Direct protein introduction into plant cells using a multi-gas plasma jet

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, CO(2) or N(2) plasma was generated using a multi-gas plasma jet. Confocal microscopy indicated that sGFP signals were observed inside of leaf cells after treatment with CO(2) or N(2) 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 CO(2) or N(2) plasma, also indicating the introduction of sGFP-fused adenylate cyclase into the cells. These results suggested that treatment with CO(2) or N(2) plasma could be a useful technique for protein introduction into plant tissues.