Extended Data Fig. 3: Analysis of pAP3 activation by LFY-UFO.
a, Description of pAP3. Top line represents WT pAP3 with regulatory regions and cis-elements. Coordinates are relative to AP3 start codon. TSS: Transcription Start Site. Orange triangle represents LFYBS. Other rows show the promoter versions used in (b) and (c). Green rectangles in swapped versions correspond to the same random sequence. b,c, pAP3 LFY-UFO response element mapping with pAP3 versions described in (a) by DLRA in Arabidopsis protoplasts. 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 test (c). One-way ANOVA was performed with data from the same effector, and stars represent a statistical difference compared to WT pAP3. Unpaired t-tests (b). (NS: p > 0.05,*: p < 0.05, **: p < 0.01; ***: p < 0.001). d, EMSA with ASK1-UFO, LFY-DBD and LUBS0 DNA probe. Different competitor DNA concentrations were tested as indicated. e, Molecular mass determination for ASK1-UFO-LFY-DBD in complex with LUBS0 DNA by SEC-MALLS (top). Elution profiles correspond to absorbance at 280 nm and 260 nm (left ordinate axis, A.U: Arbitrary Unit). The black line shows the molecular mass distribution (right ordinate axis). A mass of 102 ± 3.3 kDa was found for this ASK1-UFO-LFY-DBD-LUBS0 complex, consistent with one copy of each protein per DNA molecule (theoretical mass of 108 kDa). Coomassie-stained SDS-PAGE gel of the different SEC-MALLS fractions (bottom). Each lane corresponds to a 0.5 mL fraction. Molecular weights of the protein standards are indicated (BioRad Precision Plus). Faint bands above UFO likely correspond to contaminants. f, EMSA with ASK1-UFO, LFY-DBD and indicated DNA probes. Sequences with coordinates relative to AP3 start codon (left). Red letters indicate mutated bases. Bars under sequences represent the regions required for ASK1-UFO-LFY-DBD binding. EMSA with described DNA probes (right). Each DNA probe was mixed with the same ASK1-UFO-LFY-DBD protein mix. Note that the LUBS0 mutation also reduced pAP3 activation in protoplasts (Fig. 2b). Source data are available in Supplementary Data 4.
