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

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.

Improvement of yield and grain quality by periodic cold plasma treatment with rice plants in a paddy field

“…In agriculture, plasma treatment has a useful potential in a variety of production stages, including preplanting, preharvest, and postharvest. [ 1‐4 ] Specifically, previous studies have demonstrated the utility of cold plasma in protein induction, [ 5 ] promotion of seed germination [ 6‐9 ] and plant growth, sterilization/disinfection, [ 10,11 ] and food storage. [ 12 ] Recently, numerous studies have investigated the application of cold plasma to the growth enhancement of various plants, such as pea, [ 6 ] wheat, [ 7 ] Arabidopsis thaliana , [ 8 ] radish, [ 13‐16 ] barley, [ 17 ] tomato, [ 4,18 ] Chinese cabbage, [ 19 ] rye, [ 20 ] rice, [ 21 ] soybean, [ 22 ] and sunflower.…”

Influence of Gas Temperature in Atmospheric Non-Equilibrium Plasma on Bactericidal Effect

“…Additionally, the gas temperature of several thousand degrees causes heat damage. On the other hands, atmospheric non-equilibrium plasma has attracted attention as a new sterilization device for heat sensitive objects such as fibers, polymers, and living bodies because it has a wide antibacterial spectrum at low temperatures and shows residual toxicity unlike ethylene oxide gas Fridman et al, 2008;Graves, 2012;Takamatsu et al, 2013;Yanagawa et al, 2017 . In particular, a growing number of reports have shown evidence of bacterial inactivation on living bodies by direct plasma irradiation O Connor et al, 2014;Traba et al, 2013;van Gils et al, 2013 .…”

I-SceI Endonuclease-Mediated Plant Genome Editing by Protein Transport through a Bacterial Type III Secretion System

“…To evaluate protein transfer through the Xcc type III secretion system, we used CyaA as a reporter protein as we did previously to analyze protein introduction by plasma treatment [ 9 ]. In this previous study, the amount of cAMP produced by CyaA introduced by plasma treatment was 1.3–4.0 times greater than the amount of cAMP in a negative control; in the current study as shown in Figure 1 , the amount of cAMP produced by CyaA introduced by inoculation with Xcc carrying Xcc1_31290::CyaA was 80.3 times greater than in a negative control ( Xcc carrying an empty vector).…”

“…To evaluate protein transport into plant cells, the amount of cAMP was measured with an Amersham cAMP Biotrak Enzymeimmunoassay System according to the manufacturer’s instruction (GE Healthcare, Tokyo, Japan). Preparation of assay sample and assay method were performed as described previously [ 9 ]. Briefly, two leaf discs of 13-mm diameter from each infected leaf were prepared with a cork borer.…”

“…Cell-penetrating peptides are also useful to transfer proteins into plant cells using infiltration to leaves or stem cells, although no genome-edited plant has been produced yet [ 8 ]. We previously developed a technique to introduce protein directly into plant cells by using an atmospheric-pressure plasma [ 9 ]. In this work, we suggest a new technique for protein transfer into plant cells that uses phytopathogenic bacteria.…”