Mutations in FLS2 Ser-938 dissect signaling activation in FLS2-mediated Arabidopsis immunity

PLoS Pathog. 2013;9(4):e1003313. doi: 10.1371/journal.ppat.1003313. Epub 2013 Apr 18.

Abstract

Flagellin-sensing 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / genetics
  • Arabidopsis / immunology*
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Flagellin / immunology*
  • Flagellin / metabolism
  • Gene Expression Regulation, Plant
  • Phosphorylation
  • Plants, Genetically Modified
  • Protein Kinases / chemistry
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Serine-Threonine Kinases / metabolism*
  • Receptors, Pattern Recognition / metabolism
  • Signal Transduction

Substances

  • Arabidopsis Proteins
  • Receptors, Pattern Recognition
  • Flagellin
  • Protein Kinases
  • BAK1 protein, Arabidopsis
  • FLS2 protein, Arabidopsis
  • BIK1 protein, Arabidopsis
  • Protein Serine-Threonine Kinases

Grants and funding

This work was supported by U.S. Department of Energy (Contract No. DE-FG02-02ER15342) to AFB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.