Gene delivery into the plant mitochondria via organelle-specific peptides
- 571 Downloads
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.
KeywordsMicrospores Mitochondria mTPs Protoplasts Transformation Triticale
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.
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar
- 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 CrossRefGoogle Scholar