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Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

Bilichak A, Luu J, Eudes F - Front Plant Sci (2015)

Bottom Line: We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9).Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein.Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.

View Article: PubMed Central - PubMed

Affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada Lethbridge, AB, Canada.

ABSTRACT
Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.

No MeSH data available.


Schematic map of generated vectors used for purification of Cys-mCherry (A) and CPP-Cys-mCherry (B) proteins from bacterial culture. (C) SDS-PAGE analysis of the purified proteins: 1, Cys-mCherry; 2, penetratin-Cys-mCherry; 3, R9-Cys-mCherry; 4, transportan-Cys-mCherry; 5, MAP-Cys-mCherry; M, molecular weight protein marker.
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Figure 1: Schematic map of generated vectors used for purification of Cys-mCherry (A) and CPP-Cys-mCherry (B) proteins from bacterial culture. (C) SDS-PAGE analysis of the purified proteins: 1, Cys-mCherry; 2, penetratin-Cys-mCherry; 3, R9-Cys-mCherry; 4, transportan-Cys-mCherry; 5, MAP-Cys-mCherry; M, molecular weight protein marker.

Mentions: The mCherry protein was chosen as a cargo due to its stability, fast maturation time, high level of fluorescence, and low auto fluorescence level of microspores in the mCherry spectrum range. Four distinct CPPs were picked up for microspore transfection experiments based on their physical-chemical properties (Table 1). The nucleotide sequences coding for corresponding CPPs and 6xHis sequence were fused in-frame to the coding sequence of mCherry at the 5′- and 3′-ends, respectively, using PCR. The resulting PCR fragments were cloned into pET45b(+) plasmid (Figures 1A,B). The proteins were purified from bacterial culture with the final purity of more than 98% (Figure 1C). All mCherry fusions demonstrated same intensity of fluorescence in regard to their concentration indicating that CPPs did not affect the protein confirmation (Supplementary Figure 1). The mCherry protein bearing the cysteine at the N-terminus was used as a no-CPP control treatment for all microspore transfection experiments.


Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

Bilichak A, Luu J, Eudes F - Front Plant Sci (2015)

Schematic map of generated vectors used for purification of Cys-mCherry (A) and CPP-Cys-mCherry (B) proteins from bacterial culture. (C) SDS-PAGE analysis of the purified proteins: 1, Cys-mCherry; 2, penetratin-Cys-mCherry; 3, R9-Cys-mCherry; 4, transportan-Cys-mCherry; 5, MAP-Cys-mCherry; M, molecular weight protein marker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4552043&req=5

Figure 1: Schematic map of generated vectors used for purification of Cys-mCherry (A) and CPP-Cys-mCherry (B) proteins from bacterial culture. (C) SDS-PAGE analysis of the purified proteins: 1, Cys-mCherry; 2, penetratin-Cys-mCherry; 3, R9-Cys-mCherry; 4, transportan-Cys-mCherry; 5, MAP-Cys-mCherry; M, molecular weight protein marker.
Mentions: The mCherry protein was chosen as a cargo due to its stability, fast maturation time, high level of fluorescence, and low auto fluorescence level of microspores in the mCherry spectrum range. Four distinct CPPs were picked up for microspore transfection experiments based on their physical-chemical properties (Table 1). The nucleotide sequences coding for corresponding CPPs and 6xHis sequence were fused in-frame to the coding sequence of mCherry at the 5′- and 3′-ends, respectively, using PCR. The resulting PCR fragments were cloned into pET45b(+) plasmid (Figures 1A,B). The proteins were purified from bacterial culture with the final purity of more than 98% (Figure 1C). All mCherry fusions demonstrated same intensity of fluorescence in regard to their concentration indicating that CPPs did not affect the protein confirmation (Supplementary Figure 1). The mCherry protein bearing the cysteine at the N-terminus was used as a no-CPP control treatment for all microspore transfection experiments.

Bottom Line: We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9).Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein.Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.

View Article: PubMed Central - PubMed

Affiliation: Lethbridge Research Centre, Agriculture and Agri-Food Canada Lethbridge, AB, Canada.

ABSTRACT
Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.

No MeSH data available.