Plant Materials and Growth Conditions
Nicotiana benthamiana seeds were kindly provided by Dr. Yongjiang Zhang of the Chinese Academy of Inspection and Quarantine (CAIQ). N. benthamiana seedlings were grown at 22–25 °C with 16 hours dark period/8 hours light period and 65% humidity in small 5 inch diameter plastic pots. All seedlings were watered on daily basis and supplemented with Hoagland solution once a week when required.
Plasmids and Agrobacterium strains
The plasmids pJL TRBO-G, pCBNoX P19 and Agrobacterium tumefaciens GV3101 were kindly provided by Dr. John A. Lindbo of the Ohio State University. pJL TRBO-G, a Tobacco mosaic virus (TMV) RNA-based overexpression vector [10], was used to express GFP in N. benthamiana leaves using transient agroinfiltration. pCBNoX P19, expressing the 19 kDa silencing suppressor from Tomato bushy stunt virus (TBSV), was co-infiltrated with the pJL TRBO-G to prevent the RNAi-mediated gene silencing in plants. The plasmids were transformed into A. tumefaciens GV3101 using the freeze-thaw method.
Agroinfiltration of N. benthamiana plants
A. tumefaciens GV3101 carrying either pJL TRBO-G or pCBNoX P19, were grown in 4 ml LB media for 24 h with shaking (250 rpm) at 28°C. The cultures were then transferred into 100 ml LB media having 200 μM of acetosyringone (Sigma-Aldrich) grown overnight at 28°C with shaking (250 rpm). Cells were harvested by centrifugation at 3,000 g for 10 min and re-suspended in sterile, IM buffer (pH 5.6, 10 mM MES, 10 mM MgCl2 and200 μM acetosyringone) to achieve an OD600 of 0.4. The pJL TRBO-G expression vector was mixed in a 1:1 volume ratio with the gene-silencing suppressor (pCBNoX P19). The mixed Agrobacterium suspensions were incubated in the dark at room temperature for 2–3 h before infiltration. Then the incubated Agrobacterium suspensions were infiltrated into the abaxial surface of leaves using a 1-ml syringe without needle. The agroinfiltrated plants were incubated in the growth chamber for 4–8 days after which the leaves were harvested.
GFP imaging
The GFP fluorescence was monitored by illumination with a hand-held long-wave UV source (UVP Blak-Ray 100AP) and was photographed with a Cannon G6 digital camera. For microscopic analysis, GFP-positive leaf cells were visualized using confocal laser scanning microscopy (Leica TCS STED Microscopy).
Total soluble proteins extraction and protein quantification
Protein samples were prepared by freezing agroinfiltrated leaves in liquid nitrogen and then grinding to a fine powder with a mortar and pestle. The total soluble protein was obtained in two volumes of Extraction Buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA). Extracts were clarified by centrifugation at 12,000 g for 15 min at 4 °C. Total protein content was determined by BCA protein assay kit (Pierce). The concentration of GFP was measured by GFP ELISA Kit (Abcam).
Purification of GFP by alcohol/salt aqueous two-phase system (ATPS) and hydrophobic interaction chromatography (HIC)
0.3 ml of 5 M NaCl and 2.33 mL of saturated ammonium sulfate were added in turn to per milliliter of the total soluble protein. The entire solution was immediately mixed with one-third volume of anhydrous ethanol and vigorously shaken for 30 sec. The phases were separated by centrifugation at 3,000 g for 5 min and the upper ethanol phase was carefully collected. Then, one-fourth volume of n-butanol was added to the ethanol extracts. After shaking and centrifugation as described above, the lower aqueous phase containing GFP protein was recovered.
AKTA Purifier system (GE Healthcare) equipped with an HIC column was used for further purification of GFP protein. Ammonium sulfate was added to aqueous phase obtained from the previous step to give to final concentration of 1.7 M. The solution was filtered through a 0.22 mm filter (Millipore) and then loaded onto 4.7-ml HiScreen Capto Butyl column (GE Healthcare) equilibrated with Binding Buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1.7 M ammonium sulfate). After extensive washing with the Binding Buffer, the GFP was eluted at 1 ml/min with Elution Buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Then, the purified GFP was dialyzed with Elution Buffer.
SDS-PAGE and Western blot
Protein samples were subjected to 12% SDS-PAGE gels under reducing or non-reducing conditions with or without heat treatment of the samples according to the experimental requirement. After electrophoresis, gels were either stained with Coomassie brilliant blue or silver. For Western blot analysis, the proteins were transferred to a 0.45 μm nitrocellulose membrane (Sigma-Aldrich) using semi-dry electrophoresis transfer (Bio-Rad Trans-Blot SD system). The blot was developed with rabbit anti-GFP antiserum (Abcam) diluted 1:5,000, followed by secondary goat anti-rabbit antibody conjugated with alkaline phosphatase (Abcam, USA). Specific immunoreactive proteins were detected using a Western Blot ECL Plus kit (GE Healthcare).
Fluorescence spectroscopy
Samples were diluted in 10 mM Tris-HCl, 10 mM EDTA, pH 8.0 buffers and fluorescence spectra were recorded on a Hitachi F–4500 spectrophotometer at room temperature. Excitation spectra were measured between 350 and 500 nm with the emission wavelength fixed at 508 nm. Emission spectra were measured between 450 and 600 nm with the excitation wavelength fixed at 396 nm.