Gel imager

Restriction fragment length analysis was performed on the specific PCR, 16S rDNA sequencing and MALDI-TOF/MS confirmed isolates to observe that whether these isolates belongs to similar strains (clone of a single strain) or belongs to different strains. The primers fliC forward 5′-CTC GGA TCCAAC AGC AAC-3′ and fliC reverse 5′-TAT TGC AGG TAC CTT CAG-3′ were used for the amplification of the 1167 bp fliC gene. The PCR amplification was carried out in a 50 μl reaction volume with the following PCR conditions: initial denaturation at 95°C for 6 min and 30 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 1 min and extension at 72°C for 2 min with the final extension at 72°C for 10 min. PCR products were purified using a gel extraction purification kit (Qiagen) and concentration was determined spectrophotometrically. Restriction digestion was performed with three restriction enzymes Dde I, Msp I and Pst I (Fermentas, fast digest). Restriction digestion reaction mixture contained 7 μl of purified fliC PCR products at a conc. of 100 ng/μl and 1U of restriction enzymes. Restriction digestion reaction was carried out in 0.5 ml tubes incubated at 37°C for 2 hrs and then inactivated by keeping tubes in a dry bath at 65°C for 10 minutes. Then digested products were electrophoresed on 1.8% agarose gel and visualized under UV in an Alpha Innotech Gel Imager.

The following experimental apparatus and instruments were used in this study: 5% CO₂ cell culture incubator (Thermo, Marietta, OH, USA) and a fluorescent microscope (TE2000, Nikon, Tokyo, Japan). cDNA was prepared on a Bio-Rad T100 thermal cycler, qPCR was performed using the Bio-Rad CFX connect real-time system (USA), and the quality of RNA was measured using a spectrophotometer (UNIC2802H, Shanghai, China). The Wet/Tank Blotting Systems (Bio-rad, Hercules, CA, USA) were used to perform the Western blotting. The signal intensities were captured by a Tanon 5200 chemiluminescence/fluorescence image analysis system (Tanon, Urumqi, China). The luciferase activity and TG contents were detected by the SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA, USA). Other apparatus involved in this study included a liquid nitrogen tank (Chart/Golden Phoenix Liquid Nitrogen Container USA), Microplate Centrifuge (Tiangen Biotech Co., Ltd., Beijing, China), and Gel Imager (Alpha Innotech, San Leandro, CA, USA).

Tobacco leaf material was harvested 2–3 days after infiltration and ground in liquid nitrogen. Infiltrated leaf tissue and non-infiltrated leaf tissue (as control) was incubated in SDS-buffer (0.1 g / 1 ml buffer) for 20 min at 60°C. The PAGE run with 10 μl sample on a 10% SDS-Gel for 1 h at 20 mA. A wet-immunoblot was performed at 4°C at 100 V in 1 h on Nitrocellulose membrane activated by Blotting-buffer (20% EtOH, 25 mM Tris, 192 mM glycine). Anti-HA (1:5000, Roche) and anti-rabbit (1:10000, HRP coupled, Roche) were used to detect MDP-COL4-CFP. The anti-GFP (1:5000, rat, Jackson ImmunoResearch) and anti-rat (1:10000, HRP coupled, Roche) were used to detect MDP-COL4-CFP and MAS-COL4-YFP. Results were visualized by detection of chemiluminescence (ECL-detection kit, Roche) in a gel imager (Alpha Innotech).