Fig. 2: Yeast-based discovery of novel Atsly1 mutant proteins.
From: Evolution of a plant growth-regulatory protein interaction specificity
a, Atsly1 substitutions shown in red were detected in yeast-based screens only, those in blue in both yeast-based and in planta screens. G84D (for reference) was detected in in planta screens only (see also Fig. 1c). b, Quantified (mean ± s.d.) yeast 2-hybrid AtSLY1–GAI interactions, Atsly1 variants as in a, arranged in a gradient of increasing interactions. Red dots indicate individual values (n = 3), different letters (a–g) indicate significant differences (one-way ANOVA with Tukey’s test). c, In vitro interactions between E. coli-expressed His-tagged GAI and MBP-tagged Atsly1 variants. Anti-His confirms similar amounts of GAI in each IP reaction, while anti-MBP shows how much SLY1 (quantified against anti-His, arbitrarily set at 1.00) or sly1 variant was pulled down. d, Transgenic expression of Atsly1 variants suppresses gai phenotype. Genotypes as shown, with gai (far left) and WT (far right) (for equivalence of expression, see Extended Data Fig. 2g). Scale bar, 5 cm. e, Mean (±s.d.) plant height, genotypes as in d, with non-transgenic WT and gai. Red dots indicate individual heights (n = 10), different letters (a–c) indicate significant differences (one-way ANOVA with Tukey’s test). f, Mean (±s.d.) yeast 2-hybrid AtSLY1–RGA interaction strengths, Atsly1 variants as in a. Red dots indicate individual values (n = 3), different letters (a–d) indicate significant differences (one-way ANOVA with Tukey’s test). g, Yeast 3-hybrid analysis of affinities for GAI A and A’ forms. GA promotes GAI–GID1C binding, converting GAI from A to A’. h, In vitro analysis of interactions between E. coli-expressed His-tagged GAI and MBP-tagged Atsly1 variants in the presence and absence of GST-GID1C and GA3. Anti-His confirms that similar amounts of GAI protein were used in each IP reaction, while anti-MBP and anti-GST show how much SLY1 (quantified against anti-His, arbitrarily set at 1.00) or sly1 variant and GID1C (if present) were pulled down. GA promotes GAI–GID1C binding, converting GAI from A to A’.
