Additional file 1 of One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells
Creators: name: Maika, Jan Eric
name: Krämer, Benedikt
name: Strotmann, Vivien I.
name: Wellmer, Frank
name: Weidtkamp-Peters, Stefanie
name: Stahl, Yvonne
name: Simon, Rüdiger
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Molecular Biology
subject: Developmental Biology
subject: Cancer
subject: Statistics
subject: FOS: Mathematics
subject: Plant Biology
subject: FOS: Biological sciences
subject: Computational Biology

description: Additional file 1: Fig. S1. FLIM analysis of AP1-mV NLS-mCh and AP1-mV Ap1-mCh in N. benthamiana leaf cells. FLIM experiments were performed in N. benthamiana leaf epidermis cells. Fusion proteins were expressed from the UBQ10 promoter and imaged 3–4 days after infiltration. BINDING [%] (grey) and FRET efficiencies [%] (white) for AP1-mV, AP1-mV NLS-mCh and AP1-mV AP1-mCh. Depicted are the same data as in Fig. 1, but FRET efficiencies with BINDING below 10% were not excluded. FRET efficiencies of AP1-mV and AP1mV AP1-mV-NLS-mCh samples have a higher variance compared to the AP1-mV AP1-mCh sample. When the value for BINDING was below 10%, FRET efficiencies showed a higher tendency for values close to the limits of the fitting model (10% and 80% FRET efficiencies). (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). Fig. S2. Comparison of the One pattern analysis and a mono exponential Donor decay model. BINDING values for AP1-mV samples, fitted with the One pattern analysis or an analysis which assumes a mono exponentially decaying donor model. For the One pattern analysis, three lifetime components were fitted (see methods for details), and for the analysis which assumes a mono exponential decaying donor, two lifetime components were fitted. Due to the influence of the secondary shorter mV lifetime, the analysis assuming a monoexponentially decaying donor results in increased BINDING of more than 10% in almost one third of the images indicating false positive FRET. In contrast, using the One pattern analysis, most acquired BINDING values are below the 10% limit and are less variable. Fig. S3. Change in BINDING and FRET efficiency acquired in the same cell before and after acceptor bleaching. FLIM experiments were performed in N. benthamiana leaf cells. Fusion proteins were expressed via te UBQ10 promoter and images were acquired 3 days after infiltration. After the first time series, photobleaching of mCherry was performed with a 561nm laser at 100 % for ninety frames, followed b a second time series. Analysis was done as described in Fig. 2. Binding between AP1-mV and AP1-mCh was not detectable anymore after acceptor bleaching. (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). Fig. S4. Dependence of BINDING on acceptor concentration. B BINDING values calculated for AP1-mV AP1-mCh, and corresponding photons detected in the acceptor channel. BINDING correlates with the Acceptor concentration. Fig. S5. Standard fluorescence lifetime analysis of AP3-mVn PI-mVc samples. Donor only decays were fitted using a monoexponential decay model while decays from donor + acceptor samples were fitted using a biexponential decay model. The non-FRET sample, containing NLS-mCh, shows a significant reduction in fluorescence lifetime compared to the donor only sample (AP3-mVn PI-mVc). In contrast with the OPA, standard lifetime analysis does not reveal interaction between the NLS-mCh containing sample and the AP1-mCh containing sample. Only for the SEP3-mCh and AP1-mCh + SEP3 containing samples a significant reduction in fluorescence lifetime compared to the NLS-mCh containing sample was detectable. 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) (Number of repetitions are indicated in the bottom of the plot). Fig. S6. Average BINDING and FRET efficiencies of all FRET and negative control samples. Interacting samples are labelled in green and non-interacting samples are labelled in magenta. Occurrence of interaction was judged by a significant increase in BINDING based on Kruskal-Wallis and a Post hoc test using the criterium Fisher’s least significant difference (alpha parameter is 0.05). Error bars indicate the standard error. Fig. S7. MADS-domain protein reporters. Expression pattern of AP1-mT2 in ap1-1 (A), SEP3-GFP (B), PI-GFP in pi-1 (C) and AP3-GFP in ap3-3 (D). Expression of AP1 starts in stage 2 floral buds. In stage 3 and stage 4 buds AP1 is braoldy expressed. Expression starts to become restricted to sepals and petal initiation sites stating in stage 5 flowers. Weak expressionin of SEP3 starts in late stage 2 buds. In lates stage buds SEP3 is is mexpressed in cells of the third and fourth whorl. AP3 and PI became visible in early stage 3 florla buds and are expressed in circular pattern in the second and third whorl of stage 4 and stage 5 flowers. In stage 6 flowers expression of AP3 or PI proteins is restricted to developing stamen and peatl initiation sites. Numbers indicate floral stage as previously defined (Smyth, Bowman, and Meyerowitz 1990). (Z-stacks, Scale Bars: 50 µm). Fig. S8. Photons in donor channel in different donor only samples. Number of photons was measured in ROIs marking several nuclei in Arabidopsis samples or one nucleus in N. benthamiana samples. Nucleoli of N. benthamiana nuclei were excluded from analysis. Counts in Arabidopsis GFP reporter lines of AP3, PI and SEP3 were lower compared to counts in the AP1-mV reporter or in N. benthamiana samples. Fig. S9. Co-expression of AP1 with PI and mutant complementation of ap1-1 and pi-1 and. A–A’’: Z-stack of AP1-mV signal, AP1-mCh signal and Merged signals respectivly. B–B’’: Z-stack of AP1-mV signal, PI-mCh signal and Merged signals respectivly. (Scale Bars: 50 µm). C–F Inflocrescences of ap1-1 (C–E) or pi-1 (F) mutants complemented with AP1-mCh (C), AP1-mV (D), AP1-mT2 (E) and PI-mCh (F). Indicated MADS-domain proteins, tagged with FPs, were expressed via their endogenous promoter. All fusion proteins rescued the organ deficient phenotype of the mutants in 4 (ap1-1) or 2 (pi-1) independent lines. Table S1. Summary of the FRET-FLIM measurements for the investigation of MADS-domain protein interactions in N. benthamiana. Mean, standard deviation (SD) and standard error (SE) of FRET efficiencies were calculated after removing values with BINDING &lt; 10%. Table S2. Summary of the FRET-FLIM measurements for the investigation of MADS-domain protein interactions in Arabidopsis. Mean, standard deviation (SD) and standard error (SE) of FRET efficiencies were calculated after removing values with BINDING &lt; 10%. Table S3. Plasmids used, but not constructed during this study. Table S4. Plasmids used from the GreenGate kit. Table S5. “Entry” plasmids generated in this study. The list contains all “entry” plasmids which were used for the construction of plant expression plasmids. Plasmids were cloned by restriction ligation using BsaI. Inserts were amplified with the respective primers from the according templates and cloned in the appropriate backbone. Table S6. Plant expression plasmids constructed in this study. Plasmids were used for stable A. thaliana transformation or transient transformation of N. benthamiana. Construction of the plasmids was achieved by the GreenGate method using the appropriate Inserts and assemble them in the respective Backbone. Table S7. Oligonucleotides used in this study.