are utilized (see Step 4 in Methods), and the proportion increases further when regional conservation scores are used (from 18% to 20% to 25% of interacting residues, respectively; Figure 3). 45% of all interacting residues appeared in the top 20% of RCM scores in this analysis. The remaining high-scoring sites identified by RCM may be involved in other functional processes that are not detected in the available LRR+ligand crystal structures, such as interaction with other ligands, cofactors or co-receptor proteins (see Dis- cussion). Comparison of homologous proteins with diversified functions is also addressed below. Mutations of AtEFR at conserved locations identified by RCM often disrupt receptor function RCM was used as a de novo discovery tool in experiments on EFR, the EF-Tu receptor of plants, for which there is no known structure or ligand binding site. To identify an EFR homolog for comparison, the derived amino acid sequence of Arabidopsis EFR was used with BLASTP to query all publicly available sequences from the Brassica Genome Gateway as of April 2009, and two high-scoring matches from Brassica rapa were identified. One of these sequences was cloned and then transiently expressed in Nicotiana benthamiana leaf mesophyll tissues, which do not otherwise respond to the EF-Tu-based elf18 peptide [32]. The defense mechanisms initiated by EFR or FLS2 include production of reactive oxygen species, release of other directly antimicrobial compounds, and production of callose and lignin as part of a cell wall strengthening response that limits pathogen penetration [82]. Plant seedlings undergoing chronic defense activation display inhibition of growth after multiple days of exposure to a recog- nized MAMP; this is a sensitive and widely used assay for FLS2 or EFR activation [31,32]. The cloned Brassica rapa EFR homolog, designated BrEFR1, conferred recognition of 1 uM elf18 in an ROS assay (Figure S3). BrEFR1 subsequently was shown to complement an Arabidopsis efr2 mutant, rescuing the ability of the plants to respond to elf18 in ROS and seedling growth inhibition assays (Figure S3 and data not shown). Figure 3. Residues involved in intramolecular interactions receive high scores in conservation mapping. For each map in Figures 2 and S1 (eleven protein families), all scores were arranged in descending order and then divided into deciles. Then, across all maps, the number of occurrences of residue positions marked by asterisks (LRR+ligand contact points) were tallied for each decile. Histogram shows the frequency with which three different types of scores fell into a particular decile. Score types are raw average pairwise BLOSUM65 score (grey), weighted average pairwise BLOSUM65 scores (see ‘‘Step 4’’ in Methods) prior to adjustment based on nearby amino acids (white), and regional conservation score that serves as final RCM output (black). Note that a subset of these protein comparisons involved homologs with unclear and possibly divergent rather than fully overlapping functions, in which case LRR-ligand contact points might be predicted to score as divergent rather than as conserved (see text). doi:10.1371/journal.pone.0021614.g003 Repeat Conservation Mapping RCM was used to create a conservation map of AtEFR with BrEFR1 (Figure 4A). The map highlights several small patches of conservation on the surface of EFR, the highest scoring (most conserved) of which appear on the concave face of the receptor. The convex face also contains patches of conservation (the third and fourth columns of Figure 4A). There is a large patch of divergence in LRRs 3 through 10. Hypothesizing that the ortholog map predicts functionally significant locations, we constructed a series of double-alanine mutants within EFR along the predicted concave face (also known as the b-strand, b-turn region [1]). For each construct, we changed two of the five variable residues within a single repeat’s b-strand, b-turn region to alanine. Alleles were made that mutate sites of conservation, or as controls, sites lacking conservation. The double-alanine mutants were tested in stable transgenic Arabidopsis efr2 plants. Of the fourteen constructs tested in a seedling growth inhibition assay, four of them had no detectable response to peptide, and four constructs had a response that was weaker than a wild-type response (Figure 5). Seven of these mutants were in conserved locations on the RCM map made from the EFR orthologs, including each of the four conserved patches of the b- strand, b-turn region (Figures 4, 5). The other two alleles with mutations in conserved locations did not detectably alter function. Importantly, only one of the five mutants in any of the poorly conserved regions resulted in a discernible difference in receptor function, with response slightly less than a wild-type receptor (Figures 4, 5). These results were further supported by ROS assays in N. benthamiana (not shown) and ROS and callose assays in Arabidopsis (Figure S4). All of the EFR double-alanine mutant proteins, whether functional or not, were still present at functional levels in plants, as detected by Western blot (Figure S5). Conservation mapping of EFR and its paralogs reveals a divergent b-strand, b-turn region Use of paralogs rather than orthologs was further examined. The four Arabidopsis paralogs most similar to EFR were identified by BLAST using the Arabidopsis EFR LRR sequence to query the Arabidopsis genome. An RCM map of EFR and these paralogs was then generated (Figure 4B). Some areas are generally conserved across these paralogs, in particular along one ‘shoulder’ of the convex face of the LRR that is also conserved among EFR orthologs. However, a large portion of the concave face of the LRR region is highly divergent in this paralog map. We hypothesized that the divergent b-strand, b-turn region is responsible for recognizing distinct ligands in these receptors, since 1) the concave face of LRR regions are most often implicated in ligand binding [15]; and 2) knock-out of EFR results in a plant completely insensitive to elf18 [32], implying that these EFR paralogs are not capable of EF-Tu recognition. Based on this map, we performed site-directed randomizing mutagenesis on EFR to create libraries of concave face mutations at amino acid positions that are highly divergent (predicted to impact function), or conserved (also predicted to impact function), or neither highly conserved nor highly divergent (not predicted to impact function). Each allele library contains different mutations at a single position. These libraries were introduced into Arabidopsis efr2 plants that were then tested for response to 100 nM elf18 in a seedling growth inhibition assay. Most libraries were significantly impacted in their ability to recognize 100 nM elf18 (as compared to a wild-type receptor response) regardless of level of conservation, but all libraries still had several functional clones (Figure 6). The most significantly impacted library (LRR 2.3 (Asn 103)) was also identified in the double-alanine mutagenesis and is conserved in both RCM analyses performed (Figure 4). Overall, mutagenesis of PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e21614