Project description:Legume plants can establish symbiotic nitrogen fixation (SNF) with rhizobia mostly in root nodules, where rhizobia-infected cells are accompanied with uninfected cells in a mosaic pattern. Inside the mature nodules of legume, carbon and nitrogen nutrients between host plant cells and their resident bacteria are actively exchanged. To elucidate the metabolite dynamics relevant for SNF in nodules, three cell-types from nodule tissues of a model legume, Lotus japonicus, were isolated using laser microdissesction, and transcriptome analysis was done by an oligoarray with 60-mer length representing 21,495 genes. In our cell-type-specific profiling, many genes were identified as being expressed in nodules with spatial-specific manners. Among them, genes coding for metabolic enzymes were classified according to their function, and detailed data analysis figured out that secondary metabolic pathway was highly activated in nodule cortex. In particular, a number of metabolic genes for phenyl propanoid pathway were found as highly expressed genes accompanied with those encoding putative transporters of secondary metabolites. These data suggest the involvement of novel physiological function of phenylpropanoids in SNF. Gene expression in three different cell-types of Lotus japonicus nodule was measured. Three independent experiments were performed at each cell-types.
Project description:RNA-seq analysis was performed to know the expression profiles of Lotus japonicus genes during nodule development and nitrogen fixation.
Project description:Legumes establish endosymbiotic associations with nitrogen-fixing rhizobia, which they host inside root nodules. Here, specific physiological and morphological adaptations, such as the production of oxygen-binding leghemoglobin proteins and the formation of an oxygen diffusion barrier in the nodule periphery, are essential to protect the oxygen-labile bacterial nitrogenase enzyme. The molecular basis of the latter process remains elusive, as the identification of required genes is limited by the epistatic effect of nodule organogenesis over nodule infection and rhizobia accommodation. We overcame this by exploring the phenotypic diversity of Lotus japonicus accessions that uncouple nodule organogenesis from nodule infection when inoculated with a sub-compatible Rhizobium strain. Using comparative transcriptomics, we identified genes with functions associated with oxygen homeostasis and deposition of lipid polyesters on cell walls to be specifically upregulated in infected compared to uninfected nodules. As such hydrophobic modifications on cell walls are pivotal for creating diffusion barriers like the root endodermis, we focused on two Fatty acyl-CoA reductase genes that were specifically activated in the nodule or in the root endodermis. Mutant lines in a Fatty acyl-CoA reductase gene expressed exclusively in the nodule endodermis showed had decreased suberization of this cell layer and increased nodule permeability compared to wild type plants. Oxygen concentrations were significantly increased in the inner cortex of mutant nodules, which correlated with reduced nitrogen fixation rates, and impaired shoot growth. These results provide the first genetic evidence for the formation of the nodule oxygen diffusion barrier, a key adaptation enabling nitrogen fixation in legume nodules.
Project description:Legume plants can establish symbiotic nitrogen fixation (SNF) with rhizobia mostly in root nodules, where rhizobia-infected cells are accompanied with uninfected cells in a mosaic pattern. Inside the mature nodules of legume, carbon and nitrogen nutrients between host plant cells and their resident bacteria are actively exchanged. To elucidate the metabolite dynamics relevant for SNF in nodules, three cell-types from nodule tissues of a model legume, Lotus japonicus, were isolated using laser microdissesction, and transcriptome analysis was done by an oligoarray with 60-mer length representing 21,495 genes. In our cell-type-specific profiling, many genes were identified as being expressed in nodules with spatial-specific manners. Among them, genes coding for metabolic enzymes were classified according to their function, and detailed data analysis figured out that secondary metabolic pathway was highly activated in nodule cortex. In particular, a number of metabolic genes for phenyl propanoid pathway were found as highly expressed genes accompanied with those encoding putative transporters of secondary metabolites. These data suggest the involvement of novel physiological function of phenylpropanoids in SNF.
Project description:Acting in a partially redundant manner, NF-Ys were shown previously to regulate bacterial infection, including selection of a superior rhizobial strain, and to participate in mediating nodule structure formation. However, the exact mechanism(s) by which these transcriptional factors exert their symbiotic functions has remained elusive. Gene expression profiling for wild-type and Nuclear Factor YA (nf-ya)-A mutants in roots of Lotus japonicus were conducted 4 days after inoculation with Mesorhizobium loti in order to understand the interaction of NF-Ys and other genes in Lotus japonicus.
Project description:The casparian strip serves as a crucial diffusion barrier in the endodermis, playing a vital role in controlling the flow of substances between the root and its external environment. Here, we observed that when the casparian strip is absent, bacteria can accumulate abundantly on the roots. To investigate the impact of this bacterial accumulation on the root indued by casparian strip deficiency , we inoculated Col-0 and sgn3 myb36 roots with CHA0 in a hydroponic system. Ultimately, we found that the bacterial accumulation on the roots, resulting from the absence of the Casparian strip, strongly induces the immune response of the roots. This indicates that the casparian strip plays an important role in regulating the interaction between the root and microorganisms.
Project description:A time-course transcriptomic study was performed on mature nodules of Lotus japonicus MG-20 at 4 weeks post-inoculation (wpi) with Mesorhizobium loti MAFF303099. Plants were exposed to 10 mM KNO3 for six days and then nitrate was removed for five more days.
Project description:Legumes interact with rhizobial bacteria to form nitrogen-fixing root nodules. Host signalling following mutual recognition ensures a specific response, but is only partially understood. Focusing on the stage of epidermal infection with Mesorhizobium loti, we analysed endogenous small RNAs (sRNAs) of the model legume Lotus japonicus to investigate their involvement in host response regulation. We used Illumina sequencing to annotate the L. japonicus sRNA-ome and isolate infection-responsive sRNAs, followed by candidate-based functional characterization. Sequences from four libraries revealed 219 novel L. japonicus micro RNAs (miRNAs) from 114 newly assigned families, and 76 infection-responsive sRNAs. Unlike infection-associated coding genes such as NODULE INCEPTION (NIN), a micro RNA 172 (miR172) isoform showed strong accumulation in dependency of both Nodulation (Nod) factor and compatible rhizobia. The genetics of miR172 induction support the existence of distinct epidermal and cortical signalling events. MIR172a promoter activity followed a previously unseen pattern preceding infection thread progression in epidermal and cortical cells. Nodule-associated miR172a expression was infection-independent, representing the second of two genetically separable activity waves. The combined data provide a valuable resource for further study, and identify miR172 as an sRNA marking successful epidermal infection. We show that miR172 acts upstream of several APETALA2-type (AP2) transcription factors, and suggest that it has a role in fine-tuning AP2 levels during bacterial symbiosis.
Project description:To identify the regulatory targets of the R2R3-Myb transcription factor, LjMyb14, the gene was constitutively over-expressed in Lotus japonicus under the Lotus ubiquitin promoter. The gene expression levels of three biological replicates of the Lotus japonicus (MG20) were averaged and compared to the the gene expression levels of three independent lines of Lotus japonicus japonicus constituitively over expressing LjMyb14 using the Lotus ubiquitin promoter.