, Hsieh J-T (2006) Inhibition of mitogen-elicited signal transduction and growth in prostate cancer with a small peptide derived from the functional domain of DOC-2/DAB2 delivered by a unique vehicle. Cancer Res 66:8954–8958. https://doi.org/10.1158/0008-5472.CAN-06-1726
Zhou J, Liu L, Chen J (2010) Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation. Eukaryot Cell 9:806–814. https://doi.org/10.1128/EC.00349-09
Acknowledgements
We express our appreciation for the contributions made by Bernie Genswein for his computational assistance as well as the Kovalchuk Laboratory, Eric Amundsen and Victoria Hodgson for their technical assistance in the lab.
Author information
Authors and Affiliations
Corresponding author
Additional information
The original version of this article was revised: Copyrights of the article has been changed to “Crown”.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
MacMillan, T., Ziemienowicz, A., Jiang, F. et al. Gene delivery into the plant mitochondria via organelle-specific peptides. Plant Biotechnol Rep 13, 11–23 (2019). https://doi.org/10.1007/s11816-018-0502-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11816-018-0502-y