2.5 Bimolecular fluorescence complementation (BiFC)
For YFP fusion vector construction, the full length PpVIN2 coding
sequence (CDS) was synthesized and cloned into pSPYNE-35s at the
XbaI-BamHI restriction sites. Similarly, the PpINH1 CDS was
synthesized and cloned into pSPYCE-35s at the XbaI-BamHI sites. The
resulting plasmids were PpVIN2 -N-YFP (PpVIN2 -YN) andPpINH1 -C-YFP (PpINH1 -YC). Constructions were transiently
expressed in tobacco leaves as described by Glass et al. (2015).
Fluorescence signals and bright field images were obtained with a
confocal microscope (OLYMPUS, Japan). YFP and GFP emissions were
measured at the excitation wavelengths of 514 nm and 498 nm,
respectively.