Probing the Arabidopsis Flagellin Receptor: FLS2-FLS2 Association and the Contributions of Specific Domains to Signaling Function

Abstract

FLAGELLIN SENSING2 (FLS2) is a transmembrane receptor kinase that activates antimicrobial defense responses upon binding of bacterial flagellin or the flagellin-derived peptide flg22. We find that some Arabidopsis thaliana FLS2 is present in FLS2-FLS2 complexes before and after plant exposure to flg22. flg22 binding capability is not required for FLS2-FLS2 association. Cys pairs flank the extracellular leucine rich repeat (LRR) domain in FLS2 and many other LRR receptors, and we find that the Cys pair N-terminal to the FLS2 LRR is required for normal processing, stability, and function, possibly due to undescribed endoplasmic reticulum quality control mechanisms. By contrast, disruption of the membrane-proximal Cys pair does not block FLS2 function, instead increasing responsiveness to flg22, as indicated by a stronger oxidative burst. There was no evidence for intermolecular FLS2-FLS2 disulfide bridges. Truncated FLS2 containing only the intracellular domain associates with full-length FLS2 and exerts a dominant-negative effect on wild-type FLS2 function that is dependent on expression level but independent of the protein kinase capacity of the truncated protein. FLS2 is insensitive to disruption of multiple N-glycosylation sites, in contrast with the related receptor EF-Tu RECEPTOR that can be rendered nonfunctional by disruption of single glycosylation sites. These and additional findings more precisely define the molecular mechanisms of FLS2 receptor function.

Chimeric FLS2 Receptors Reveal the Basis for Differential Flagellin Perception in Arabidopsis and Tomato

“…An earlier study by Ali et al (2007), using bimolecular fluorescence complementation and fluorescence resonance energy transfer in Arabidopsis protoplasts, could find no evidence for homodimer formation of FLS2. By contrast, in a recent report using FLS2 constructs with two distinct tags, such dimers could be coimmunoprecipitated (Sun et al, 2012). Apparently, these FLS2 dimers are present irrespective of the presence of the flg22.…”

CRT1 is a nuclear-translocated MORC endonuclease that participates in multiple levels of plant immunity

“…As FLS2 is predominantly localized in the PM before activation 34 , while the majority of CRT1 is in endomembranes 13 , an interesting question is where these immune proteins interact. Analyses of endoglycosidase H sensitivity 35 have suggested that FLS2, whether expressed in N. benthamiana or Arabidopsis, is present in both the endoplasmic reticulum and PM 36,37 . Moreover, a portion of FLS2 was found in endomembranes 36 , suggesting the CRT1-FLS2 interaction may occur in this compartment.…”

Site-SpecificN-Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae

“…Moreover, even in the case of the one N-glycosylation site, Asn-389 in the N389-C390-T391A motif, whose elimination did abrogate the SI response, our results indicated that it was the structural disruption caused by the T391A mutation rather than the lack of an N-glycan chain at this site that caused breakdown of SI. Thus, SRKb is more similar to the A. thaliana FLS2 (Sun et al, 2012) and the Medicago truncatula NFP (Lefebvre et al, 2012) receptor-like kinases, which are insensitive to disruption of its N-glycosylation sites, than to the A. thaliana EFR and tomato (Solanum lycopersicum) Cf-9 receptors, which are inactivated by disruption of even single N-glycosylation sites (van der Hoorn et al, 2005;Häweker et al, 2010;Saijo, 2010).…”