Rhizosphere frame system enables nondestructive live-imaging of legume-rhizobium interactions in the soil.

Most plants interact with various soil microorganisms as they grow through the soil. Root nodule symbiosis by legumes and rhizobia is a well-known phenomenon of plant-microbe interactions in the soil. Although microscopic observations are useful for understanding the infection processes of rhizobia, nondestructive observation methods have not been established for monitoring interactions between rhizobia and soil-grown roots. In this study, we constructed Bradyrhizobium diazoefficiens strains that constitutively express different fluorescent proteins, which allows identification of tagged rhizobia by the type of fluorophores. In addition, we constructed a plant cultivation device, Rhizosphere Frame (RhizoFrame), which is a soil-filled container made of transparent acrylic plates that allows observation of roots growing along the acrylic plates. Combining fluorescent rhizobia with RhizoFrame, we established a live imaging system, RhizoFrame system, that enabled us to track the nodulation processes with fluorescence stereomicroscope while retaining spatial information about roots, rhizobia, and soil. Mixed inoculation with different fluorescent rhizobia using RhizoFrame enabled the visualization of mixed infection of a single nodule with two strains. In addition, observation of transgenic Lotus japonicus expressing auxin-responsive reporter genes indicated that RhizoFrame system could be used for a real-time and nondestructive reporter assay. Thus, the use of RhizoFrame system is expected to enhance the study of the spatiotemporal dynamics of plant-microbe interactions in the soil.

Mutants of Lotus japonicus deficient in flavonoid biosynthesis.

Spatiotemporal features of anthocyanin accumulation in a model legume Lotus japonicus (Regel) K.Larsen were elucidated to develop criteria for the genetic analysis of flavonoid biosynthesis. Artificial mutants and wild accessions, with lower anthocyanin accumulation in the stem than the standard wild type (B-129 'Gifu'), were obtained by ethyl methanesulfonate (EMS) mutagenesis and from a collection of wild-grown variants, respectively. The loci responsible for the green stem of the mutants were named as VIRIDICAULIS (VIC). Genetic and chemical analysis identified two loci, namely, VIC1 and VIC2, required for the production of both anthocyanins and proanthocyanidins (condensed tannins), and two loci, namely, VIC3 and VIC4, required for the steps specific to anthocyanin biosynthesis. A mutation in VIC5 significantly reduced the anthocyanin accumulation. These mutants will serve as a useful system for examining the effects of anthocyanins and proanthocyanidins on the interactions with herbivorous pests, pathogenic microorganisms and nitrogen-fixing symbiotic bacteria, Mesorhizobium loti.

Requirements of Qa-SNARE LjSYP132s for Nodulation and Seed Development in Lotus japonicus.

SNAREs (soluble N-ethyl maleimide-sensitive factor attachment protein receptors) mediate membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s are the members of Qa-SNAREs in Lotus japonicus. Two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis detected strong expression of LjSYP132s in infected root nodules and seeds by posttranscriptional modification. In either LjSYP132a or LjSYP132b silenced roots (RNAi-LjSYP132a, RNAi-LjSYP132b), the infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR showing that both genes were expressed at the early stage of infection. However, during the nodulation, only LjSYP132a was induced. LjSYP132s protein was observed in the Mesorhizobium loti-inoculated roots of mutants, nfr1, castor and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the peribacteroid membrane suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting that LjSYP132b has a role in pollen tube growth and nutrient transport in the plasma membrane of seeds.

Kinase activity-dependent stability of calcium/calmodulin-dependent protein kinase of Lotus japonicus.

MAIN CONCLUSION: Accumulation of calcium/calmodulin-dependent protein kinase (CCaMK) in root cell nucleus depends on its kinase activity but not on nuclear symbiotic components crucial for nodulation. Plant calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator of symbioses with rhizobia and arbuscular mycorrhizal fungi as it decodes symbiotic calcium signals induced by microsymbionts. CCaMK is expressed mainly in root cells and localizes to the nucleus, where microsymbiont-triggered calcium oscillations occur. The molecular mechanisms that control CCaMK localization are unknown. Here, we analyzed the expression and subcellular localization of mutated CCaMK in the roots of Lotus japonicus and found a clear relation between CCaMK kinase activity and its stability. Kinase-defective CCaMK variants showed lower protein levels than the variants with kinase activity. The levels of transcripts driven by the CaMV 35S promoter were similar among the variants, indicating that stability of CCaMK is regulated post-translationally. We also demonstrated that CCaMK localized to the root cell nucleus in several symbiotic mutants, including cyclops, an interaction partner and phosphorylation target of CCaMK. Our results suggest that kinase activity of CCaMK is required not only for the activation of downstream symbiotic components but also for its stability in root cells.

The Mycoheterotrophic Symbiosis Between Orchids and Mycorrhizal Fungi Possesses Major Components Shared with Mutualistic Plant-Mycorrhizal Symbioses.

