FLS2-BAK1 extracellular domain interaction sites required for defense signaling activation

• Published in: PloS one Vol. 9; no. 10; p. e111185
• Main Authors: Koller, Teresa, Bent, Andrew F
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 30.10.2014; Public Library of Science (PLoS)
• Subjects: Amino Acid Substitution; Amino acids; Arabidopsis; Arabidopsis - chemistry; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; BASIC BIOLOGICAL SCIENCES; Biocompatibility; Biology and Life Sciences; Biomedical materials; C-Terminus; co-immunoprecipitation; Conservation; Crystal defects; Crystal structure; Crystallography, X-Ray; Damage patterns; Flagellin; Flowers & plants; Immunoprecipitation; In vivo methods and tests; Kinases; leaves; Leucine; Ligands; Moisture content; Mutagenesis; Mutation; Mutation, Missense; nicotiana; Nicotiana - chemistry; Nicotiana - enzymology; Nicotiana - genetics; Plant cells; Plant pathology; protein interactions; Protein Kinases - chemistry; Protein Kinases - genetics; Protein Kinases - metabolism; Protein Serine-Threonine Kinases - chemistry; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; Protein Structure, Quaternary; Protein Structure, Tertiary; Proteins; Receptors; Receptors, Cell Surface; Residues; seedings; Signal Transduction - physiology; Signaling; Site-directed mutagenesis; Structure-function relationships; United States > US; Wisconsin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0111185

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.