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

Nitrogen-fixing root nodules on legumes result from two developmental processes, bacterial infection and nodule organogenesis. To balance symbiosis and plant growth, legume hosts restrict nodule numbers through an inducible autoregulatory process. Here, we present a mechanism where repression of a negative regulator ensures symbiotic susceptibility of uninfected roots of the host We show that microRNA miR2111 undergoes shoot-to-root translocation to control rhizobial infection through posttranscriptional regulation of the symbiosis suppressor TOO MUCH LOVE in roots. miR2111 maintains a susceptible default status in uninfected hosts and functions as an activator of symbiosis downstream of LOTUS HISTIDINE KINASE1-mediated cytokinin perception in roots and HYPERNODULATION ABERRANT ROOT FORMATION1, a shoot factor in autoregulation. The miR2111- node ensures activation of feedback regulation to balance infection and nodulation events.

Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host-symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant-fungal and plant-bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales.actinorhizal symbioses ͉ Casuarina glauca ͉ mycorrhizae ͉ signaling R oot endosymbioses are associations between plants and soil microorganisms involving intracellular accommodation of microbes within host cells. The most widespread of these associations is arbuscular mycorrhiza (AM), which is formed by the majority of land plants with fungi belonging to the phylum Glomeromycota (1). In contrast, nitrogen-fixing nodulation symbioses of plant roots and bacteria are restricted to four orders of eurosid dicots (2). Actinorhiza, formed by members of the Fagales, Rosales, and Cucurbitales with Gram-positive Frankia bacteria, differs from the interaction of legumes with Gramnegative rhizobia in several morphological and cytological aspects (3). Although these differences suggest independent regulatory mechanisms, the close relatedness of nodulating lineages indicates a common evolutionary basis of root nodulation symbioses (2). In the legume-rhizobia interaction, among the key factors mediating recognition between the plant and the bacteria are Nod factors (NFs). NFs are bacterial lipochitooligosaccharides with an N-acetylglucosamine backbone (4). The perception of NFs induces a series of responses in host roots, including ion flux changes and membrane depolarization, rhythmic calcium oscillations in and around the nucleus (calcium spiking), cytoskeletal modifications and root hair curling, and activation of cortical cell divisions (5). Extensive mutant screenings performed in legumes led to the identification of several loci involved in this signaling cascade, and recently most of the corresponding genes were identified by map-based approaches (6). In Lotus japonicus, two genes, NFR1 and NFR5 encoding receptor-like serine/threonine kinases with L...