Abstract
Protein introduction into cells is more difficult in plants than in mammalian cells, although it was reported that protein introduction was successful in shoot apical meristem and leaves only together with a cell-penetrating peptide. In this study, we tried to introduce superfolder green fluorescent protein (sGFP)-fused to adenylate cyclase as a reporter protein without a cell-penetrating peptide into the cells of tobacco leaves by treatment with atmospheric non-thermal plasmas. For this purpose, CO2 or N2 plasma was generated using a multi-gas plasma jet. Confocal microscopy indicated that sGFP signals were observed inside of leaf cells after treatment with CO2 or N2 plasma without substantial damage. In addition, the amount of cyclic adenosine monophosphate (cAMP) formed by the catalytic enzyme adenylate cyclase, which requires cellular calmodulin for its activity, was significantly increased in leaves treated with CO2 or N2 plasma, also indicating the introduction of sGFP-fused adenylate cyclase into the cells. These results suggested that treatment with CO2 or N2 plasma could be a useful technique for protein introduction into plant tissues.
Highlights
Protein introduction into organic cells is a valuable technique for basic research and for industrial uses
To select suitable gas sources for protein introduction by plasma treatment, a solution with purified cell-penetrating peptides (CPPs) R8 (RRRRRRRR)-fused superfolder green fluorescent protein (sGFP)-CyaA (His-sGFP-CyaA-R8) (Fig 2A) was applied to leaf pieces that had been treated with various gas plasmas controlled at approximately 20–30 ̊C
We demonstrated that His-sGFP-CyaA protein was introduced into cells of tobacco leaves (Figs 7 and 8), which have cell walls in addition to cell membranes using temperature controlled non-thermal atmospheric plasma generated with CO2 or N2 gas source
Summary
Protein introduction into organic cells is a valuable technique for basic research and for industrial uses. Protein introduction techniques such as transfection using cell-penetrating factors dependent on endocytosis and bacterial delivery have already been used, and various protein transfection reagents are available commercially [1,2,3]. There have been numerous reports using protein introduction techniques such as genome editing and functional inhibition of proteins in mammalian systems [2,3,4]. A previous report showed that florigen, which is a small protein that promotes flowering, was introduced into shoot apical meristems by soaking with cell-penetrating peptides (CPPs) [5]. The techniques using CPPs still have limitations in depending of the tissues and/or methods involved. Novel techniques for protein introduction into plant cells are needed in order expected to be developed to advance plant research
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