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. 2019 Nov 25;8:e50253. doi: 10.7554/eLife.50253

Figure 1. Experimental approach for the ancestral resurrection experiment.

(A) The sequence of the putative last common ancestor of SRK03 and SRK28 was inferred by a phylogenetic approach using codon-based models implemented in PAML. Four different versions of SRKa were defined due to inference uncertainty at two aa positions. (B) SRK03 and SRK28 sequences were cloned from A. halleri DNA BAC clones, whereas SRKa sequences were obtained by gene synthesis. (C) Representation of the controlled cross program to decipher the specificity of SRKa.

Figure 1.

Figure 1—figure supplement 1. Three models for the emergence of new self-incompatibility specificities.

Figure 1—figure supplement 1.

Docking interaction between ligands and receptors are represented by oriented arrows. (A) In the compensatory mutation model, a non-functional self-compatible intermediate (R1/L2) segregates transiently and is rapidly compensated, creating a novel specificity (R2/L2), while the ancestral one (R1/L1) remains unchanged over time. (B) In the turnover model, slight functional variants with different affinity between them (R1’/L1’ and R1’’/L1’’) segregate in the population and give rise to new specificities (R2/L2 and R3/L3). Dotted lines correspond to weaker affinity interactions. (C) In the promiscuous model, the intermediate receptor (R1,2) has widened up its specificity spectrum enabling it to recognize another potential ligand (L2) while maintaining its capacity to recognize its original ligand (L1). Emergence of the new ligand then favours narrowing of specificity the dual-receptor (R1,2 → R2).

Figure 1—figure supplement 2. Maximum likelihood phylogenetic tree based on the 17 SRK alleles used for SRKa construction.

Figure 1—figure supplement 2.

The Bayesian reconstruction displays an identical topology. Values at the branches represent the bootstrap percentage/posterior probabilities. The node representing the reconstructed ancestral SRK allele (SRKa) is shown in red. Asterisks highlight alleles with a complete sequence available for ancestral reconstruction.

Figure 1—figure supplement 3. Schematic representation of the molecular constructs used for A. thaliana transformation.

Figure 1—figure supplement 3.

AhSRK03 and AhSRK28 constructs were cloned using a unique genomic fragment starting around −2 Kb and ending 1 Kb after the stop codon. SRKa constructs were each cloned using 3 DNA fragments (amplified promoter and kinase domain of AhSRK03 or AhSRK28 and the synthesized ancestral S-domain). The different parts of the construct were concatenated using the Multisite Gateway Technology at the positions indicated on the figure. SRK03p:GFP and SRK28p:GFP constructs were generated using the same promoter sequence as in the SRKa constructs, but were introduced into the pKGWFS7.0 destination vector.