Achlorophylous and early developmental stages of chorolophylous orchids are highly dependent on carbon and other nutrients provided by mycorrhizal fungi, in a nutritional mode termed mycoheterotrophy. Previous findings have implied that some common properties at least partially underlie the mycorrhizal symbioses of mycoheterotrophic orchids and that of autotrophic arbuscular mycorrhizal (AM) plants; however, information about the molecular mechanisms of the relationship between orchids and their mycorrhizal fungi is limited. In this study, we characterized the molecular basis of an orchid-mycorrhizal (OM) symbiosis by analyzing the transcriptome of Bletilla striata at an early developmental stage associated with the mycorrhizal fungus Tulasnella sp. The essential components required for the establishment of mutual symbioses with AM fungi or rhizobia in most terrestrial plants were identified from the B. striata gene set. A cross-species gene complementation analysis showed one of the component genes, calcium and calmodulin-dependent protein kinase gene CCaMK in B. striata, retains functional characteristics of that in AM plants. The expression analysis revealed the activation of homologs of AM-related genes during the OM symbiosis. Our results suggest that orchids possess, at least partly, the molecular mechanisms common to AM plants.

Rhizobial infection does not require cortical expression of upstream common symbiosis genes responsible for the induction of Ca(2+) spiking.

For the establishment of an effective root nodule symbiosis, a coordinated regulation of the infection processes between the epidermis and cortex is required. However, it remains unclear whether the symbiotic genes identified so far are involved in epidermal and/or cortical infection, e.g. epidermal and cortical infection thread formation or cortical cell division. To analyze the symbiotic gene requirements of the infection process, we have developed an epidermis-specific expression system (pEpi expression system) and examined the symbiotic genes NFR1, NFR5, NUP85, NUP133, CASTOR, POLLUX, CCaMK, CYCLOPS, NSP1 and NSP2 for involvement in the infection process in the epidermis and cortex. Our study shows that expression of the upstream common symbiosis genes CASTOR, POLLUX, NUP85 and NUP133 in the epidermis is sufficient to induce formation of infection threads and cortical cell division, leading to the development of fully effective nodules. Our system also shows a requirement of CCaMK, CYCLOPS, NSP1 and NSP2 for the entire nodulation process, and the different contributions of NFR1 and NFR5 to cortical infection thread formation. Based on these analyses using the pEpi expression system, we propose a functional model of symbiotic genes for epidermal and cortical infection.

Rhizobial and fungal symbioses show different requirements for calmodulin binding to calcium calmodulin-dependent protein kinase in Lotus japonicus.

Ca(2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is a key regulator of root nodule and arbuscular mycorrhizal symbioses and is believed to be a decoder for Ca(2+) signals induced by microbial symbionts. However, it is unclear how CCaMK is activated by these microbes. Here, we investigated in vivo activation of CCaMK in symbiotic signaling, focusing mainly on the significance of and epistatic relationships among functional domains of CCaMK. Loss-of-function mutations in EF-hand motifs revealed the critical importance of the third EF hand for CCaMK activation to promote infection of endosymbionts. However, a gain-of-function mutation (T265D) in the kinase domain compensated for these loss-of-function mutations in the EF hands. Mutation of the CaM binding domain abolished CaM binding and suppressed CCaMK(T265D) activity in rhizobial infection, but not in mycorrhization, indicating that the requirement for CaM binding to CCaMK differs between root nodule and arbuscular mycorrhizal symbioses. Homology modeling and mutagenesis studies showed that the hydrogen bond network including Thr265 has an important role in the regulation of CCaMK. Based on these genetic, biochemical, and structural studies, we propose an activation mechanism of CCaMK in which root nodule and arbuscular mycorrhizal symbioses are distinguished by differential regulation of CCaMK by CaM binding.

The recent evolution of a symbiotic ion channel in the legume family altered ion conductance and improved functionality in calcium signaling.

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wild-type and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.

Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation.

Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

A rice gene for microbial symbiosis, Oryza sativa CCaMK, reduces CH4 flux in a paddy field with low nitrogen input.

Plants have mutualistic symbiotic relationships with rhizobia and fungi by the common symbiosis pathway, of which Ca(2+)/calmodulin-dependent protein kinase (encoded by CCaMK) is a central component. Although Oryza sativa CCaMK (OsCCaMK) is required for fungal accommodation in rice roots, little is known about the role of OsCCaMK in rice symbiosis with bacteria. Here, we report the effect of a Tos17-induced OsCCaMK mutant (NE1115) on CH4 flux in low-nitrogen (LN) and standard-nitrogen (SN) paddy fields compared with wild-type (WT) Nipponbare. The growth of NE1115 was significantly decreased compared with that of the WT, especially in the LN field. The CH4 flux of NE1115 in the LN field was significantly greater (156 to 407% in 2011 and 170 to 816% in 2012) than that of the WT, although no difference was observed in the SN field. The copy number of pmoA (encodes methane monooxygenase in methanotrophs) was significantly higher in the roots and rhizosphere soil of the WT than in those of NE1115. However, the mcrA (encodes methyl coenzyme M reductase in methanogens) copy number did not differ between the WT and NE1115. These results were supported by a (13)C-labeled CH4-feeding experiment. In addition, the natural abundance of (15)N in WT shoots (3.05‰) was significantly lower than in NE1115 shoots (3.45‰), suggesting greater N2 fixation in the WT because of dilution with atmospheric N2 (0.00‰). Thus, CH4 oxidation and N2 fixation were simultaneously activated in the root zone of WT rice in the LN field and both processes are likely controlled by OsCCaMK.

