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Genetic Transformation of Plant Mitochondria with mTP-DNA Nano-Complexes
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Summary
Here, we present a protocol for gene delivery into plant cell mitochondria. This new method utilizes cell penetrating peptides with mitochondria targeting properties and a mitochondria-optimized aadA:gfp reporter cassette. It is suitable for transient and stable, organelle-specific gene transfer and expression in somatic and germ cells.
Abstract
We report a peptide based in vivo plant cell mitochondrial genetic engineering method. Peptide vectors were created by selecting mitochondrial protein sorting signal sequences that contain similar physical and chemical properties as a cell-penetrating peptide (CPP). In the presence of exogenous double-stranded DNA (dsDNA) the peptide vectors called mitochondrial targeting peptides (mTPs) form peptide-nucleic acid nanoparticles. The nanoparticles when incubated with plant cells first cross the cellular membrane and then cross the outer and inner mitochondrial membranes to deliver exogenous DNA into the mitochondria of plant cells. The exogenous DNA integrates into the mitochondrial genome and the transfected plant cells can be cultured into plantlets. A linear double stranded gene construct was delivered into protoplasts and microspores of AC Ultima spring triticale (X. Triticosecale Wittmack) plants using the mTP peptide vector system to study in vivo mitochondrial transfection of plant cells and transient mitochondrial gene expression.
Introduction
Mitochondria are organelles that supply energy for metabolic activities in eukaryotic cells. In plants, the mitochondria are involved not only with energy conversion during aerobic respiration and carbon metabolism in the Krebs cycle, but also to chloroplast function and stress responses1. The ability to manipulate the mitochondrial genome will facilitate the study of plant primary and secondary metabolism. In addition, plant mitochondrial genetic engineering has many important applications in agricultural crop breeding and hybrid maintenance due to the mitochondria being linked to cytoplasmic male sterility2.
Protocol
1. Preparation of culture media and cells
- Stock solutions
- NPB-99 Macro-salts (10x). Prepare 1 L of 10x Macro-salts stock solution by dissolving 14.15 g of KNO3, 2.32 g of (NH4)2SO4, 2.0 g of KH2PO4, 0.83 g of CaCl2·2H2O and 0.93 g of MgSO4·7H2O in 500 mL of ddH2O. Stir the solution at room temperature and bring the volume to 1 L with ddH2O.<.......
Representative Results
DNA binding and protection properties of mTPs
The ability of mTPs to non-covalently bind nucleic acids is an essential property for translocation of dsDNA cargoes into the plant mitochondria. The minimum peptide amount needed to bind to and completely saturate the linearized dsDNA in preparation for microspore transfection was determined in a gel mobility shift assay and a nuclease protection assay. Titration of the mTP peptide was performed by adding increasing amounts of the.......
Discussion
Genetic engineering of mitochondria is a key tool in plant biology and biotechnology but has been very limited in its utility so far due to restrictions in DNA delivery into mitochondrial organelles. Biolistics and electroporation have proven to be popular methods of gene delivery into eukaryotic and prokaryotic cells40, but there are limitations when applying these technologies to in vivo plant mitochondrial genetic transformation. Frequently mitochondrial transformation rates are low
Acknowledgements
The authors thank Bernie Genswein for his computational assistance as well as the Kovalchuk Laboratory, Eric Amundsen and Victoria Hodgson for their technical assistance in the lab. This work was supported by Agriculture and Agri-Food Canada and Agriculture Funding Consortium (Alberta Innovates/Biosolutions, Alberta Wheat Commission and Saskatchewan Wheat Development Commission).
....Materials
| Name | Company | Catalog Number | Comments |
| Chemicals | |||
| (NH4)2SO4 | Sigma | A3920 | Component of 10x Macrosalts solution |
| 2,4-Dichlorophenoxyacetic acid (2,4-D) | Sigma | D8407 | Component of NPB-99 medium |
| Agar | Sigma | A7921 | Solidifying agent 2; Component of rooting medium |
| a-ketoglutaric acid | Sigma | K1750 | Component of GEM medium |
| Arabinogalactan (Larcoll) | Sigma | L0650 | Component of NPB-99 and GEM media |
| Ascorbic acid | Sigma | A4544 | Component of 100x GEM vitamins solution |
| Betaine chloride | Sigma | B7045 | Component of 100x GEM vitamins solution |
| Biotin | Sigma | B3399 | Component of 100x GEM vitamins solution |
| CaCl2, 2H2O | Sigma | C7902 | Component of 10x Macrosalts solution |
| Choline-HCL | Sigma | C-1879 | Component of 100x GEM vitamins solution |
