Figure 1. LRR structure and an outline of conservation mapping procedure. (A) Left: a representative LRR domain (left), P. vulgaris PGIP2, which forms the regular spiral pattern typical of LRRs. Right: a single 24 amino acid repeat of the LRR, surrounded by circles designating the residues of the LRR consensus amino acid sequence (xxLxxLxxLxxLxLxxNxLxGxIP). Note that the consensus residue side chains (orange) form the core of the protein, whereas the variable residues (green) are solvent-exposed. (B) Schematic representation of the conservation mapping procedure, using the example of PGIP1-4. See Methods and Text S1 for a detailed description of the procedure. doi:10.1371/journal.pone.0021614.g001 xxLxxLxxLxxLxLxxNxLxGxIP). The solenoid is curved so that a convex and a concave face are created. The concave face is largely composed of b-strands, and often forms the ligand binding site for LRR domains [15]. Deviations from the repeat consensus can result in intervening segments that interrupt adjacent solenoid regions, providing structural flexibility to the LRR. An LRR domain may be relatively brief (e.g. two or three repeats), or quite long (30 repeats or more). The consensus residues (mostly leucine Repeat Conservation Mapping or other hydrophobic amino acids) are usually buried in the core of the solenoid, while residues at the variable positions are predominantly solvent exposed (Figure 1A). The LRR structure efficiently creates a large surface-to-volume ratio protein domain that tolerates a wide variety of surface compositions, encoded in a condensed genomic space [16]. LRRs play a central role in the receptors that mediate two major branches of the plant immune system [17]. Many plant cell surface receptors for microbe- or pathogen-associated molecular pattern (MAMP/PAMP) molecules, which confer recognition of conserved microbial molecular motifs, contain LRRs as the bulk of their extracellular domain and a protein kinase intracellular domain [18–20]. The transmembrane LRR-kinase family of receptors is expansive in plants (over 200 different protein types in Arabidopsis, over 400 in rice [21]), and their functions extend well beyond immunity to include prominent roles in plant growth and development pathways [22,23]. Plant MAMP receptors carry clear structural and functional analogies to (but apparent evolu- tionary independence from) animal MAMP receptors, the Toll- like receptors (TLRs), which also contain LRR domains [24]. LRRs are also central to recognition specificity in the large, diverse family of plant intracellular nucleotide binding (NB)-LRR proteins known as resistance (or ‘‘R’’) proteins. These R proteins initiate strong defense responses upon recognition of specific pathogen effector molecules or upon recognition of a host protein alteration caused by a specific pathogen effector [11]. Plant R proteins are structurally similar to animal nucleotide-binding leucine-rich re- peat (NLR) proteins that play significant recognition roles in the mammalian immune response [25–28]. Two plant LRR-kinase MAMP receptors that have been a particular focal point for research are ELONGATION FACTOR TU (EF-Tu) RECEPTOR (EFR) and the flagellin receptor FLAGELLIN-SENSING 2 (FLS2) [29–33]. FLS2 orthologs can be found in a wide range of monocotyledonous and dicotyledon- ous plant species, whereas EFR appears to be limited to the mustard family Brassicaceae. FLS2 can be activated by flagellin or by synthetic peptides that represent the highly conserved minimal recognition domain of flagellin, such as the 22 amino-acid flg22 peptide [29]. Similarly, EFR responds to conserved peptides from the recognized domain of bacterial EF-Tu, such as the elf18 peptide [32]. There is evidence that the LRR domains of these proteins directly interact with MAMP ligands [32,34–36], but the large size of their LRR domains (22 repeats for EFR, 28 for FLS2) leaves open the possibility that these LRRs also mediate inter- action with other ligands, co-receptor proteins and/or cofactors. FLS2, EFR and other MAMP receptors can be significant barriers to microbial infection [32,37–42]. Intriguingly, transgenic tomato plants expressing Arabidopsis EFR are more resistant to the plant pathogenic bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, implying that many of the mechanisms for down- stream defense signaling from MAMP receptors are conserved across diverse plant species [38,43]. Protein functional site prediction is already possible using a number of computational methods, each of which has certain advantages and limitations. Functional sites are often detectable as the sets of amino acids that have been most conserved or diver- sified among a set of homologous proteins [44–46]. Some com- putational methods require only primary sequences to look for conserved codons or amino acids within a sequence alignment, such as database searching for protein motifs (e.g., [47]) or align- ment with homologous sequences (e.g., CLUSTAL). In contrast, positive/purifying selection analysis (e.g., Ka/Ks or dN/dS ratio; [46]) looks for amino acids that have undergone selection, either purifying or diversifying (positive), based on identification of PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e21614