Transient expression in N. benthamiana and confocal laser
scanning microscopy
The preparation of Agrobacterium and transient expression
in N. benthamiana was performed as previously described (Li,
2011). The vector pB7FWG2:35S:TaA6b-GFP was transformed
into A. tumefaciens GV3101. A. tumefaciens GV3101
harboring the binary vector was grown in LB-broth media containing
50 mg/L of spectinomycin, 30 mg/L gentamycin, 30 mg/L of rifampin to the
stationary phase at 28 °C. After centrifugation, A.
tumefaciens was resuspended in the infiltration medium (10 mM
MgCl2, 100 μM acetosyringone) to a final
OD600 of 0.5. The suspension was infiltrated with a 1-mL
needleless syringe into the abaxial surface of 4-week-old leaves
of N. benthamiana . Protein localization was analyzed 24-48 hours
after infiltration by confocal laser scanning microscope (TCS SP8,
Leica). GFP was excited at 488 nm and the fluorescence detected between
498-525 nm.