Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity.

Vijayata Singh,Amy Marshall-Colon,Jae H Lee,Man-Ho Oh,Steven C Huber,Benjamin Schwessinger,Youfu Zhao,Stuti Shrivastava,Cyril Zipfel,Sang Y Kim,Artemis Perraki

doi:10.3389/fpls.2017.01273

Abstract

The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F) transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F)-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F) in the bak1-4 null mutant. Neither BAK1 (Y610F) transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F)-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL) inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F)-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F) transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background) and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive oxygen species production, mitogen-activated protein kinase activation, and seedling growth inhibition. These new results do not support any involvement of Tyr-610 phosphorylation in either BR or immune signaling.

Highlights

Receptor kinases (RKs) play important roles in perceiving extracellular signals and activating downstream signaling via auto- and trans-phosphorylation reactions (Clouse, 2011)
Because conclusions about the physiological role of BRASSINOSTEROID-INSENSITIVE 1 (BRI1)-ASSOCIATED KINASE1 (BAK1) Tyr-610 had not been properly established, we generated new transgenic plants expressing BAK1 (Y610F)-Flag driven by the native promoter in the double null mutant bak1-4 bkk1-1 background
The homozygous bak1-4 bkk1-1 double mutant is seedling lethal (He et al, 2007) but can be rescued by expression of functional BAK1. This system was used previously to determine the functional role of various serine and threonine phosphosites in BAK1 (Wang et al, 2008) and was used in the present study to elucidate the role of Tyr-610 in vivo

Summary

Introduction

Receptor kinases (RKs) play important roles in perceiving extracellular signals and activating downstream signaling via auto- and trans-phosphorylation reactions (Clouse, 2011). Using the recombinant cytoplasmic domain of BRI1, autophosphorylation on tyrosine residues was identified and Tyr-831, located in the juxtamembrane domain, was established as a major site of autophosphorylation both in vitro and in vivo (Oh et al, 2009). BAK1, which functions as a co-receptor kinase with BRI1 in BR signaling, and with FLS2 in flg signaling (Ma et al, 2016), was shown to have dual specificity and Tyr-610 in the carboxyterminal domain was identified as a major phosphotyrosine site (Oh et al, 2010). Studies with previously produced and characterized transgenic plants expressing BAK1-Flag in the bak bkk double null mutant background (Wang et al, 2008) established that phosphorylation of Tyr-610 occurs in vivo in a BL-dependent manner. In continuing studies with the BAK1 (Y610F)-Flag transgenic plants, we came to realize that the plants generated in the original study were not correctly genotyped, resulting in retraction of the original publication

Methods

Results

Conclusion