Fig. 1: Mutant gar2 alleles suppress the gai phenotype.
From: Evolution of a plant growth-regulatory protein interaction specificity
a, gar2 alleles variably suppress gai-conferred dwarfism. gai (gai GAR2) on far left, WT (GAI GAR2) on far right. Bolt stems cut from vegetative rosette. Scale bar, 5 cm. b, Mean (±s.d.) plant heights, genotypes as in a; red dots indicate individual heights (n = 10), different letters (a–e) indicate significant differences (one-way ANOVA with Tukey’s test). c, Amino acid substitutions encoded by gar-2 alleles. Conserved F-box, GGF and LSL domains (positions 1–151) are indicated. d, Yeast 2-hybrid analysis of gai–Atsly1 interactions. Top line (SD/-LW) confirms double transformation (bait and prey constructs), bottom line (SD/-LWAH/X) indicates interaction: no detectable interaction in the absence of bait (empty vector), baseline gai-SLY1 interaction (medium blue), stronger gai–Atsly1gar2 interactions. e, Mean (±s.d.) yeast 2-hybrid interaction strengths, mutants as in d; note gradient of increasing interaction strength correlating with increase in plant height (b). ND, not detected; red dots indicate individual values (n = 3), different letters (a–d) indicate significant differences (one-way ANOVA with Tukey’s test). f, In vitro analysis of interactions between E. coli-expressed His-tagged gai and MBP-tagged Atsly1 variants. Anti-His serves as control and confirms that similar amounts of gai protein were used to ‘pull down’ a SLY1 or sly1 variant protein in each immunoprecipitation (IP) reaction, while anti-MBP shows how much SLY1 (quantified against anti-His, arbitrarily set at 1.00) or sly1 variant was pulled down. The increasing amounts of MBP-SLY1, MBP-sly1gar2-2, MBP-sly1gar2-1 and MBP-sly1gar2-3 detected suggests that the height (b) and yeast-based interaction (e) gradients reflect a gradient of increasing affinity. g, Destruction rates of E. coli-expressed His-gai in plant extracts quantified against Actin control (arbitrarily set at 1.00 for timepoint 0), genotypes as shown. While His-gai is appreciably degraded in the gai (SLY1) control by 60 min, gar2 variant alleles confer faster degradation, with gar2-3 being the fastest, consistent with the affinity gradient (f). 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.
