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Lysin-motif receptor-like kinases (LysM-RLKs) are involved in the recognition of microbe-associated molecular patterns to initiate pattern-triggered immunity (PTI). LysM-RLKs are also required for recognition of microbe-derived symbiotic signal molecules upon establishing mutualistic interactions between plants and microsymbionts. A LysM-RLK CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) plays central roles both in chitin-mediated PTI and in arbuscular mycorrhizal symbiosis, suggesting the overlap between immunity and symbiosis, at least in the signal perception and the activation of downstream signal cascades. In this study, we screened for the interacting proteins of Nod factor Receptor1 (NFR1), a CERK1 homolog in the model legume Lotus japonicus , and obtained a protein orthologous to NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13 (NHL13), a protein involved in the activation of innate immunity in Arabidopsis thaliana , which we named LjNHL13a. LjNHL13a interacted with NFR1 and with the symbiosis receptor kinase SymRK. LjNHL13a also displayed positive effects in nodulation. Our results suggest that NHL13 plays a role both in plant immunity and symbiosis, possibly where they overlap. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The establishment of the legume-rhizobia symbiosis, termed the root-nodule symbiosis (RNS), requires elaborate interactions at the molecular level. The host plant-derived transcription factor NODULE INCEPTION (NIN) is known to be crucial for RNS, regulating associated processes such as alteration of root hair morphology, infection thread formation, and cell division during nodulation. This emphasizes the importance of the precise spatiotemporal regulation of NIN expression for the establishment of RNS; however, the detailed role of NIN promoter sequences in this process remains unclear. The daphne mutant, a nin mutant allele containing a chromosomal translocation approximately 7 kb upstream of the start codon, does not form nodules but does form infection threads, indicating that the region within 7 kb of the NIN start codon contributes to NIN expression during infection thread formation. CYCLOPS binds to a CYCLOPS response element ( CYC-RE ) in the NIN promoter, and cyclops mutants are defective in infection thread formation. Here, we performed complementation analysis in nin mutants, using various truncated forms of the NIN promoter, and found that the CYC-RE is important for infection thread formation. Additionally, the CYC-RE deletion mutant, generated through CRISPR/Cas9 technology, displayed a significant reduction in infection thread formation, indicating that the CYC-RE is important for the fine-tuning of NIN expression during this process. However, the fact that infection thread formation is not completely abolished in the CYC-RE deletion mutant suggests that cis and trans factors other than CYCLOPS and the CYC-RE may cooperatively regulate NIN expression for the induction of infection thread formation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Sinorhizobium fredii HH103-Rif r , a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rif r invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rif r . A S. fredii HH103-Rif r nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rif r mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rif r fla mutants nodulated L. burttii. None of the S. fredii HH103-Rif r surface-polysaccharide mutants gained effective nodulation with L. japonicus.

Legume plants engage in intimate relationships with rhizobial bacteria to form nitrogen-fixing nodules, root-derived organs that accommodate the microsymbiont. Members of the Nuclear Factor Y (NF-Y) gene family, which have undergone significant expansion and functional diversification during plant evolution, are essential for this symbiotic liaison. Acting in a partially redundant manner, NF-Y proteins were shown, previously, to regulate bacterial infection, including selection of a superior rhizobial strain, and to mediate nodule structure formation. However, the exact mechanism by which these transcriptional factors exert their symbiotic functions has remained elusive. By carrying out detailed functional analyses of Lotus japonicus mutants, we demonstrate that LjNF-YA1 becomes indispensable downstream from the initial cortical cell divisions but prior to nodule differentiation, including cell enlargement and vascular bundle formation. Three affiliates of the SHORT INTERNODES/STYLISH transcription factor gene family, called STY1, STY2, and STY3, are demonstrated to be among likely direct targets of LjNF-YA1, and our results point to their involvement in nodule formation.

