Naoya Takeda

The Graduate University for Advanced Studies | SOKENDAI · National Institute for Basic Biology

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeat...

Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with a wide range of terrestrial plants, including rice. However, the mechanisms underlying the initiation of AM symbiosis have yet to be elucidated, particularly in nonleguminous plants. We previously demonstrated that OsCERK1, a lysin motif (LysM) receptor-like kinase essential for...

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,...

Legumes and nitrogen‐fixing rhizobial bacteria establish root nodule symbiosis, which is orchestrated by several plant hormones. Exogenous addition of biologically active gibberellic acid (GA) is known to inhibit root nodule symbiosis. However, the precise role of GA has not been elucidated because of the trace amounts of these hormones in plants a...

[This corrects the article DOI: 10.1371/journal.pgen.1007865.].

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the fo...

lan nodulation phenotype of later nodulation stages and inoculation of M. loti nodC mutants. (A) The numbers of sites of cortical cell proliferation (CCP) and of nodule primordia (NP), and mature nodules in DR5:GFP-NLS/WT MG-20 and in the DR5:GFP-NLS/lan plants at 45 dai (n = 11 plants). CCP was identified by GFP-NLS signals that were expressed und...

Close-up images showing effect of the lan mutation in early nodule development. (A and B) Nodule primordia formed on 4 dai DR5:GFP-NLS/WT MG-20 (A) and on 11 dai DR5:GFP-NLS/lan (B) roots. (C and D) Merged images of patterns of rhizobial invasion and auxin response of nodule primordia formed on 4 dai DR5:GFP-NLS/WT MG-20 (C) and on 11 dai DR5:GFP-N...

Complementation analysis. (A) Average nodule number in WT and the lan mutant with transgenic roots containing respective constructs at 14 dai (n = 17–22 plants). NP, nodule primrdia. (B-F) Representative transgenic hairy roots of WT MG-20 (B) or lan (C-F) constitutively expressing GUS (B and F), LjLAN (C), AtMED2 (D), LjLANΔC (E) at 14 dai. The det...

Patterns of calcium spiking. Transgenic hairy roots containing the nuclear-localized yellow-chameleon (YC2.60) construct were analyzed. Nod-factor (A and B) or water (C and D) were applied to WT MG-20 (A and C) and lan (B and D) roots. In this experimental condition, Nod factor treatment generated calcium spiking in 14/48 WT MG-20 and 29/104 lan ro...

Exon-intron structure of the LjLAN gene and the site of mutation in the lan mutant. Boxes indicate exons. Initiation codon (ATG) of LjLAN is marked in magenta. Splice (GT) and acceptor (AG) site of intron is marked in blue. Thick arrows indicate locations of primer sets used for RT-PCR analysis in Fig 4A. The position of introns in LjLAN in WT was...

LjLAN expression pattern. (A) RT-PCR analysis of the LjLAN gene. LjUBQ was used as the RNA loading control. The locations of primer sets used for PCR is shown in S5 Fig. cDNA was prepared from total RNAs roots (0), and in inoculated roots at 1, 7 and 14 dai. (B) Real-time RT-PCR analysis of LjLAN in lan and the LORE1-tagged line of lan (Plant ID: 3...

Primers used in this work. (XLS)

Map-based cloning of LjLAN. The lan locus was mapped using F2 population derived from a cross between lan and Gifu B-129 plants. 108 F2 plants that exhibited the nodulation-deficient phenotype were used for this analysis. Arrow indicates the LAN candidate gene (chr3.CM0112.280.r2.d) found in the L. japonicus genomic sequence database. The primers u...

LjLAN is a putative orthologue of AtMED2/29/32. (A) Phylogenetic tree of LjLAN-related proteins. Full-length amino acids sequences were compared and the tree was constructed by neighbor-joining methods. Numbers indicate bootstrap values. (B) Amino acid alignment of the LjLAN-related proteins. The amino acid residues with 100% homology among the pro...

Spatial expression patterns of LjLAN during nodulation. (A-E) GUS staining pattern of WT MG-20 transgenic hairy roots containing the ProLjLAN:GUS plus construct at 4 dai (A-D) and 9 dai (E). Closed and open arrowheads respectively indicate nodulation foci and curled root hair. Arrows indicate lateral roots. Scale bars: 1 mm (A, E); 100 μm (B-D). (T...

AM symbiosis phenotypes of LORE1-tagged lines of lan. (A and B) R. irregularis colonization ratio of hyphae (A) and arbuscules (B) at 21 dai. (n = 6 plants). Error bars indicate SD. (TIF)

The effect of the lan mutation on shoot and root growth. (A) WT MG-20 (left) and lan (right) plants at 14 days after germination (dag). (B and C) shoot (B) and primary root (C) length at 14 dag. Plants were grown in the soil that contained enough nutrients in the absence of rhizobia and AMF. (D and E) Root hairs phenotype of WT MG-20 (D) and lan (E...

Shoot and root growth of LORE1-tagged lines of lan. (A) WT Gifu (left) and 30008618 (right) plants at 14 days after germination (dag). (B and C) shoot (B) and primary root (C) length at 14 dag. Plants were grown in the soil that contained enough nutrients in the absence of rhizobia and AMF. Scale bar: 1 cm. Error bars indicate SD. (TIF)

Model for the position of LjLAN-mediated regulation in root nodule symbiosis. Perception of rhizobia-derived Nod factor by its receptors elicits Nod factor signaling. Consequently, calcium signaling is induced, which is decoded by LjCCaMK. LjCCaMK then activates LjCYCLOPS, which directly induces LjNIN expression. As normal calcium spiking pattern w...

