Temperature-controlled atmospheric-pressure plasma treatment induces protein uptake via clathrin-mediated endocytosis in tobacco cells

Abstract

Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescenttagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N 2 plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N 2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma

“…We also showed that an sGFP fusion protein was introduced into plant cells upon treatment with Ar plasma, although the results observed using Ar plasma were not better than those observed using N 2 or CO 2 plasma [ 13 ]. The mechanism underlying introduction of proteins by plasma into cells via endocytosis is almost similar in both plants and mammalian cells; however, previous studies have used Ar or air plasma for the treatment of mammalian cells [ 14 , 23 , 24 ]. These results imply that plasma generated by other gases, such as Ar, may work to introduce Cas9/sgRNA into plant cells, although plasma generated from different gas sources produce qualitatively and quantitatively distinct reactive species that can influence protein uptake [ 25 , 26 ].…”