Extended Data Figure 6: LIMYB, which belongs to the MYB domain-containing superfamily, interacts with RPL10 in the yeast two-hybrid system.

a, LIMYB and RPL10 interact in yeast. LIMYB was expressed in yeast as a GAL4 activation domain (AD) fusion (pAD–LIMYB), and RPL10 was expressed as a GAL4 binding domain (BD) fusion (pBD–RPL10). The interactions between the tested proteins were examined by monitoring His prototrophy. b, The interactions were further confirmed by measuring the expression activity of the β-galactosidase reporter enzyme for the second reporter gene, β-galactosidase. The interaction between pAD–RPL10 and pBD–NIK1 was monitored as a positive control. Means ± 95% confidence intervals (n = 3) based on bootstrap resampling replicates of three technical replicates are shown. c, LIMYB harbours two MYB domains. The position of these MYB domains is indicated in the schematic representation of the LIMYB primary structure. d, Sequence identity of the closest related LIMYB (At5g05800) homologues. e, Dendrogram of MYB domain-containing proteins from Arabidopsis. The MYB family sequences were retrieved from the Agris database (http://arabidopsis.med.ohio-state.edu). The alignment was performed by Maft aligner software using full-length sequences, and the tree was built by Fasttree software (the bootstrap values are indicated close to the branch divisions). The arrow indicates LIMYB (At5g05800). f, g, The negative controls used in the BiFC analysis. f, Confocal fluorescent image of SPYNE + LIMYB:SPYCE, LIMYB:SPYNE + SPYCE, SPYNE + RPL10:SPYCE and RPL10:SPYNE + SPYCE, as indicated in the figure. We used a BiFC assay to determine whether RPL10 and LIMYB interact in the nuclei of plant cells. The formation of a RPL10–LIMYB complex occurred in the nuclei of transfected cells independent of the orientation of the LIMYB or RPL10 fusions (amino terminus or carboxy terminus of YFP; Fig. 2a), and the reconstituted fluorescent signal was much higher than that of the background (control panels with combinations of the protein fusions with empty vectors). The figure displays representative samples from three independent biological repeats. Scale bars, 20 µm. g, The C-terminal (SPYCE) and N-terminal region (SPYNE) of YFP accumulates detectably in co-transfected leaves. Total protein extracts from leaves co-transfected with the indicated constructs were immunoblotted with anti-YFP serum. The arrow indicates the position of RPL10–SPYCE and RPL10–SPYNE fusions, and arrowheads indicate the positions of the C-terminal (SYYCE) and N-terminal (SPYNE) regions of YFP.