Functional analysis of Arabidopsis LysM-RLKs and related kinases in chitin perception and defense signaling
Plant Cell and Developmental Biology
Final Report Abstract
One focus of our studies within this project was on the analysis of Arabidopsis LysM-RLKs LYK5 and LYK4. We could show that lyk5 and lyk5 lyk4 double mutant plants were impaired in chitininduced 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 and co-localization studies combined with 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. In a preceding yeast two-hybrid screen the RLCK CERK1-INTERACTING LysM-RLK-LIKE 1 (CLR1) was identified as a putative interactor of the CERK1 kinase domain. 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. 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 canonical chitin signalling. 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.
Publications
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The role of the putative receptor-like cytoplasmic kinase (RLCK) CIR1 in chitin signalling. XVI International Congress on Molecular Plant- Microbe Interactions (6.-10. July 2014, Rhodes)
Ziegler, Y., Petutschnig, E.K., Lipka, V.
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Chitininduced and CERK1-dependent endocytosis of the LysM-RLKs LYK4 and LYK5. International Conference on Arabidopsis Research 2015 (5.-9. July 2015, Paris)
Erwig, J., Petutschnig, E.K., Ghareeb, H., Hacke, R. Kopischke, M., Matei, A., Lipka, V.
