Repeat Conservation Mapping Figure 8. Conservation mapping using different EFR ortholog sets. (A) RCM map for ten Brassicaceae EFR orthologs. All sequences were confirmed as functional orthologs. (B) RCM map of two EFR orthologs, from distantly related Eruca sativa and Erysimum raulinii. (C) RCM map of two EFR orthologs, both from Erysimum raulinii. Maps are as described in Figure 2. doi:10.1371/journal.pone.0021614.g008 in RCM, indicating that these positions are not conserved. These amino acids could be responsible for differences in specificity of the compared proteins rather than any shared activity. Even in cases where two related proteins have extremely similar functions, some variable residues are to be expected at functional sites as these could be responsible for fine-tuning of receptor function, such as through ligand interaction kinetics. As a separate issue, there were many residues that scored highly in RCM that did not interact with ligand in the available crystal structure. While these could be false positives, many of them are likely responsible for functions not detectable in the crystal structure (i.e., interaction with molecules not present in the crystal structure). The regions highlighted by RCM but not matching known LRR+ligand interaction sites are intriguing targets for future study. In LRR domains, disruption of single solvent-exposed residues very frequently does not disrupt function (e.g., [35,88]). We were pleased to find seven different double-alanine mutants of EFR that were each severely disrupted in function when compared to a wild- type receptor. These mutants were in four distinct conserved regions on the surface of EFR, one of which overlaps with a region identified by [36] as important for EFR function. A full-length protein can be detected for these different EFR LRR mutation alleles (Figure S5), but that leaves many other possible activities that could be disrupted. Some of these patches may fold together to form a shared ligand-binding domain, but alternatively, three of the four sites may be involved in other processes such as receptor dimerization, interaction with co-receptors, or protein localization. Experiments are in progress to investigate the functions that are disrupted when these different regions are mutated. One prediction is that different function-blocking mutations in the same region (such as in the concave face of LRRs 11–12, or LRRs 16–17) will cause the same type of functional disruption. Interestingly, although seven of the nine double-alanine mutants made in conserved EFR regions disrupted function, two of the mutants behaved in a manner identical to wild-type. This demon- strates that not all residues in a conserved region are essential for function. It is important to note that the score placed at any residue position is a regional conservation score for a 565 window, and may be elevated due to the presence of multiple conserved residues surrounding a relatively less conserved center residue. Examination of the single-residue conservation scores (as in Figure S2) may help to identify the most significant residues in a conserved region. Alternatively, the conserved regions not func- tionally disrupted by mutations may be functionally important in ways that would be not detected using the ligand that we utilized. As a third option, it is also possible that RCM detects a certain amount of ‘noise’ that represents relatively unselected stochastic conservation. Prediction of functionally significant EFR sites was originally performed with only two sequences. When we identified a further seven functional orthologs of EFR and created a conservation map with these additional sequences, the same sites were identified, probably with more precision. Use of two sequences could be misleading when the two sequences are very similar (see also the FLS2 maps of Figure S6). Additional sequences may be a means of increasing the power or reliability of RCM, but even a pairwise comparison can be informative. We chose to focus on the b-strand, b-turn region of EFR because of the propensity of ligand binding sites to occur in this region [15]. However, in a few cases crystal structures have identified important residues outside the concave face of the protein, such as in the case of TLR3. The regions outside the concave face that were highlighted by the present conservation mapping effort may also be important for receptor function. For example, [89] recently identified a site of glycosylation, N143, on the convex face of the LRR of EFR that, when mutated, results in a severely impaired receptor. This residue occurs in the fourth repeat within the large conserved patch identified in both the ortholog and paralog conservation maps of EFR (Figure 4). We were surprised that the most divergent region RCM identified among related but functionally distinct EFR paralogs, centered on the concave face of the fifth through ninth repeats, contained residues with an intermediate rather than a strong impact elf18 perception (Figure 4B and Figure 6). We infer that these paralogs are functionally distinct from EFR because none of the four paralogs confer elf18 recognition when they remain wild- type in an efr2 mutant. This failure to see a strong impact on EFR function, when testing many of the site-directed random mutations of single residues in this most divergent region, may have occurred because the divergent sites we tested are more important for the gain of function of one or more of the other paralogs while being relatively unimportant for the responsiveness of EFR to EF-Tu. It PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e21614