In many legumes, roots that are exposed to light do not form nodules. Here, we report that blue light inhibits nodulation in Lotus japonicus roots inoculated with Mesorhizobium loti. Using RNA interference, we suppressed the expression of the phototropin and cryptochrome genes in L. japonicus hairy roots. Under blue light, plants transformed with an empty vector did not develop nodules, whereas plants exhibiting suppressed expression of cry1 and cry2 genes formed nodules. We also measured rhizobial growth to investigate whether the inhibition of nodulation could be caused by a reduced population of rhizobia in response to light. Although red light had no effect on rhizobial growth, blue light had a strong inhibitory effect. Rhizobial growth under blue light was partially restored in signature-tagged mutagenesis (STM) strains in which LOV-HK/PAS- and photolyase-related genes were disrupted. Moreover, when Ljcry1A and Ljcry2B-silenced plants were inoculated with the STM strains, nodulation was additively increased. Our data show that blue light receptors in both the host plant and the symbiont have a profound effect on nodule development. The exact mechanism by which these photomorphogenetic responses function in the symbiosis needs further study, but they are clearly involved in optimizing legume nodulation.

We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

During development of legume root nodules, rhizobia and their host plant cells undergo profound differentiation, which is underpinned by massive changes in gene expression in both symbiotic partners. Oxygen concentrations in infected and surrounding uninfected cells drop precipitously during nodule development. To assess what effects this has on plant and bacterial cell differentiation and gene expression, we used a leghemoglobin-RNA-interference (LbRNAi) line of Lotus japonicus, which is devoid of leghemoglobins and has elevated levels of free-oxygen in its nodules. Bacteroids in LbRNAi nodules showed altered ultrastructure indicating changes in bacterial differentiation. Transcript analysis of 189 plant and 192 bacterial genes uncovered many genes in both the plant and bacteria that were differentially regulated during nodulation of LbRNAi plants compared with the wild type (containing Lb and able to fix nitrogen). These included fix and nif genes of the bacteria, which are involved in microaerobic respiration and nitrogen fixation, respectively, and plant genes involved in primary and secondary metabolism. Metabolite analysis revealed decreased levels of many amino acids in nodules of LbRNAi plants, consistent with the defect in symbiotic nitrogen fixation of this line.

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.

Lotus japonicus determinate nodules differ greatly from indeterminate nodules in their organogenesis and morphological characteristics, whereas Lupinus albus lupinoid nodules share features of determinate and indeterminate nodules. The mitotic inhibitor Ccs52A is essential for endoreduplication and ploidy-dependent cell enlargement during symbiotic cell differentiation in Medicago truncatula indeterminate nodules. ccs52A homolog genes were isolated from lupin and lotus nodules; the deduced Ccs52A proteins showed high sequence similarity with other Cdh-1-type activators of the anaphase-promoting complex and were grouped with A-type Ccs52 proteins from different plants. In lupin, ccs52A expression was restricted to the earlier stages of nodule development, whereas ccs52A transcripts accumulated in lotus nodule primordia and, to a lesser extent, in mature nodules. Nodule development in Lupinus albus involved a progressive increase in nuclear and cellular size and ploidy level; similarly, Lotus japonicus nodules contained polyploid nuclei and enlarged cells in the infected zone. Nevertheless, in situ hybridization experiments showed the highest ccs52A expression in the inner cortex cells of the lupin nodule primordium, probably associated to the increased size of these cells in mature nodules. In view of our results, Ccs52A-mediated endoreduplication appears to be a universal mechanism required for nodule cell differentiation during the establishment of nitrogen-fixing symbioses.

The role of Mesorhizobium loti surface polysaccharides on the nodulation process is not yet fully understood. In this article, we describe the nodulation phenotype of mutants affected in the synthesis of lipopolysaccharide (LPS) and beta(1,2) cyclic glucan. M. loti lpsbeta2 mutant produces LPS with reduced amount of O-antigen, whereas M. loti lpsbeta1 mutant produces LPS totally devoid of O-antigen. Both genes are clustered in the chromosome. Based on amino acid sequence homology, LPS sugar composition, and enzymatic activity, we concluded that lpsbeta2 codes for an enzyme involved in the transformation of dTDP-glucose into dTDP-rhamnose, the sugar donor of rhamnose for the synthesis of O-antigen. On the other hand, lpsbeta1 codes for a glucosyltransferase involved in the biosynthesis of the O-antigen. Although LPS mutants elicited normal nodules, both show reduced competitiveness compared with the wild type. M. loti beta(1-2) cyclic glucan synthase (cgs) mutant induces white, empty, ineffective pseudonodules in Lotus tenuis. Cgs mutant induces normal root hair curling but is unable to induce the formation of infection threads. M. loti cgs mutant was more sensitive to deoxycholate and displayed motility impairment compared with the wild-type strain. This pleiotropic effect depends on calcium concentration and temperature.

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.