Although most higher plants establish a symbiosis with arbuscular mycorrhizal fungi, symbiotic nitrogen fixation with rhizobia is a salient feature of legumes. Despite this host range difference, mycorrhizal and rhizobial invasion shares a common plant-specified genetic programme controlling the early host interaction. One feature distinguishing legumes is their ability to perceive rhizobial-specific signal molecules. We describe here two LysM-type serine/threonine receptor kinase genes, NFR1 and NFR5, enabling the model legume Lotus japonicus to recognize its bacterial microsymbiont Mesorhizobium loti. The extracellular domains of the two transmembrane kinases resemble LysM domains of peptidoglycan- and chitin-binding proteins, suggesting that they may be involved directly in perception of the rhizobial lipochitin-oligosaccharide signal. We show that NFR1 and NFR5 are required for the earliest physiological and cellular responses to this lipochitin-oligosaccharide signal, and demonstrate their role in the mechanism establishing susceptibility of the legume root for bacterial infection.

Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia. Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules. The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed. Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip. Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia. To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition. We show that nin is required for the formation of infection threads and the initiation of primordia. NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.

Plants belonging to the legume family develop nitrogen-fixing root nodules in symbiosis with bacteria commonly known as rhizobia. The legume host encodes all of the functions necessary to build the specialized symbiotic organ, the nodule, but the process is elicited by the bacteria. Molecular communication initiates the interaction, and signals, usually flavones, secreted by the legume root induce the bacteria to produce a lipochitin-oligosaccharide signal molecule (Nod-factor), which in turn triggers the plant organogenic process. An important determinant of bacterial host specificity is the structure of the Nod-factor, suggesting that a plant receptor is involved in signal perception and signal transduction initiating the plant developmental response. Here we describe the cloning of a putative Nod-factor receptor kinase gene (NFR5) from Lotus japonicus. NFR5 is essential for Nod-factor perception and encodes an unusual transmembrane serine/threonine receptor-like kinase required for the earliest detectable plant responses to bacteria and Nod-factor. The extracellular domain of the putative receptor has three modules with similarity to LysM domains known from peptidoglycan-binding proteins and chitinases. Together with an atypical kinase domain structure this characterizes an unusual receptor-like kinase.

Most higher plant species can enter a root symbiosis with arbuscular mycorrhizal fungi, in which plant carbon is traded for fungal phosphate. This is an ancient symbiosis, which has been detected in fossils of early land plants. In contrast, the nitrogen-fixing root nodule symbioses of plants with bacteria evolved more recently, and are phylogenetically restricted to the rosid I clade of plants. Both symbioses rely on partially overlapping genetic programmes. We have identified the molecular basis for this convergence by cloning orthologous SYMRK ('symbiosis receptor-like kinase') genes from Lotus and pea, which are required for both fungal and bacterial recognition. SYMRK is predicted to have a signal peptide, an extracellular domain comprising leucine-rich repeats, a transmembrane and an intracellular protein kinase domain. Lotus SYMRK is required for a symbiotic signal transduction pathway leading from the perception of microbial signal molecules to rapid symbiosis-related gene activation. The perception of symbiotic fungi and bacteria is mediated by at least one common signalling component, which could have been recruited during the evolution of root nodule symbioses from the already existing arbuscular mycorrhiza symbiosis.

Legume root nodules originate from differentiated cortical cells that reenter the cell cycle and form organ primordia. We show that perception of the phytohormone cytokinin is a key element in this switch. Mutation of a Lotus japonicus cytokinin receptor gene leads to spontaneous development of root nodules in the absence of rhizobia or rhizobial signal molecules. The mutant histidine kinase receptor has cytokinin-independent activity and activates an Escherichia coli two-component phosphorelay system in vivo. Mutant analysis shows that cytokinin signaling is required for cell divisions that initiate nodule development and defines an autoregulated process where cytokinin induction of nodule stem cells is controlled by shoots.

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.

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Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.