Lysin motif (LysM) is a universal carbohydrate-binding module that is found in all kingdoms of life. In leguminous plants, LysM receptor-like kinases (LysM-RLKs) are known to be involved in Rhizobia-Legume symbiosis (RLS) and perceive rhizobia molecular signals on their extracellular LysM domains and initiate downstream symbiotic signalling via the intracellular kinase domain. In the model legume Lotus japonicus, two LysM-RLKs, Nod factor receptor 1 (NFR1) and Nod factor receptor 5 (NFR5) perceive a decorated lipochitooligosaccharide called Nodulation factor (Nod Factor) and initiate a downstream symbiotic response. Rhizobial exopolysaccharide (EPS) is another signalling molecule that is recognized by Exopolysaccharide receptor 3 (EPR3) that is also crucial for recognizing the correct symbiont and establishing downstream symbiosis responses. These three receptors are important for establishing functional nodules on the plant’s roots that houses rhizobia bacteria for nitrogen fixation. In L. japonicus, there are 17 members of the LysM-RLK receptor family compared to just 5 members in A. thaliana, which does not form any symbioses. This highlights the importance of studying the LysM-RLKs in legumes to better understand how legumes can functionally distinguish mutualistic microbes from pathogens.
Using in vitro biochemical and structural tools in conjunction with in planta genetic and functional screens, the role of two LysM-RLKs in L. japonicus have been uncovered.
The receptors that perceive chitin, a pathogen-associated molecule pattern, and mediate chitin-induced defence response are known for both A. thaliana and rice. These receptors involve LysM-RLKs. However, the receptors in legumes still remain unknown. Genetic screens and in planta experiments indicate a LysM-RLK, LYS6, as a receptor that perceives chitin and mounts chitin-induced defence responses in roots. Based on this, quantitative binding experiments using Microscale Thermophoresis (MST) were performed that experimentally affirm LYS6 as a chitin binder. In addition, a crystal structure of LYS6 ectodomain was determined. Based on the high structural similarity to established plant chitin receptors, the crystal structure further indicates the role of LYS6 as a chitin binder.
Another LysM-RLK from Lotus, LYS1, was found to act in concert with NFR1 and NFR5 for the development of symbiotic root nodules from in planta experiments. Using two different quantitative binding techniques, MST and Biolayer Interferometry (BLI), LYS1 was shown to bind Nod factors but not chitin. Additionally, kinase assays were performed showing that LYS1 has an active kinase and is able to phosphorylate the NFR5 kinase domain in vitro. These findings, together with the subtle phenotype observed with LYS1, suggest a more complex pathway rather than “On-off” switch mediated by NFR1 and NFR5.