Analysis of the subcellular behavior of Arabidopsis thaliana LysM-proteins and their role in plant innate immunity

Abstract

Health and survival of all higher eukaryotic organisms depend on efficient pathogen detection and rapid activation of defense mechanisms. Plants detect potential pathogens by recognizing conserved microbial molecules, so-called microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs), via pattern recognition receptors (PRRs). Recognition of MAMPs/PAMPs initiates defense signaling which leads to the establishment of plant innate immunity. The fungal polysaccharide chitin is perceived through lysin motif receptor-like kinases (LysM-RLKs) and receptor-like proteins (LysM-RLPs) which are thought to form receptor complexes for signal transduction. This study focuses on the analysis of Arabidopsis CERK1, a LysM-RLK essential for the perception of chitin, and the LysM-RLKs LYK5 and LYK4, which contribute to chitin signaling. lyk5 and lyk5 lyk4 double mutant plants were impaired in chitin-induced CERK1 phosphorylation but not MAPK activation. To quantify the effect of LYK5 and LYK4 disruption on immune responses chitin-induced marker gene expression was tested. lyk5 and lyk5 lyk4 plants showed moderately but significantly reduced expression of WRKY30, WRKY33 and WRKY53 upon chitin stress. To investigate ligand-induced spatial dynamics, the subcellular behavior of CERK1 and LYK5 in response to chitin was tested. Both LysM-RLKs localized to the plasma membrane and showed constitutive endomembrane trafficking, but only LYK5 underwent clear chitin-induced relocalization into mobile intracellular vesicles. Inhibitor approaches, co-localization studies and quantitative confocal microscopy demonstrated that chitin perception transiently induces the internalization of LYK5 into endocytic compartments that traffic along the cytoskeleton. In vitro phosphorylation assays revealed that LYK5 and LYK4 are substrates of CERK1 phosphorylation. CERK1-dependent and chitin-specific LYK5 phosphorylation was detected in planta. Interestingly, plants that lack CERK1 or express an enzymatically inactive CERK1 variant did not exhibit chitin-induced endocytosis of LYK5. Together, these results suggest that chitin-induced phosphorylation of LYK5 by CERK1 triggers LYK5 endocytosis. LYM2, a LysM-RLP with chitin binding activity, represents another putative component of the Arabidopsis chitin recognition complex. However, lym2 mutants show no defects in canonical chitin signaling. Confocal laser scanning microscopy showed plasma membrane localization of LYM2. Upon chitin elicitation LYM2 specifically relocalizes into plasmodesmata (PD) in a CERK1-independent manner. Surprisingly, lyk5 lyk4 lym2 triple mutant plants were not viable, potentially suggesting an involvement of these proteins in plant developmental processes. The results of this work contribute to a better understanding of the role of LYK5, LYK4 and LYM2 in CERK1-mediated chitin signaling and shed light on their subcellular behavior.