Expression patterns of LjLAN and LjLAN LIKE. (A and B) Real-time RT-PCR analysis of LjLAN (A) and LjLAN LIKE (B) expression in reproductive and vegetative organs. Each cDNA sample was prepared from total RNA derived from the flower, leaf, stem, shoot apex, non-inoculated (-) and 1 dai (+) roots. (C) Real-time RT-PCR analysis of LjLAN expression in...

Nodulation phenotypes of LORE1-tagged lan lines and lan plants created by the CRISPR/Cas9 genome editing system. (A) A schematic diagram of LORE1 insertion site in the LORE1-tagged line of lan (Plant ID: 30008618). (B) Amino acid alignment of several truncated LjLAN used in this study. The amino acids sequence of LjLAN in 30008618 and two lan CRISP...

AM symbiosis phenotypes of lan cyclops double mutant. (A and B) R. irregularis colonization ratio of hyphae (A) and arbuscules (B) at 28 dai. (n = 8 plants). Error bars indicate SD. Columns with the same lower-case letter indicate no significant difference (Tukey’s test, P < 0.05). (TIF)

Arbuscular mycorrhizal fungus (AMF) species are some of the most widespread symbionts of land plants. Our much improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated a discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or 11 copies of complete...

Arbuscular mycorrhizal fungus (AMF) species are one of the most widespread symbionts of land plants. Our substantially improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or eleven copies o...

Lotus japonicus THIC is expressed in all organs, and the encoded protein catalyzes thiamine biosynthesis. Loss of function produces chlorosis, a typical thiamine-deficiency phenotype, and mortality. To investigate thiamine's role in symbiosis, we focused on THI1, a thiamine-biosynthesis gene expressed in roots, nodules, and seeds. The thi1 mutant h...

Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculat...

Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were e...

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from t...

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a C...

Arbuscular mycorrhiza (AM) is a mutualistic plant-fungal interaction that confers great advantages for plant growth. Arbuscular mycorrhizal (AM) fungi enter the host root and form symbiotic structures that facilitate nutrient supplies between the symbionts. The phytohormones gibberellins (GAs) are known to inhibit AM fungal infection. However, our...

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. H...

Arbuscular mycorrhizal symbiosis (AMS) and root nodule symbiosis (RNS) share several common symbiotic components, and many of the common symbiosis mutants block the entry of symbionts into the roots. We recently reported that CERBERUS (an E3 ubiquitin ligase) and NSP1 (a GRAS family transcription factor), required for RNS, also modulate AMS develop...

Arbuscular mycorrhizal symbiosis (AMS) and root nodule symbiosis (RNS) are mutualistic plant-microbe interactions that confer nutritional benefits to both partners. Leguminous plants possess a common genetic system for intracellular symbiosis with AM fungi and with rhizobia. Here we show that CERBERUS and NSP1, which respectively encode an E3 ubiqu...

The interaction of legumes with N2-fixing bacteria collectively called rhizobia results in root nodule development. The number of nodules formed is tightly restricted through the systemic negative feedback control by the host called autoregulation of nodulation (AON). Here, we report the characterization and gene identification of TOO MUCH LOVE (TM...

During the course of evolution, mainly leguminous plants have acquired the ability to form de novo structures called root nodules. Recent studies on the autoregulation and hormonal controls of nodulation have identified key mechanisms and also indicated a possible link to other developmental processes, such as the formation of the shoot apical meri...

The common symbiosis pathway is at the core of symbiosis signaling between plants and soil microbes. In this pathway, calcium- and calmodulin-dependent protein kinase (CCaMK) plays a crucial role in integrating the signals both in arbuscular mycorrhizal symbiosis (AMS) and in root nodule symbiosis (RNS). However, the molecular mechanism by which CC...

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 fl...

The subtilisin-like serine protease SbtM1 is strongly and specifically induced during arbuscular mycorrhiza (AM) symbiosis in Lotus japonicus. Another subtilase gene, SbtS, is induced during early stages of nodulation and AM. Transcript profiling in plant symbiosis mutants revealed that the AM-induced expression of SbtM1 and the gene family members...

Legumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of the calcium and calmodulin-dependent kinase (CCaMK), a central regulatory component of the plant's common symbiosis signaling network. We performed a microscopy...

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 biologicall...

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of t...

The brush mutant of Lotus japonicus exhibits a temperature-dependent impairment in nodule, root, and shoot development. At 26 degrees C, brush formed fewer nodules, most of which were not colonized by rhizobia bacteria. Primary root growth was retarded and the anatomy of the brush root apical meristem revealed distorted cellular organization and re...

The mechanism underlying perinuclear calcium spiking induced during legume root endosymbioses is largely unknown. Lotus japonicus symbiosis-defective castor and pollux mutants are impaired in perinuclear calcium spiking. Homology modeling suggested that the related proteins CASTOR and POLLUX might be ion channels. Here, we show that CASTOR and POLL...

Proteases catalyze the hydrolysis of peptide bonds in proteins/peptides inside or outside of cells. They play important roles in development and responses to environmental stresses. In arbuscular mycorrhiza (AM), symbiosis-induced protease genes were found by large-scale transcriptome analyses in different plant species, suggesting that proteolytic...

Nitrogen fixation in nodules provides leguminous plants with an ability to grow in nitrogen-starved soil. Infection of the host plants by microsymbionts triggers various physiological and morphological changes during nodule formation. In Lotus japonicus, expression of early nodulin (ENOD) genes is triggered by perception of bacterial signal molecul...

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 th...