Repeat Conservation Mapping Figure 4. Conservation mapping of EFR. RCM maps of LRR domains (as described in Figure 2) depicting: (A) Conservation of AtEFR and a Brassica rapa homolog shown to have EFR activity. (B) Conservation mapping of AtEFR and its four most closely related Arabidopsis EFR paralogs, which fail to confer elf18 recognition in an Arabidopsis efr2 mutant. The pairs of X symbols mark sites of double-alanine mutations, and O symbols mark sites of site-directed randomizing mutagenesis. The asterisks mark predicted sites of N-glycosylation as reported in [89]. Both (A) and (B) show the same set of X, O and * symbols. Based on data from Figures 5 and 6 and [89], symbols are white if the mutation disrupts EFR function, black if it does not, and grey for a partial/intermediate impact (less responsive than 95% of positive controls transformed with wild-type EFR but more responsive than 95% of empty-vector efr2 negative controls; or statistically different from both wild-type and empty vector controls). doi:10.1371/journal.pone.0021614.g004 regions conserved among EFR orthologs often broke function while mutagenesis of regions divergent among EFR paralogs had a less severe impact. Conservation mapping highlights an additional region of functional significance in AtFLS2 RCM was further tested as a discovery tool through work with a second protein, FLS2. FLS2 has been deeply studied but there is no solved structure or definitively determined extracellular ligand binding site [33]. To identify additional important regions of the FLS2 LRR we created various RCM maps of AtFLS2, for example in comparison to eight Brassicaceae orthologs from plants that respond to flg22 (Figure 7A; see also [35]). Within the b- strand/b-turn region there were two main areas of conservation. One of these was located at LRRs 9–13, in agreement with previous findings that this is an important region for flg22 binding and recognition [35]. Another conserved area was persistently observed at LRRs 22–26. To investigate their role in flg22 perception, five solvent-exposed residues in this region were mutagenized by site-directed randomizing mutagenesis. These libraries were used to transform mutant Arabidopsis fls2-101 plants that were then tested for response to flg22 peptide in a seedling growth inhibition assay. Alteration of Y629 significantly impacted flg22 responsiveness, and alteration of S633 also caused a detectable depletion of FLS2 activity (Figure 7B). RCM using many or few EFR and FLS2 orthologs, from closely or distantly related species Changes in RCM output for a given protein, in response to varied types of input, was further examined after isolation of additional EFR orthologs from other Brassicaceae. EFR homologs were isolated by PCR from plant accessions that exhibited a response to elf18. A function in EF-Tu sensing was confirmed for nine homologous LRR domains (one each from Brassica aucheri, Brassica rapa, Brassica napus, Eruca sativa, Biscutella auriculata and two EFR sequences each from Enarthrocarpus arcuatus and Erysimum raulinii), using ROS assays for responsiveness to elf18 after transient expression in N. benthamiana leaves (data not shown). These confirmed homologs and the EFR sequence from Arabidopsis thaliana Col-0 were analyzed by RCM. The resulting map (Figure 8A) is notably similar to the map generated with only two EFR sequences (Figure 4). However, the map generated with ten sequences more clearly delineates the most consistently conserved clusters, which included the concave face regions confirmed to be required for EFR function (Figures 4, 5, 6). Additional RCM maps were generated using smaller subsets of these EFR proteins based on the overall relatedness of different Brassicaceae species [83]. One analysis used EFR sequences from two of the most distantly related species of our sample set, E. sativa and E. raulinii (Figure 8B), while another analysis used two different EFR sequences obtained from a single E. raulinii plant (Figure 8C). PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e21614