The recombinant plasmids (35Sprom::SCI1-GFP and the nuclear markers AtFibrillarin-mRFP and AtCoilin-mRFP—Koroleva et al., 2009 (link)) were transformed into A. tumefaciens strain C58C1(pGV2260) and infiltrated on leaves of 5–6 weeks old N. benthamiana plants, as previously described (Bracha et al., 2002 (link)). Fluorescence detection was observed 24–72 h following infiltration. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) and examined under a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems—Germany). GFP excitation was done with an argon laser at 488 nm, and the spectral detector was set between 500 and 550 nm, while RFP excitation was done with a laser at 543 nm and a spectral detector set between 630 and 680 nm. The fluorescence of DAPI was obtained with excitation at 340–380 nm and captured at 425–450 nm. Image analysis was carried out with Leica LCS, ImageJ, and Illustrator CS6.
To verify interaction partners in planta, the coding sequences of SCI1, 14-3-3A1 and 14-3-3D2 were subcloned into pK7m34GW or pH7m34GW vectors with nGFP or cGFP fragments, under the control of 35S promoter (Karimi et al., 2007 (link)). These split-GFP Gateway-based vectors were infiltrated in N. benthamiana leaves and used for BiFC analysis, as Bracha-Drori et al. (2004) (link) described.
To verify interaction partners in planta, the coding sequences of SCI1, 14-3-3A1 and 14-3-3D2 were subcloned into pK7m34GW or pH7m34GW vectors with nGFP or cGFP fragments, under the control of 35S promoter (Karimi et al., 2007 (link)). These split-GFP Gateway-based vectors were infiltrated in N. benthamiana leaves and used for BiFC analysis, as Bracha-Drori et al. (2004) (link) described.