Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar, and rice callus

Keiji Numata,Taku Demura,Kazusato Oikawa,Yoko Horii,Misato Ohtani,Yu Miyagi

doi:10.1038/s41598-018-29298-6

Keiji Numata, Taku Demura + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-29298-6

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Abstract

Cell-penetrating peptides (CPPs) are used for various applications, especially in the biomedical field. Recently, CPPs have been used as a part of carrier to deliver proteins and/or genes into plant cells and tissues; hence, these peptides are attractive tools for plant biotechnological and agricultural applications, but require more efficient delivery rates and optimization by species before wide-scale use can be achieved. Here, we developed a library containing 55 CPPs to determine the optimal CPP characteristics for penetration of BY-2 cells and leaves of Nicotiana benthamiana, Arabidopsis thaliana, tomato (Solanum lycopersicum), poplar (hybrid aspen Populus tremula × tremuloides line T89), and rice (Oryza sativa). By investigating the cell penetration efficiency of CPPs in the library, we identified several efficient CPPs for all the plants studied except rice leaf. In the case of rice, several CPPs showed efficient penetration into rice callus. Furthermore, we examined the relationship between cell penetration efficiency and CPP secondary structural characteristics. The cell penetration efficiency of Lys-containing CPPs was relatively greater in plant than in animal cells, which could be due to differences in lipid composition and surface charge of the cell membranes. The variation in optimal CPPs across the plants studied here suggests that CPPs must be optimized for each plant species and target tissues of interest.

Highlights

BP100 (KKLFKKILKYL), which we previously used in a Cell-penetrating peptides (CPPs) system to deliver genes into plant leaf cells of A. thaliana and N. benthamiana[19], was used as the model CPP and Peptide No 1
We evaluated the relationship between cell penetration efficiency into BY-2 cells and the number of Lys or Arg residues in CPP sequences (Fig. S6)
We evaluated the effect of net charge of CPPs at pH 7 on the cell penetration efficiency into BY-2 cells (Fig. 6a)

Summary

Results and Discussion

The cell penetration efficiency of CPP into BY-2 cells was determined based on the ratio of numbers of cell with and without TAMRA signals of CPP by confocal laser scanning microscopy (CLSM) (Fig. 2). CPP concentration and approximately 380 cells/μL, which provided approximately 50% cell penetration efficiency of BP100 as the baseline, to examine the efficiencies of other CPPs. The peptide library was assayed with BY-2 cells under the optimal conditions determined above Based on CLSM images, the cell penetration efficiencies of the 55 CPPs into BY-2 suspension culture cells were determined and compared to that of BP100 (Fig. 5a). CPPs (Fig. 1a and Table 1), those eight efficient CPPs did not show high helix content This tendency related to the secondary structures indicates that predominant helical structures are not essential to penetrate into plant cells.

GTKMIFVGIKKKEERADLIAYLKKA hLF peptide

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