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Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers.

Chuah JA, Yoshizumi T, Kodama Y, Numata K - Sci Rep (2015)

Bottom Line: Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide.We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency.Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

View Article: PubMed Central - PubMed

Affiliation: Enzyme Research Team, Biomass Engineering Program Cooperation Division, Center for Sustainable Resource Science, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

ABSTRACT
Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

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Related in: MedlinePlus

GFP expression in the mitochondria of A. thaliana leaf 12 h after transfection.(a) Western blot analysis of crude mitochondrial extracts from leaves previously infiltrated with naked pDNA, or complexes of MTPKH (N/P 0.5) or CPP-MTPKH (N/P 0.5 for each peptide) with pDNA. Data is representative of two independent experiments. (b) Confocal laser scanning microscope observation of epidermal cells of a leaf previously infiltrated with CPP-MTPKH-pDNA complexes (N/P 0.5 for each peptide). Enlarged images of several mitochondria with GFP expression are shown in the extreme right panel. Scale bars indicate 10 μm.
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f3: GFP expression in the mitochondria of A. thaliana leaf 12 h after transfection.(a) Western blot analysis of crude mitochondrial extracts from leaves previously infiltrated with naked pDNA, or complexes of MTPKH (N/P 0.5) or CPP-MTPKH (N/P 0.5 for each peptide) with pDNA. Data is representative of two independent experiments. (b) Confocal laser scanning microscope observation of epidermal cells of a leaf previously infiltrated with CPP-MTPKH-pDNA complexes (N/P 0.5 for each peptide). Enlarged images of several mitochondria with GFP expression are shown in the extreme right panel. Scale bars indicate 10 μm.

Mentions: As a final step, the efficiency of our designed peptide-based carrier was verified by delivery of pDNA containing a gene that encodes another well-known reporter, the green fluorescent protein (GFP), also under the control of the same mitochondrial COX-2 promoter. For comparison, leaves were infiltrated with either naked pDNA, MTPKH-pDNA or CPP-MTPKH-pDNA, and mitochondrial expression of GFP was subsequently detected by Western blotting using an anti-GFP antibody. Of the three different formulations, only the CPP-MTPKH combination mediated pDNA delivery and significant levels of GFP expression, as evident from the band corresponding to the size of 27 kDa GFP (Fig. 3a). Confocal laser scanning microscopy provided visual confirmation of GFP expression, localized in the mitochondria of epidermal cells of leaves transfected using the optimized peptide-pDNA formulation (MTPKH and CPP at N/P 0.5 each) (Fig. 3b).


Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers.

Chuah JA, Yoshizumi T, Kodama Y, Numata K - Sci Rep (2015)

GFP expression in the mitochondria of A. thaliana leaf 12 h after transfection.(a) Western blot analysis of crude mitochondrial extracts from leaves previously infiltrated with naked pDNA, or complexes of MTPKH (N/P 0.5) or CPP-MTPKH (N/P 0.5 for each peptide) with pDNA. Data is representative of two independent experiments. (b) Confocal laser scanning microscope observation of epidermal cells of a leaf previously infiltrated with CPP-MTPKH-pDNA complexes (N/P 0.5 for each peptide). Enlarged images of several mitochondria with GFP expression are shown in the extreme right panel. Scale bars indicate 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291575&req=5

f3: GFP expression in the mitochondria of A. thaliana leaf 12 h after transfection.(a) Western blot analysis of crude mitochondrial extracts from leaves previously infiltrated with naked pDNA, or complexes of MTPKH (N/P 0.5) or CPP-MTPKH (N/P 0.5 for each peptide) with pDNA. Data is representative of two independent experiments. (b) Confocal laser scanning microscope observation of epidermal cells of a leaf previously infiltrated with CPP-MTPKH-pDNA complexes (N/P 0.5 for each peptide). Enlarged images of several mitochondria with GFP expression are shown in the extreme right panel. Scale bars indicate 10 μm.
Mentions: As a final step, the efficiency of our designed peptide-based carrier was verified by delivery of pDNA containing a gene that encodes another well-known reporter, the green fluorescent protein (GFP), also under the control of the same mitochondrial COX-2 promoter. For comparison, leaves were infiltrated with either naked pDNA, MTPKH-pDNA or CPP-MTPKH-pDNA, and mitochondrial expression of GFP was subsequently detected by Western blotting using an anti-GFP antibody. Of the three different formulations, only the CPP-MTPKH combination mediated pDNA delivery and significant levels of GFP expression, as evident from the band corresponding to the size of 27 kDa GFP (Fig. 3a). Confocal laser scanning microscopy provided visual confirmation of GFP expression, localized in the mitochondria of epidermal cells of leaves transfected using the optimized peptide-pDNA formulation (MTPKH and CPP at N/P 0.5 each) (Fig. 3b).

Bottom Line: Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide.We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency.Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

View Article: PubMed Central - PubMed

Affiliation: Enzyme Research Team, Biomass Engineering Program Cooperation Division, Center for Sustainable Resource Science, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

ABSTRACT
Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

Show MeSH
Related in: MedlinePlus