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.

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro.

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

Understanding Nod factor signalling paves the way for targeted engineering in legumes and non-legumes.

Legumes evolved LysM receptors for recognition of rhizobial Nod factors and initiation of signalling pathways for nodule organogenesis and infection. Intracellularly hosted bacteria are supplied with carbon resources in exchange for fixed nitrogen. Nod factor recognition is crucial for initial signalling, but is reiterated in growing roots initiating novel symbiotic events, and in developing primordia until symbiosis is well-established. Understanding how this signalling coordinates the entire process from cellular to plant level is key for de novo engineering in non-legumes and for improved efficiency in legumes. Here we discuss how recent studies bring new insights into molecular determinants of specificity and sensitivity in Nod factor signalling in legumes, and present some of the unknowns and challenges for engineering.

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.

Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid.

The pepper (Capsicum annuum) resistance gene bacterial spot3 (Bs3) is transcriptionally activated by the matching Xanthomonas euvesicatoria transcription-activator-like effector (TALE) AvrBs3. AvrBs3-induced Bs3 expression triggers a rapid and local cell death reaction, the hypersensitive response (HR). Bs3 is most closely related to plant flavin monooxygenases of the YUCCA (YUC) family, which catalyze the final step in auxin biosynthesis. Targeted mutagenesis of predicted NADPH- and FAD-cofactor sites resulted in Bs3 derivatives that no longer trigger HR, thereby suggesting that the enzymatic activity of Bs3 is crucial to Bs3-triggered HR. Domain swap experiments between pepper Bs3 and Arabidopsis (Arabidopsis thaliana) YUC8 uncovered functionally exchangeable and functionally distinct regions in both proteins, which is in agreement with a model whereby Bs3 evolved from an ancestral YUC gene. Mass spectrometric measurements revealed that expression of YUCs, but not expression of Bs3, coincides with an increase in auxin levels, suggesting that Bs3 and YUCs, despite their sequence similarity, catalyze distinct enzymatic reactions. Finally, we found that expression of Bs3 coincides with increased levels of the salicylic acid and pipecolic acid, two compounds that are involved in systemic acquired resistance.

An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus.

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs31EBE ), and fused this engineered promoter with multiple EBEs (ProBs314EBE ) to either the ß-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs31EBE and ProBs314EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs314EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs314EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants.