Extended Data Fig. 8: The LFY K249 is essential for LFY-UFO-LUBS complex formation.
a, Structure of LFY-DBD29. Residues were colored by conservation using Consurf with default parameters85. K249 residues on each LFY monomer are represented as sticks and indicated with arrows. Note that the K249-containing loop is highly conserved. b,c, Promoter activation measured by DLRA in Arabidopsis protoplasts with indicated effectors (right). EV = Empty Vector (pRT104-3xHA). Tested promoters are indicated below each graph. Note that for 3xHA-LFY + UFO-3xFLAG on pAG only n = 3 biological replicates are shown. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4 unless specified). One-way ANOVA with Tukey’s multiple comparisons tests (b) or Welch’s ANOVA with Games-Howell post-hoc test (c). In (c), stars above bars represent a statistical difference compared to GFP. Other comparisons are indicated with brackets. (NS: p > 0.05,*: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001). d, Effect of the LFYK249R mutation on LFY-UFO interaction in Y2H. EV = Empty Vector. LFY-40 is a LFY version lacking the first 40 aa and better tolerated by yeast cells. Values correspond to the different dilutions (OD = 7, 0.7 and 0.07). Top picture corresponds to the non-selective plate lacking Leucine and Tryptophan (SD -L-W), and bottom picture corresponds to the selective plate lacking Leucine, Tryptophan, Histidine and Adenine (SD -L-W-A-H). Pictures were taken at day + 4. e, EMSA with DNA probes corresponding to pAP3 DEE LFYBS and pAP3 LUBS1 and indicated proteins. pAP3 DEE LFYBS DNA probe was used as a control for binding on canonical LFYBS. f, WB after DNA elution during ampDAP-seq experiment. After DNA elution, 20 µL of 1X SDS-PAGE Protein Sample Buffer was added to the remaining beads to run WB. Each lane represents one replicate. g, Reproducibility of ampDAP-seq experiments with LFYK249R (left) and LFYK249R-UFO (right) through the comparison of replicates datasets 2 by 2. h, Comparison of peak coverage in LFYK249R (y-axis, this study) and LFY (x-axis)48 ampDAP-seq experiments. i, Integrated Genome Browser (IGB) view of pAP3 showing LFY ChIP-seq in inflorescences (light blue)25 or seedlings (dark blue)26, LFY-UFO ampDAP-seq (yellow; this study), LFY ampDAP-seq (pink)48 and LFYK249R ampDAP-seq (purple; this study). Numbers indicate read number range. j, Pictures of WT and representative transgenic plants expressing 35S::LFY or 35S::LFYK249R (scale bar, 1 cm). The white arrows indicate ectopic rosette flowers. 35S::LFY was obtained previously26. 42 T1 plants expressing 35S::LFYK249R were analyzed; the percentage of plants with a LFY overexpressing phenotype is comparable to the one obtained with 35S::LFY26. Source data are available in Supplementary Data 4.
