scholarly journals Responses of a Model Legume Lotus japonicus to Lipochitin Oligosaccharide Nodulation Factors Purified from Mesorhizobium loti JRL501

2001 ◽  
Vol 14 (7) ◽  
pp. 848-856 ◽  
Author(s):  
Shinobu Niwa ◽  
Masayoshi Kawaguchi ◽  
Haruko Imaizumi-Anraku ◽  
Svetlana A. Chechetka ◽  
Masumi Ishizaka ◽  
...  
Keyword(s):  
High Performance ◽  
Molecular Genetic ◽  
Lotus Japonicus ◽  
Cytoplasmic Bridge ◽  
Cortical Cells ◽  
Model Legume ◽  
Mesorhizobium Loti ◽  
Root Hair Deformation ◽  
Early Nodulin ◽  
Microbe Interactions

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetyl-glucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10-7 to 10-9 M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.

scholarly journals Lotus japonicus Contains Two Distinct ENOD40 Genes That Are Expressed in Symbiotic, Nonsymbiotic, and Embryonic Tissues

2000 ◽  
Vol 13 (9) ◽  
pp. 987-994 ◽  
Author(s):  
Emmanouil Flemetakis ◽  
Nektarios Kavroulakis ◽  
Nicolette E. M. Quaedvlieg ◽  
Herman P. Spaink ◽  
Maria Dimou ◽  
...  
Keyword(s):  
Root Nodule ◽  
Vascular System ◽  
Lotus Japonicus ◽  
Mrna Accumulation ◽  
Mesorhizobium Loti ◽  
Embryonic Tissues ◽  
Early Nodulin ◽  
Gene Transcripts ◽  
Young Seed

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.

scholarly journals Analysis of Promoter Activity of the Early Nodulin Enod40 in Lotus japonicus

2005 ◽  
Vol 18 (5) ◽  
pp. 414-427 ◽  
Author(s):  
Mette Grønlund ◽  
Andreas Roussis ◽  
Emmanouil Flemetakis ◽  
Nicolette E. M. Quaedvlieg ◽  
Helmi R. M. Schlaman ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Lotus Japonicus ◽  
Root Hairs ◽  
Early Time ◽  
Cellular Level ◽  
Nod Factors ◽  
Root Cortex ◽  
Model Legume ◽  
Early Nodulin ◽  
Nodulin Genes

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

scholarly journals Whole-Genome Sequence of the Nitrogen-Fixing Symbiotic Rhizobium Mesorhizobium loti Strain TONO

2016 ◽  
Vol 4 (5) ◽  
Author(s):  
Yoshikazu Shimoda ◽  
Hideki Hirakawa ◽  
Shusei Sato ◽  
Kazuhiko Saeki ◽  
Makoto Hayashi
Keyword(s):  
Comparative Genomics ◽  
Genome Sequence ◽  
Lotus Japonicus ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Nitrogen Fixing ◽  
Model Legume ◽  
Mesorhizobium Loti ◽  
Symbiotic Properties

Mesorhizobium loti is the nitrogen-fixing microsymbiont for legumes of the genus Lotus . Here, we report the whole-genome sequence of a Mesorhizobium loti strain, TONO, which is used as a symbiont for the model legume Lotus japonicus . The whole-genome sequence of the strain TONO will be a solid platform for comparative genomics analyses and for the identification of genes responsible for the symbiotic properties of Mesorhizobium species.

scholarly journals Expression Islands Clustered on the Symbiosis Island of the Mesorhizobium loti Genome

2004 ◽  
Vol 186 (8) ◽  
pp. 2439-2448 ◽  
Author(s):  
Toshiki Uchiumi ◽  
Takuji Ohwada ◽  
Manabu Itakura ◽  
Hisayuki Mitsui ◽  
Noriyuki Nukui ◽  
...  
Keyword(s):  
Acid Metabolism ◽  
Symbiotic Nitrogen Fixation ◽  
Transcriptional Profiling ◽  
Lotus Japonicus ◽  
Model Legume ◽  
Mesorhizobium Loti ◽  
Genome Region ◽  
Transcriptional Dynamics ◽  
Rhizobial Symbiosis ◽  
Highly Expressed Genes

