The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.BiFC ͉ map-based cloning ͉ plant-microbe symbiosis ͉ protein phosphorylation ͉ protein-protein interaction L egume plants can establish endosymbiotic interactions with nitrogen-fixing rhizobia and phosphate-delivering arbuscular mycorrhiza (AM) fungi. Plant root hairs form a tight curl in which rhizobia are entrapped. From this closed infection pocket, the bacteria are guided by plant membrane-delimited infection threads (ITs) into the root nodule, a specialized organ developed by the plant to provide an optimized environment for nitrogen fixation (1). AM fungal hyphae are guided through epidermal and cortical cells toward the inner cortex (2), where arbuscules, highly branched intracellular symbiotic structures, are formed (3). Intracellular infection by rhizobia and AM fungi is preceded by an exchange of specific signaling molecules. Rhizobia produce lipochito-oligosaccharides (Nod factors) that activate host plant responses including root hair deformation, and preinfection thread formation, which are structures that determine the path of IT growth through the root (4), and initiation of cortical cell division (1). One of the earliest plant responses to stimulation by Nod factors is Ca 2ϩ -spiking, which consists of perinuclear oscillations of calcium concentration in root cells (5). In the legume

Symbiotic root nodules are beneficial to leguminous host plants; however, excessive nodulation damages the host because it interferes with the distribution of nutrients in the plant. To keep a steady balance, the nodulation programme is regulated systemically in leguminous hosts. Leguminous mutants that have lost this ability display a hypernodulating phenotype. Through the use of reciprocal and self-grafting studies using Lotus japonicus hypernodulating mutants, har1 (also known as sym78), we show that the shoot genotype is responsible for the negative regulation of nodule development. A map-based cloning strategy revealed that HAR1 encodes a protein with a relative molecular mass of 108,000, which contains 21 leucine-rich repeats, a single transmembrane domain and serine/threonine kinase domains. The har1 mutant phenotype was rescued by transfection of the HAR1 gene. In a comparison of Arabidopsis receptor-like kinases, HAR1 showed the highest level of similarity with CLAVATA1 (CLV1). CLV1 negatively regulates formation of the shoot and floral meristems through cell-cell communication involving the CLV3 peptide. Identification of hypernodulation genes thus indicates that genes in leguminous plants bearing a close resemblance to CLV1 regulate nodule development systemically, by means of organ-organ communication.

Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin-oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYMdependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calciumspiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.