Subcellular localization and BiFC assays
For subcellular localization analysis of 35S::CPT3 , A. tumefaciens cells carrying the vectors CPT3-GFP and cd3-954 (ER-CFP, used as an organelle marker) were introduced into the abaxial side ofNicotiana benthamiana leaves. A BiFC assay was performed based on split EYFP. EYFP was fused to the C-terminus of CPT3 and the N-terminus of Lew1, resulting in the expression of CPT3:EYFPC and Lew1:EYFPN. CPT3:EYFPC was co-infiltrated with Lew1:EYFPN into the abaxial side ofN. benthamiana leaves. A positive fluorescence signal (EYFP) is indicative of the restoration of EYFP due to the heterodimerization of CPT3 with Lew1.
The transient expression of CPT3, ER-CFP, and CPT3/Lew1-YFP fusion proteins was observed under a Nikon C1 confocal system built on TE2000E with 408, 488 and 543 nm laser excitations for CFP (450/35 nm emission filter) and GFP (515/30 nm emission filter), respectively.