Synergistic N2-fixation and salt stress mitigation in soybean through dual inoculation of ACC deaminase-producing Pseudomonas and Bradyrhizobium.

We investigated the potential dual application of two Bradyrhizobium strains (B. diazoefficiens USDA110 and B. ottawaense SG09) and plant growth-promoting bacteria, PGPB (Pseudomonas spp.: OFT2 and OFT5), to improve nodulation and N2-fixation in soybean plants. The growth-promoting effects of dual inoculation were observed on plant growth, physiology, and nodulation of soybean under normal conditions compared with plants individually inoculated with either USDA110 or SG09. Both OFT2 and OFT5 promoted N2-fixation by 11% and 56%, respectively, when dual inoculation with USDA110 and by 76% and 81%, respectively, when dual inoculation with SG09. Salinity stress significantly reduces soybean growth, physiology, nutrient uptake, nodulation, and N2-fixation. However, these adverse effects were attenuated by the dual inoculation of PGPB and rhizobia depending on the combination of inoculants. In particular, dual inoculation of PGPB with SG09 was more effective in enhancing the salt tolerance of soybean by reducing salt-induced ethylene production and improving nutrient uptake. However, no such effect was observed with the combined inoculation of USDA110 and OFT5. An effective symbiotic association between SG09 and two Pseudomonas bacteria can be considered a beneficial approach to improving the symbiotic efficiency of nodulation and mitigating salinity stress in soybeans.

Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression.

N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil.

Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development.

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.

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts.

In legumes, Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca(2+) spiking, one of the earliest cellular responses to microbial signals. A gain-of-function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain-of-function CCaMK(T265D) suppresses loss-of-function mutations of common symbiosis genes required for the generation of Ca(2+) spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca(2+) spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK(T265D) induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.

The genotype of the calcium/calmodulin-dependent protein kinase gene (CCaMK) determines bacterial community diversity in rice roots under paddy and upland field conditions.

The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria, including endophytes and epiphytes, were examined by using a Tos17 rice mutant line under paddy and upland field conditions. Roots were sampled at the flowering stage and were subjected to clone library analyses. The relative abundance of Alphaproteobacteria was noticeably decreased in R plants under both paddy and upland conditions (0.8% and 3.0%, respectively) relative to those in D plants (10.3% and 17.4%, respectively). Population shifts of the Sphingomonadales and Rhizobiales were mainly responsible for this low abundance in R plants. The abundance of Anaerolineae (Chloroflexi) and Clostridia (Firmicutes) was increased in R plants under paddy conditions. The abundance of a subpopulation of Actinobacteria (Saccharothrix spp. and unclassified Actinosynnemataceae) was increased in R plants under upland conditions. Principal coordinate analysis revealed unidirectional community shifts in relation to OsCCaMK gene dosage under both conditions. In addition, shoot length, tiller number, and plant weight decreased as the OsCCaMK gene dosage decreased under upland conditions. These results suggest significant impacts of OsCCaMK on both the diversity of root-associated bacteria and rice plant growth under both paddy and upland field conditions.

Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots.

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.

CYCLOPS, a mediator of symbiotic intracellular accommodation.

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.

Gibberellin controls the nodulation signaling pathway in Lotus japonicus.

Root nodule formation is regulated by several plant hormones, but the details of the regulation of the nodulation signaling pathway are largely unknown. In this study, the role of gibberellin (GA) in the control of root nodule symbiosis was investigated at the physiological and genetic levels in Lotus japonicus. Exogenous application of biologically active GA, GA(3), inhibited the formation of infection threads and nodules, which was counteracted by the application of a biosynthesis inhibitor of GA, Uniconazole P. Nod factor-induced root hair deformation was severely blocked in the presence of GA, which was phenocopied by nsp2 mutants. The number of spontaneous nodules triggered by the gain-of-function mutation of calcium/calmodulin-dependent kinase (CCaMK) or the lotus histidine kinase 1 (LHK1) was decreased upon the addition of GA; moreover, the overexpression of the gain-of-function mutation of L. japonicus, SLEEPY1, a positive regulator of GA signaling, resulted in a reduced nodule number, without other aspects of root development being affected. These results indicate that higher GA signaling levels specifically inhibit the nodulation signaling pathway. Nod factor-dependent induction of NSP2 and NIN was inhibited by exogenous GA. Furthermore, the cytokinin-dependent induction of NIN was suppressed by GA. From these results, we conclude that GA inhibits the nodulation signaling pathway downstream of cytokinin, possibly at NSP2, which is required for Nod factor-dependent NIN expression. These results clarify the roles of GA in the nodulation signaling pathway, and in relation to the cytokinin signaling pathway for nodulation in L. japonicus.

CERBERUS, a novel U-box protein containing WD-40 repeats, is required for formation of the infection thread and nodule development in the legume-Rhizobium symbiosis.

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus, cerberus, which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti. Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.