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

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

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

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 fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N2 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 N2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.

Resin Cement-Zirconia Bond Strengthening by Exposure to Low-Temperature Atmospheric Pressure Multi-Gas Plasma.

The purpose of this study was to investigate the effect of gas species used for low-temperature atmospheric pressure plasma surface treatment, using various gas species and different treatment times, on zirconia surface state and the bond strength between zirconia and dental resin cement. Three groups of zirconia specimens with different surface treatments were prepared as follows: untreated group, alumina sandblasting treatment group, and plasma treatment group. Nitrogen (N2), carbon dioxide (CO2), oxygen (O2), argon (Ar), and air were employed for plasma irradiation. The bond strength between each zirconia specimen and resin cement was compared using a tension test. The effect of the gas species for plasma irradiation on the zirconia surface was investigated using a contact angle meter, an optical interferometer, an X-ray diffractometer, and X-ray photoelectric spectroscopy. Plasma irradiation increased the wettability and decreased the carbon contamination on the zirconia surface, whereas it did not affect the surface topography and crystalline phase. The bond strength varied depending on the gas species and irradiation time. Plasma treatment with N2 gas significantly increased bond strength compared to the untreated group and showed a high bond strength equivalent to that of the sandblasting treatment group. The removal of carbon contamination from the zirconia surface and an increase in the percentage of Zr-O2 on the zirconia surface by plasma irradiation might increase bond strength.