Previously, site-directed mutagenesis has been employed for functional analysis of eLRR-containing cell surface receptors. For example, van der Hoorn et al analyzed a number of sitedirected mutants of Cf-9 and demonstrated that conserved Trp and Cys residues present in the N- and C-terminal eLRR flanking regions are important for Cf-9 activity. Similarly, recently reported site-directed mutations proved that the Cys residues in the Nterminal flanking region of the FLS2 eLRRs are required for protein stability and function. However, as these Trp or Cys residues are conserved in many other plant eLRR proteins as well, they likely contribute to the conformation and stability of the protein rather than to ligand specificity. In addition, another site-directed mutagenesis strategy focused on putative NS 3763 N-linked glycosylation sites, which frequently occur in the eLRR domain of cell surface receptors. Through Asn to Asp substitution, van der Hoorn et al demonstrated that four glycosylation sites contribute to Cf-9 functionality. These four sites are located in putative a-helixes that are exposed at the convex surface of the Cf-9 eLRR domain and are also conserved in many plant eLRR proteins. Glycosylation may contribute to protein conformation, facilitate interactions with the cell wall, or protect proteins from degradation. However, it seems unlikely that these putative glycosylation sites contribute to ligand specificity of Cf-9. Most of the Ve1 glycosylation sites are located at convex face of the eLRR domain, and thus they were not specifically targeted in our study. To the best of our knowledge, no examples of ligand perception at convex side of the eLRR domain have been reported. Moreover, N-linked glycosylation was determined to make only subtle quantitative contributions to FLS2 functionality. In contrast, alanine scanning mutagenesis on the concave b-sheet surface across the Arabidopsis FLS2 eLRR domain identified eLRR9-eLRR15 as contributors to flagellin perception. To identify eLRRs that are required for Ve1 ligand recognition, we focused our attention on the concave b-sheet surface and evaded conserved hydrophobic leucine residues in bsheets that are likely involved in framework of protein. A doublealanine scanning was performed in which two of the five variable, solvent exposed residues in a single eLRR repeat were mutated. Mutagenesis of two non-adjacent amino acids increases the chance of substituting functionally important residues. In this study, we showed that mutant alleles that reveal compromised Ve1 function are restricted to three consecutive eLRR regions, eLRR1-eLRR8, eLRR20-eLRR23 and eLRR32- eLRR37. This is consistent with previously studies, in which Elrr function was found to be determined by solvent-exposed residues in clustered LRRs of the concave b-sheet surface. For example, domain swaps of Narciclasine tomato Cfs revealed that eLRR13-eLRR16 of Cf-4 contribute to ligand specificity, while ligand specificity of Cf-9 is determined by eLRR10-eLRR16. In addition, photoaffinity labelling showed that BAM1 directly interacts with the small peptide ligand CLE9 at the eLRR6�CeLRR8 region.