Nanobody-driven signaling reveals the core receptor complex in root nodule symbiosis.

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals.

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors.

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.

The Arabidopsis thaliana LysM-containing Receptor-Like Kinase 2 is required for elicitor-induced resistance to pathogens.

In Arabidopsis thaliana, perception of chitin from fungal cell walls is mediated by three LysM-containing Receptor-Like Kinases (LYKs): CERK1, which is absolutely required for chitin perception, and LYK4 and LYK5, which act redundantly. The role in plant innate immunity of a fourth LYK protein, LYK2, is currently not known. Here we show that CERK1, LYK2 and LYK5 are dispensable for basal susceptibility to B. cinerea but are necessary for chitin-induced resistance to this pathogen. LYK2 is dispensable for chitin perception and early signalling events, though it contributes to callose deposition induced by this elicitor. Notably, LYK2 is also necessary for enhanced resistance to B. cinerea and Pseudomonas syringae induced by flagellin and for elicitor-induced priming of defence gene expression during fungal infection. Consistently, overexpression of LYK2 enhances resistance to B. cinerea and P. syringae and results in increased expression of defence-related genes during fungal infection. LYK2 appears to be required to establish a primed state in plants exposed to biotic elicitors, ensuring a robust resistance to subsequent pathogen infections.

Ligand-recognizing motifs in plant LysM receptors are major determinants of specificity.

Plants evolved lysine motif (LysM) receptors to recognize and parse microbial elicitors and drive intracellular signaling to limit or facilitate microbial colonization. We investigated how chitin and nodulation (Nod) factor receptors of Lotus japonicus initiate differential signaling of immunity or root nodule symbiosis. Two motifs in the LysM1 domains of these receptors determine specific recognition of ligands and discriminate between their in planta functions. These motifs define the ligand-binding site and make up the most structurally divergent regions in cognate Nod factor receptors. An adjacent motif modulates the specificity for Nod factor recognition and determines the selection of compatible rhizobial symbionts in legumes. We also identified how binding specificities in LysM receptors can be altered to facilitate Nod factor recognition and signaling from a chitin receptor, advancing the prospects of engineering rhizobial symbiosis into nonlegumes.

An EFR-Cf-9 chimera confers enhanced resistance to bacterial pathogens by SOBIR1- and BAK1-dependent recognition of elf18.

The transfer of well-studied native and chimeric pattern recognition receptors (PRRs) to susceptible plants is a proven strategy to improve host resistance. In most cases, the ectodomain determines PRR recognition specificity, while the endodomain determines the intensity of the immune response. Here we report the generation and characterization of the chimeric receptor EFR-Cf-9, which carries the ectodomain of the Arabidopsis thaliana EF-Tu receptor (EFR) and the endodomain of the tomato Cf-9 resistance protein. Both transient and stable expression of EFR-Cf-9 triggered a robust hypersensitive response (HR) upon elf18 treatment in tobacco. Co-immunoprecipitation and virus-induced gene silencing studies showed that EFR-Cf-9 constitutively interacts with SUPPRESSOR OF BIR1-1 (SOBIR1) co-receptor, and requires both SOBIR1 and kinase-active BRI1-ASSOCIATED KINASE1 (BAK1) for its function. Transgenic plants expressing EFR-Cf-9 were more resistant to the (hemi)biotrophic bacterial pathogens Pseudomonas amygdali pv. tabaci (Pta) 11528 and Pseudomonas syringae pv. tomato DC3000, and mounted an HR in response to high doses of Pta 11528 and P. carotovorum. Taken together, these data indicate that the EFR-Cf-9 chimera is a valuable tool for both investigating the molecular mechanisms responsible for the activation of defence responses by PRRs, and for potential biotechnological use to improve crop disease resistance.