Extended Data Fig. 2: Yeast-based discovery of novel Atsly1 mutant proteins.
From: Evolution of a plant growth-regulatory protein interaction specificity
a, Combined EP-PCR/yeast-2-hybrid screen for Atsly1 variants having increased AtGAI affinity. Different Atsly1 amino acid substitution variants (represented by different colours) are generated via error prone PCR, then used as bait against an AtGAI prey in a yeast-2-hybrid screen. Colonies exhibiting increased interaction are blue (rather than white; see also b). DNA sequencing then reveals interaction-increasing amino acid substitutions (see Methods). b, Example yeast-2-hybrid screen plate showing a blue colony (also in close-up panel) amongst whites (also in close-up). c, Yeast-2-hybrid analysis of interactions between GAI and Atsly1 variants. Top line (SD/-LW) confirms double transformation with bait and prey constructs, bottom line (SD/-LWAH/X) indicates interaction: baseline interaction between GAI and SLY1 (medium blue), stronger interactions between GAI and Atsly1 variants. d, Accumulation of immuno-detectable AtSLY1 and Atsly1 variants in yeast. GAI is tagged with HA, SLY1 and sly1 variants are tagged with Myc. RPT5 and Ponceau S provide loading controls. e, GAI compared with C-terminally truncated GAI-NT2. f, SLY1 and sly1 variant interactions with GAI and GAI-NT2 (as shown). AtSLY16 and all Atsly1 variants interact with GAI but not with GAI-NT2. The increased interaction of Atsly1 variants is thus not due to increased general ‘stickiness’. g, Mean relative AtSLY1 mRNA abundances in selected transgenic lines (genotypes as shown), with non-transgenic WT and gai. Red dots indicate individual values (n = 3), error bars indicate standard deviation, different letters (a-c) indicate significant difference (one-way ANOVA with Tukey’s test). h, Abundance of immuno-detectable gai (quantified against Actin, arbitrarily set at 1.00) or GAI and sly1 (or SLY1) in plant extracts (genotypes as shown). Anti-Actin and Ponceau S staining serve as loading controls. i, Interactions between AtSLY1 and Arabidopsis DELLAs GAI, RGA, RGL1, RGL2 and RGL3. j, Interactions between AtSLY1 or Atsly1 variants and RGA.
