generously allowed regions and 0.8% (four residues) in non- allowed regions. EFR and FLS2 Constructs The protein coding sequence of Arabidopsis thaliana EFR up to but not including the stop codon, along with native promoter sequence (1091 bp upstream of the start codon = 1074 bp upstream of the transcription start site), was amplified from Col- 0 accession genomic DNA using the primers (CACCGGGT- TTTTGTTTATTCAAAGATGGG and CATAGTATGCATG- TCCGTATTTAACATCC) and cloned into pENTR/D-TOPO (Invitrogen). Mutations to EFR were made in this construct via PCR as in [35], using mutagenic primers and a high-fidelity DNA polymerase (typically Pfu Ultra II (Stratagene)), followed by DpnI treatment to digest template and transformation of the linear product into E. coli. Mutations were verified by DNA sequencing. Site-directed randomizing mutagenesis was performed as de- scribed in [35] using mutagenic ,30 nt PCR primers in which only one codon was mutagenized using the degenerate codon NNB. Similarly, double-alanine mutants were created with a mutagenic primer of ,30 nt with two selected codons mutated to alanine. To clone BrEFR1, primers (CACCATGAAGCCGT- TTCTTTCAATTGCTCTACTCATG and CATTGTATGCA- TGTCCGCGCTTAACATCC) were designed to amplify the full- length best hit of Arabidopsis EFR, minus stop codon for fusion to C-terminal tag, for insertion into pENTR/D-TOPO. To clone EFR from other species, genomic DNA was extracted and the EFR LRR-encoding domain was amplified using primers based on the EFR sequences of Arabidopsis and Brassica rapa. Restriction enzyme sites (SbfI and FseI) flanking the LRR domain were engineered by site-directed mutagenesis into the Arabidopsis EFR promoter+cod- ing region in the pENTR/D-TOPO vector. The Arabidopsis EFR LRR-encoding domain was then cut out of the vector. The LRR- encoding domains of the Brassicaceae EFR genes were amplified using species-specific primers with the restriction enzyme sites at the 59-ends and cloned into TOPO vectors, then cut out of the TOPO vectors and ligated into the Arabidopsis EFR gene lacking the LRR domain. Sequences in pENTR/D-TOPO were moved, by LR Clonase II reaction (Invitrogen), into the Gateway vector pGWB13 (if using native EFR promoter) or pGWB14 (if using CaMV 35S promoter), fusing the EFR amino acid sequence to a C-terminal HA tag [61]. Constructs were then moved into Agrobacerium tumefaciens GV3101 by electroporation and used to transform homozygous Arabidopsis efr2 plants (SALK 068675c) using the floral dip method or for transient expression in Nicotiana benthamiana via Agrobacterium infiltration. Site-directed randomizing mutagenesis of FLS2 was as described in [35]. Constructs were transformed into homozygous fls2-101 plants. All new DNA and derived amino acid sequences are deposited at Genbank under accession numbers JN002095-JN002103. Receptor function assays Seedling growth inhibition assays were performed as described [35], with appropriate selection for transgenic seedlings prior to use. ROS assays were performed on leaf discs taken from 4- to 8- week old transgenic Arabidopsis or from 4- to 6-week old N. benthamiana leaves infiltrated two days prior with A. tumefaciens containing an EFR, FLS2 or corresponding empty vector con- struct. Leaf discs were floated on 1% DMSO overnight and then treated with 1 mM peptide (or no peptide in the case of mock) in the presence of 1 mg/mL luminol and 1 mg/mL horseradish peroxidase. Luminescence was measured on a Synergy HT Microplate Reader (Bio-Tek) for 30 minutes following addition of peptide [32,62,63]. For callose deposition assays, seedlings were Repeat Conservation Mapping grown for 5 days on 0.56 MS agar and then transferred to liquid 0.56 MS (500 ml per well in 24-well plate) with elf18 or flg22 peptide at the indicated concentrations. After 24 hours in liquid, seedlings were fixed with 2% formaldehyde/5% acetic acid/60% ethanol (FAA), cleared overnight in 95% ethanol, stained with 0.01% aniline blue and viewed under an epifluorescence micro- scope to visualize callose deposits [62,63]. For receptor protein detection, six to eight 3-week old seedlings were ground in 26SDS buffer (2 mL buffer per g tissue), boiled, and centrifuged to remove particulates. 50 ml per sample was separated by SDS-PAGE, blotted onto a PVDF membrane, and detected using an anti-HA antibody conjugated to horseradish peroxidase (Roche), made visible using the ECL Plus Kit (Amersham). Results Rationale and description of LRR conservation mapping approach LRRs have a regular structure in which a single repeat forms one turn within the overall super-helical structure. Across numerous solved LRR structures, the LRR consensus residues form the buried core of this configuration (e.g., Figure 1A) [1]. Because of the regularity of these LRR structures, two assumptions can be made for conservation mapping: 1) consensus residues are not on the protein surface; and 2) the repetitive structure of LRRs allows prediction of relative amino acid positions in the tertiary structure without requiring a crystal structure or a detailed homology model of the protein in question. Assumption 1 allows elimination of the highly conserved but functionally less revealing consensus residues from the analysis; assumption 2 allows pre- diction and assessment of spatially adjacent groups of surface residues that are not adjacent in the primary amino acid sequence. Following this rationale, we devised and implemented Repeat Conservation Mapping (RCM), a set of algorithms to identify predicted functional sites of LRR domains. RCM accomplishes this by identifying the extent of conservation of different amino acid patches on the predicted surface of LRR domains (see also program description in Text S1). As an example, the lower-right element of Figure 1B shows RCM output for an extensively studied plant LRR domain- containing protein, POLYGALACTURONASE INHIBITOR PROTEIN (PGIP). PGIP was the first LRR-containing plant protein to have its structure solved [56]. In bean (Phaseolis vulgaris), there are four PGIPs, designated PGIP1-4, which have varied but overlapping specificities for different polygalacturonases (PGs) [64]. The RCM map for these four paralogs highlights several divergent and conserved patches (Figure 1B). Interestingly, the three positions in PvPGIP2 that, when mutated to alanine, have a significant negative impact on inhibitor activity [55], are all located in divergent patches identified by conservation mapping, perhaps indicating that these residues are also responsible for differences in receptor specificity. Indeed, a single amino acid that can switch specificity between PGIP1 and PGIP2 is also found in this divergent region [65]. Validation: Conservation mapping highlights functionally significant regions of LRR domains RCM highlights, among a group of homologous proteins, regions that are highly conserved or highly divergent on the surface of the LRR domain. To verify that RCM identifies significant functional sites, we utilized RCM to analyze all proteins in PDB that, as of October 2010, had a structure for an LRR domain interacting with another protein or a ligand, and for which at least one functional homolog could be identified. Two PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e21614