Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling    

Abstract

The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation between BRI1 and BAK1(SERK3) in the natural habitat of both leucine-rich repeat receptor-like kinases using comparative colocalization analysis and fluorescence lifetime imaging microscopy. We show that activation of BR signaling by exogenous ligand application resulted in both elevated colocalization between BRI1 and BAK1(SERK3) and an about 50% increase of receptor heterooligomerization in the PM of live Arabidopsis root epidermal cells. However, large populations of BRI1 and BAK1(SERK3) colocalized independently of BRs. Moreover, we could visualize that approximately 7% of the BRI1 PM pool constitutively heterooligomerizes with BAK1(SERK3) in live root cells. We propose that only small populations of PMlocated BRI1 and BAK1(SERK3) receptors participate in active BR signaling and that the initiation of downstream signal transduction involves preassembled BRI1-BAK1(SERK3) heterooligomers.

An Overdose of the Arabidopsis Coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 or Its Ectodomain Causes Autoimmunity in a SUPPRESSOR OF BIR1-1-Dependent Manner

“…Plasmolysis experiments on Arabidopsis epidermal cells showed a retraction of the fluorescence signal associated with the cell membrane (Fig. 6C), supporting the proper localization of ECD-TM at the plasma membrane, as reported previously for BAK1 Nam and Li, 2002;Bücherl et al, 2013). To determine if ECD-TM was able to interact with PRRs, it was coexpressed with myc-tagged FLS2 in N. benthamiana leaves.…”

Phytosulfokine Regulates Growth in Arabidopsis through a Response Module at the Plasma Membrane That Includes CYCLIC NUCLEOTIDE-GATED CHANNEL17, H+-ATPase, and BAK1

“…For BRI1, the beststudied LRR-RLK binding of the ligand brassinosteroid was shown to release the inhibitory protein BRI1 kinase inhibitor 1 (BKI1), which initiates a series of transphosphorylations between BRI1 and BAK1 that result in receptor activation (Jiang et al, 2013). Using live-cell imaging, BRI1 was shown to interact with BAK1 also in the absence of a ligand, and signal initiation was proposed to take place from preassembled heterodimeric receptor complexes (Bücherl et al, 2013). Our finding that BAK1 binds to PSKR1 with high affinity is therefore not surprising and supports the idea that PSKR1 shares regulatory elements with related LRR-RLKs such as BRI1.…”

“…The brassinosteroid receptor BRI1 is known to form a complex with a coreceptor, the BRI1-associated receptor kinase 1 (BAK1) (Chinchilla et al, 2009;Bücherl et al, 2013). BAK1 is a promiscuous kinase that associates not only with BRI1 but also with receptors implicated in plant immunity, such as the flagellin receptor FLS2 (Postel et al, 2010), and BAK1 is also a target of the bacterial effector proteins AvrPto and AvrPtoB (Shan et al, 2008).…”

Retraction

“…LecRK-VI.2 does not modulate flg22-mediated association of FLS2 and BAK1 (Singh et al, 2012). The heterodimerization between FLS2 and BAK1 occurs within seconds (Schulze et al, 2010) with BAK1 acting as coreceptor for flg22 (Sun et al, 2013), indicating that both LRR-RLKs most likely exist in close proximity at the plasma membrane, as recently suggested in the case of BAK1 and BRI1 (Bücherl et al, 2013). We thus propose that the plasma membrane-localized IOS1 is required for promoting rapid FLS2-BAK1 complex formation upon flg22 binding.…”