Fig. 11
From: One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells
Larger complex formation between AP1, AP3, PI and SEP3 proteins in N. benthamiana. Higher order complex formation of MADS-box proteins was analysed by a combination of BiFC with FRET-FLIM. Complemented mV by the n-terminal and c-terminal part of mV tagged to AP3 and PI respectively served as the donor in FRET-FLIM experiments. All MADS-domain protein fused to FP were expressed via the UBQ10 promoter. Untagged SEP3 was expressed from the same T-DNA as AP1-mCh using the XVE < < oLexA-35S estradiol inducible system. Images were acquired three days after infiltration and SEP3 was induced one day before imaging. BINDING [%] (grey) and FRET efficiencies [%] (white) for AP3-mVn PI-mVc, AP3-mVn PI-mVc NLS-mCh, AP3-mVn PI-mVc SEP3-mCh, AP3-mVn PI-mVc AP1-mCh and AP3-mVn PI-mVc AP1-mCh + SEP3. Analysis was done as described in Fig. 5. SEP3 together with AP3/PI displayed increased BINDING (23.21% ± 7.56) with a FRET efficiency of 38.57% ± 9.50. Affinity of AP1 for AP3/PI was low (8.40% ± 5.66 BINDING), but strongly increased in presence of SEP3 (28.80% ± 12.36 BINDING). Statistical groups were assigned after multiple comparison with Kruskal–Wallis and a Post hoc test using the criterium Fisher’s least significant difference (alpha parameter is 0.05) (Dashed blue line marks the BINDING cut-off of 10%; number of repetitions are indicated below BINDING values and number of images with BINDING above 10% are indicated below the FRET efficiency values in the bottom of the plot)