The role of the putative receptor-like cytoplasmic kinase CLR1 in chitin signalling

Abstract

Plants detect potential pathogens by perception of conserved microbe-associated molecular patterns (MAMPs) through plasma membrane-localized receptors. Signalling initiated by these receptors is a key process of plant innate immunity. Typically, binding of MAMPs to the surface-exposed ectodomains of receptor components induces formation of homo- or heteromeric receptor complexes. These may consist of receptor-like kinases (RLKs), receptor-like proteins (RLPs) as well as receptor-like cytoplasmic kinases (RLCKs) which lack an extracellular ligand-binding domain. This study focuses on a potential heteromeric signalling complex involving the Arabidopsis lysin motif (LysM)-RLK CERK1 (Chitin Elicitor Receptor Kinase1), which mediates chitin-induced signalling and defence responses. In a preceding yeast two-hybrid screen the RLCK CERK1-INTERACTING LysM-RLK-LIKE RLCK1 (CLR1) was identified as a putative interactor of the CERK1 kinase domain. When taking a closer look at the amino acid sequence of CLR1, it becomes obvious that the sequence shares high homology with the kinase domains of Arabidopsis LysM-RLKs. Data obtained in this study suggest that the CLR1 sequence annotated by TAIR10 seems to be not correct and the protein likely starts 23 amino acids C terminal of the annotated start, thus exposing a predicted N-myristoylation motif. In vitro phosphorylation assays showed that the CERK1 kinase domain can directly phosphorylate CLR1 in vitro. This finding was supported by the fact that CLR1 fusion proteins stably expressed in Arabidopsis plants showed chitin-induced and CERK1-dependent phosphorylation. Thus, CLR1 represents a phosphorylation substrate of CERK1 in vitro and in vivo. This phosphorylation seemed to be independent of the N-terminal myristoylation of CLR1. Microsomal fractionations and subcellular localization studies in transgenic plants suggested that the majority of the CLR1 protein is soluble, but a membrane-associated CLR1 subpopulation is present in plant cells. Three independent T-DNA insertion lines were isolated and characterized with regard to chitin signalling and immunity to fungal and bacterial pathogens. The clr1 T-DNA lines showed reduced chitin-induced ROS generation, MAPK activation and defence gene expression, suggesting that CLR1 plays a role in chitin signalling. The severity of the phenotype depended on the position of the T-DNA. clr1 plants were not impaired in resistance against fungal pathogens, but showed a subtly enhanced sensitivity to bacterial infection. Since the CLR1 promoter showed high activity in hydathodes, CLR1 could be involved in selectively restricting pathogen entry through these constitutively open vents.