Extended Data Fig. 5: pAP3 LUBS are required for LFY-UFO-dependent activation.
a, EMSA with indicated probes and proteins. LUBS3 is the third highest-score pAP3 LUBS. Because LUBS0 is bound with a lower affinity by LFY-UFO compared to LUBS1 and LUBS2, we then focused on LUBS1 and LUBS2. b, EMSA with pAP3 LUBS1 and LUBS2 DNA probes and indicated proteins. LFYH383A-R386A (LFYHARA) is a LFY mutated version affected in its ability to dimerize29,51. Note the absence of the complex with a slower mobility on LUBS1 with LFYHARA. c, EMSA with pAP3 LUBS1 and LUBS2 DNA probes and indicated proteins. LFY* refers to either in vitro-produced 5xmyc-LFY (top) or recombinant 6xHis-LFY-DBD (bottom). Note the difference of complex size between UFO and UFOΔFbox. d, Same as in (c) except that UFO-3xFLAG and UFO∆Fbox-3xFLAG were produced in vitro. Note that in vitro produced UFO-3xFLAG and UFO∆Fbox-3xFLAG behave similarly as recombinant UFO versions. e, EMSA with indicated proteins and DNA probes corresponding to pAP3 LUBS1 (left) and LUBS2 (right), WT or with URM mutated. f, Promoter activation measured by DLRA in Arabidopsis protoplasts with indicated effectors. Different promoter versions were tested as indicated under x-axis. Either 2 bp (high-informative CA) or 6 bp (whole URM) of pAP3 LUBS1 and LUBS2 URM were mutated. Data represent averages of independent biological replicates and are presented as mean ± SD, each dot representing one biological replicate (n = 4). One-way ANOVA with Tukey’s multiple comparisons tests. One-way ANOVA were performed with data from the same effector and stars represent a statistical difference compared to WT pAP3 promoter. (NS: p > 0.05,*: p < 0.05, **: p < 0.01, ***: p < 0.001 and ****: p < 0.0001). g, In vivo analysis of pAP3LUBS1-2m::GUS fusions. Same as in Fig. 3d, except that staining incubation time was increased to 17 h (4 h incubation in Fig. 3d). Representative pictures are shown (top scale bar, 100 µm, bottom scale bar, 50 µm). The faint AP3 pattern suggests that other LUBS (such as LUBS0) may take over but less efficiently. Note that with this staining incubation time, all plants expressing pAP3::GUS showed a highly saturated staining. Source data are available in Supplementary Data 4.
