Xu, Guangyuan (2016-08). Genetic Dissection and Functional Analysis of Suppressors of Receptor-Like Kinase BAK1 Family-Mediated Cell Death. Doctoral Dissertation.
Precise control of cell death is critical for all organismal survival. Arabidopsis receptor-like kinase BAK1 and SERK4 redundantly and negatively regulate cell death with elusive mechanisms. By deploying a genetic screen for suppressors of cell death triggered by virus-induced gene silencing of BAK1/SERK4 on Arabidopsis knockout mutant collections, we identified STT3a, which is involved in N-glycosylation modification and cyclic nucleotide-gated channel 20, a nonselective cation channel, as important regulators as bak1/serk4 cell death. Systematic investigation of glycosylation pathway and ER quality control (ERQC) components revealed distinct and overlapping mechanisms operating BAK1/SERK4- and their interacting protein BIR1-regulated cell death. Genome-wide 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 among client proteins of protein glycosylation involved in BAK1/SERK4-regulated cell death. In addition, our genetic screen revealed that CNGC20 plays an important role in bak1/serk4 cell death. Interestingly, in vivo and in vitro assays indicated that BAK1 directly interacts with and phosphorylates membrane-resident CNGC20. Mass spectrometry and mutagenesis analysis revealed that specific phosphorylation sites of CNGC20 by BAK1 are indispensable for its function in cell death control. In conclusion, our findings unravel novel components and provide mechanistic insights into plant cell death regulation.