| Citric acid | Sigma | C4540 | Component of GEM medium |
| CoCl, 6H2O | Fisher | C371 | Component of 100x Microsalts solution |
| CuSO4, 5H2O | Fisher | C489 | Component of 100x Microsalts solution |
| FeSO4, 7H2O | Sigma | F8263 | Component of 100x Fe-EDTA solution |
| Ficoll | Sigma | F4375 | Component of NPB-99 culture medium |
| Folic Acid | Sigma | F8509 | Component of 100x GEM vitamins solution |
| Fumaric acid | Sigma | F8509 | Component of GEM medium |
| Gelrite | Sigma | G1910 | Solidifying agent 1; Component of GEM medium |
| Glutamine | Sigma | G8540 | Component of NPB-99 and GEM media |
| H3BO3 | Sigma | B9545 | Component of 100x Microsalts solution |
| KH2PO4 | Sigma | P5655 | Component of 10x Macrosalts solution |
| KH2PO4 | Sigma | P9791 | Component of 10x Macrosalts solution |
| KI | Sigma | P8166 | Component of 100x Microsalts solution |
| Kinetine | Sigma | K0753 | Component of NPB-99 medium |
| KNO3 | Sigma | P8291 | Component of 10x Macrosalts solution |
| Malic acid | Sigma | M1000 | Component of GEM medium |
| Maltose | Sigma | M5895 | Component of NPB-99 and GEM media |
| MgSO4, 7H2O | Sigma | M5921 | Component of 10x Macrosalts solution |
| MitoTracker Orange CM-H2TMRos | Invitrogen | M7511 | Mitochondria-specific dye for confocal microscopy analysis |
| MnSO4, H2O | Sigma | M7899 | Component of 100x Microsalts solution |
| Myo-Inositol | Sigma | I3011 | Component of NPB-99 and GEM media |
| Na2EDTA | Sigma | E5134 | Component of 100x Fe-EDTA solution |
| NH4NO3 | Sigma | A3795 | Component of rooting medium |
| Nicotinic acid | Sigma | N0761 | Component of 100x NPB-99 and GEM vitamins solutions |
| Pantothenate | Sigma | A7219 | Component of 100x GEM vitamins solution |
| Phenylacetic acid (PAA) | Sigma | P6061 | Component of NPB-99 and GEM media |
| Plant preservative mixture (PPM) | Plant Cell Technologies | Component of GEM medium | |
| Pyridoxine- HCl | Sigma | P8666 | Component of 100x NPB-99 and GEM vitamins solutions |
| Pyruvic acid | Sigma | P2256 | Component of GEM medium |
| Riboflavin | Sigma | C4540 | Component of 100x GEM vitamins solution |
| Ribose | Sigma | R7500 | Component of GEM medium |
| Spermidine | Sigma | 5292 | Component of GEM medium |
| Spermine | Sigma | S4264 | Component of GEM medium |
| Streptomysin sulphate | Sigma | S9137 | Selection agent |
| Succinic acid | Sigma | S7501 | Component of GEM medium |
| Sucrose | Sigma | S7903 | Component of GEM medium and rooting medium |
| Thiamine-HCl | Sigma | T1270 | Component of 100x NPB-99 and GEM vitamins solutions |
| U2.5 Amino Acid Mixture | sigma | U-7756 | Component of GEM medium |
| Xylose | Sigma | X1500 | Component of GEM medium |
| ZnSO4, 7H2O | Sigma | Z1001 | Component of 100x Microsalts solution |
| Kits & Enzymes | |||
| All Prep DNA/RNA mini kit | Qiagen | 80204 | gDNA and RNA extraction |
| DNeasy Plant Mini Kit | Qiagen | 69106 | gDNA isolation |
| GeneRuler 1 kb DNA Lader | Thermo Scientific | 11823963 | DNA size marker for agarose elecrtophoresis |
| GeneRuler Low Range DNA Lader | Thermo Scientific | 10212840 | DNA size marker for agarose elecrtophoresis |
| pCR 2.1 Vector TA Cloning Kit | Invitrogen | K204001 | Cloning of PCR amplicons |
| Plasmid Maxi kit | Qiagen | 12163 | Plasmid isolation |
| QIAquick Gel extraction kit | Qiagen | 28706 | Extraction of PCR products from agarose gels |
| QIAquick PCR purification kit | Qiagen | 28104 | Purification of linearized plazmid |
| QuantiTect SYBR Green PCR Master Mix | Qiagen | 204143 | Real time RT-PCR |
| RNase-free DNase set | Qiagen | 79256 | Dnase I treatment |
| SuperScript III First-Strand Synthesis Kit | Invitrogen | 18080051 | cDNA synthesis |
| Taq DNA polymerase kit | Qiagen | 201205 | Standard PCR |
| Other materials | |||
| 150 mm Petri dishes | ThermoFisher | 150350 | Microspore culture, multi 35 mm Petri dish container |
| 35 mm Petri dishes | Fisher Scientific | 0875100A | Microspore culture |
| 60 mm Petri dishes | Fisher Scientific | 0875113A | Microspore culture, water reservoir |
| 90 mm Petri dishes | ThermoFisher | 101IRR20 | Embryo germination |
| Cell Lyzer | Bertin Technologies | Disruption and homogenization of plant cells and tissues | |
| Magenta vessels | Sigma | V 8505 | Plantlet rooting |
| Semi-transparent flexible film (Parafilm) | ThermoFisher | 5833-0001 | Plate sealing |
| Sylvania Gro-lux wide spectrum bulbs (40 watts) | Sylvana | Growth chamber lights | |
| Vacucap 60PF 0.2 UM | VWR | CA28139-704 | Filter sterilization of media and solutions |
References
- Millar, A. H., et al. Organization and Regulation of Mitochondrial Respiration in Plants. Annual Review of Plant Biology. 62 (1), 79-104 (2011).
- Frei, U., Peiretti, E. G., Wenzel, G., Janick, J. Significance of cytoplasmic DNA in plant ....
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