title: FLS2-BAK1 extracellular domain interaction sites required for defense signaling activation.
abstract: Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.
authors.affiliation: Department of Plant Pathology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.
authors.forename: Teresa
authors.initials: T
authors.lastname: Koller
authors.affiliation: Department of Plant Pathology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Bent
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D020125:Mutation, Missense; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface; D015398:Signal Transduction / Q000502:physiology*; D014026:Nicotiana / Q000737:chemistry / Q000201:enzymology / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; C512592:PEPR1 protein, Arabidopsis; D011956:Receptors, Cell Surface; D011494:Protein Kinases; C463660:BAK1 protein, Arabidopsis; C411853:FLS2 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases