Fig. 2
Identification of variable lymphocyte receptors that bind the Pseudomonas syringae effector HopM1. a and b yeast-surface display of HopM1-specific VLRBs. The x-axis represents Alexa Fluor® 488 (conjugated to α-c-Myc antibody) fluorescence while the y-axis shows phycoerythrin (conjugated to streptavidin) fluorescence of individual yeast cells. Fluorescence was detected using BD Accuri C6 flow cytometer. The number highlighted in bold indicates the percentage of yeast cells with detectable HopM11–300 binding. a Lower-affinity binding HopM1-specific VLRB; 50 nM biotinylated HopM1. b Higher-affinity binding HopM1-specific VLRB; 50 nM biotinylated HopM1. c Amino acid alignment of the three different high-affinity HopM1-specific VLRB sequences and of Toll-like receptor 5 (TLR5; a mammalian immune receptor that recognizes bacterial flagellin)-specific VLRB. Alignment was generated using MegAlign (DNASTAR) and graphed using Boxshade. Highlighted in a white background are amino acids that are different. Amino acid position is shown on the upper right corner. A red bar over the sequence identifies the leucine-rich repeat (LRR) domains identified. In yellow and green, are the N-terminal and C-terminal LRR domains, respectively, which are characterized by the presence of disulfide bonds. The domains were identified using the SMART tool [59]. d 3-D structure model of HopM1-specific VLRB. LRR domains are highlighted in red, the N-terminal LRR domain in yellow, and the C-terminal LRR domain in green. Modeling was performed with SWISS-MODEL using the structure of a previously crystalized VLRB protein (3g3aA) [16]