Plant Pattern-Recognition Receptor Activation and Exploration of Techniques to Engineer Novel Ligand Specificities

Pattern-recognition receptors (PRRs) localized at the plasma membrane of plant cells monitor the apoplastic space for pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) and apoplastic effectors. In my thesis research I studied different aspects of the two Arabidopsis PRRs EFR and FLS2. Both EFR and FLS2 are leucine-rich repeat (LRR) receptor-like kinases (RLKs). First I cloned, sequenced and functionally tested in chimeric receptors the LRR-encoding domains of nine novel EFR alleles from seven Brassicaceae species. The sequences were subjected to Repeat Conservation Mapping (RCM), a computational program developed in our lab that identifies conserved patches on the predicted surface of LRR domains. This analysis revealed conserved clusters in the LRR domain of EFR, which are candidate functionally important sites. Additional work used RCM to compare the last seven LRRs of the BAK1-interacting LRR-RLKs EFR, FLS2, BRI1 and PEPR1, and revealed a conserved putative BAK1 ectodomain interaction site. However, site-directed mutagenesis of the putative BAK1 interaction site in the FLS2 ectodomain did not disrupt interaction with BAK1 and analogous mutagenesis in EFR resulted in receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. I then performed In vivo tests of the newly published FLS2-BAK1-flg22 ectodomain co-crystal structure, with the resulting data supporting models in which FLS2 signaling is initiated by formation of a ligand mediated FLS2 -- BAK1 ectodomain complex.;In an effort to mold our constantly growing knowledge about the plant immune system into agriculturally useful tools to improve crop health, I also explored two approaches to engineer novel PRRs. The first approach sought to use yeast surface display to evolve the endogenous LRR domains of EFR and FLS2 towards novel ligand binding capacities. The second approach used a LRR-based variable lymphocyte receptor (VLR) of Petromyzon marinus as template for rational design of novel ligand binding sites in FLS2. Both approaches are compromised by the subsequently discovered finding that PRR activation requires a SERK protein ectodomain as a co-receptor. The research projects presented here further elucidate PRR signaling activation and present possible technologies for engineering novel PRRs to improve crop health.