Specific control of Arabidopsis BAK1/SERK4-regulated cell death by protein glycosylation

Abstract

Precise control of cell death is essential for the survival of all organisms. Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate cell death through elusive mechanisms. By deploying a genetic screen for suppressors of cell death triggered by virus-induced gene silencing of BAK1/SERK4 on Arabidopsis knockout collections, we identified STT3a, a protein involved in N-glycosylation modification, as an important regulator of bak1/serk4 cell death. Systematic investigation of glycosylation pathway and endoplasmic reticulum (ER) quality control (ERQC) components revealed distinct and overlapping mechanisms of cell death regulated by BAK1/SERK4 and their interacting protein BIR1. Genomewide transcriptional analysis revealed the activation of members of cysteine-rich receptor-like kinase (CRK) genes in the bak1/serk4 mutant. Ectopic expression of CRK4 induced STT3a/N-glycosylation-dependent cell death in Arabidopsis and Nicotiana benthamiana. Therefore, N-glycosylation and specific ERQC components are essential to activate bak1/serk4 cell death, and CRK4 is likely to be among client proteins of protein glycosylation involved in BAK1/SERK4-regulated cell death.

A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death

“…As has been reported previously, silencing Arabidopsis Col-0 plants with VIGS constructs containing GFP does not affect Pst growth ( Fig. 2D; de Oliveira et al, 2016). Silencing of CRK22 and CRK28 in the crk29-1 knockout resulted in significantly higher bacterial titers compared with Col-0 control plants 3 d post inoculation (Fig.…”

“…We have demonstrated that CRK28 is both glycosylated and phosphorylated in planta. Recently, CRK4 was demonstrated to be glycosylated, and STAUROSPORIN AND TEMPERATURE SENSITIVE3 N-glycosylation was required for CRK4-induced cell death and protein accumulation in Arabidopsis (de Oliveira et al, 2016). Glycosylation also has been shown to be important for the function of multiple immune receptors, including FLS2, EFR, and Cf proteins (van der Hoorn et al, 2005;Nekrasov et al, 2009;Saijo et al, 2009;Häweker et al, 2010;Liebrand et al, 2012).…”

“…The construct pYL156-CRK28/22 contains two fragments targeting CRK28 and CRK22, respectively. The binary TRV vectors, pTRV-RNA1, pYL156-GFP, pYL156-Cla1, and pYL156-CRK28/22, were electroporated into A. tumefaciens strain GV3101, and VIGS was performed as described previously on 2-week-old plants (de Oliveira et al, 2016). The pYL156-Cla1-silenced plants exhibit a leaf-bleaching phenotype, which was used as a visual marker for silencing efficiency (de Oliveira et al, 2016).…”

“…The binary TRV vectors, pTRV-RNA1, pYL156-GFP, pYL156-Cla1, and pYL156-CRK28/22, were electroporated into A. tumefaciens strain GV3101, and VIGS was performed as described previously on 2-week-old plants (de Oliveira et al, 2016). The pYL156-Cla1-silenced plants exhibit a leaf-bleaching phenotype, which was used as a visual marker for silencing efficiency (de Oliveira et al, 2016). One pair of fully expanded leaves on the 4-week-old plants was inoculated with Pst DC3000, and bacterial titers were determined 3 d post infiltration.…”

The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1

“…The very similar phenotype of both double 360 mutants confirms this observation, explaining the bak1 bir3 double mutant phenotype 361 as a cell death response caused by the absence of BAK1 and BKK1 (He et al,362 transcript levels were not altered in the bir3 mutant compared to wild type 364 (Supplemental Figure 19 A, B). Yamada et al (2016) reported that depletion of BAK1 365 primes PEPR-mediated cell death responses, and de Oliveira et al (2016) suggested 366 that cysteine-rich receptor kinases (CRK) might be client proteins of protein 367 glycosylation involved in BAK1-regulated cell death. Therefore, sensitizing pro-death 368…”