ABSTRACT Rhizobia are symbiotic nitrogen-fixing soil bacteria that are associated with host legumes. The establishment of rhizobial symbiosis requires signal exchanges between partners in microaerobic environments that result in mutualism for the two partners. We developed a macroarray for Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, and monitored the transcriptional dynamics of the bacterium during symbiosis, microaerobiosis, and starvation. Global transcriptional profiling demonstrated that the clusters of genes within the symbiosis island (611 kb), a transmissible region distinct from other chromosomal regions, are collectively expressed during symbiosis, whereas genes outside the island are downregulated. This finding implies that the huge symbiosis island functions as clustered expression islands to support symbiotic nitrogen fixation. Interestingly, most transposase genes on the symbiosis island were highly upregulated in bacteroids, as were nif, fix, fdx, and rpoN. The genome region containing the fixNOPQ genes outside the symbiosis island was markedly upregulated as another expression island under both microaerobic and symbiotic conditions. The symbiosis profiling data suggested that there was activation of amino acid metabolism, as well as nif-fix gene expression. In contrast, genes for cell wall synthesis, cell division, DNA replication, and flagella were strongly repressed in differentiated bacteroids. A highly upregulated gene in bacteroids, mlr5932 (encoding 1-aminocyclopropane-1-carboxylate deaminase), was disrupted and was confirmed to be involved in nodulation enhancement, indicating that disruption of highly expressed genes is a useful strategy for exploring novel gene functions in symbiosis.

scholarly journals Classical and Molecular Genetics of the Model Legume Lotus japonicus

1997 ◽  
Vol 10 (1) ◽  
pp. 59-68 ◽  
Author(s):  
Qunyi Jiang ◽  
Peter M. Gresshoff
Keyword(s):  
Genetic Analysis ◽  
Recombinant Inbred Lines ◽  
Molecular Genetic ◽  
Lotus Japonicus ◽  
Dna Amplification ◽  
Molecular Genetic Analysis ◽  
Genetic Dissection ◽  
Model Legume ◽  
Sexual Hybridization ◽  
Leaf Coloration

The model legume Lotus japonicus was demonstrated to be amenable to classical and molecular genetic analysis, providing the basis for the genetic dissection of the plant processes underlying nodulation and nitrogen fixation. We have developed an efficient method for the sexual hybridization of L. japonicus and obtained F1 progeny derived from a cross of L. japonicus B-129-S9 Gifu × B-581 Funakura. Over half of the cross-pollinations resulted in fertile hybrid seed, which were confirmed morphologically and by single arbitrary primer DNA amplification polymorphisms using the DAF technique. Molecular and morphological markers segregated in true Mendelian fashion in a F2 population of 100 plants. Several DAF loci were linked using the MAPMAKER software to create the first molecular linkage groups of this model legume. The mapping population was advanced to generate a set of immortal recombinant inbred lines (F6; RILs), useful for sharing plant material fixed genetically at most genomic regions. Morphological loci for waved stem shape (Ssh), dark leaf color (Lco), and short flowering period (Fpe) were inherited as single dominant Mendelian loci. DAF markers were dominant and were detected between Gifu and Funakura at about one per primer, suggesting that the parents are closely related. One polymorphism (270G generated by single octomer primer 8.6m) was linked to a morphological locus controlling leaf coloration. The results demonstrate that (i) Lotus japonicus is amenable to diploid genetic analysis, (ii) morphological and molecular markers segregate in true diploid fashion, (iii) molecular polymorphisms can be obtained at a reasonable frequency between the related Gifu and Funakura lines, and iv) the possibility exists for map-based cloning, marker assisted selection and mapping of symbiotic mutations through a genetic and molecular map.

Improved procedures for in vitro regeneration and for phenotypic analysis in the model legume Lotus japonicus

2005 ◽  
Vol 32 (6) ◽  
pp. 529 ◽  
Author(s):  
Ani Barbulova ◽  
Enrica D'Apuzzo ◽  
Alessandra Rogato ◽  
Maurizio Chiurazzi
Keyword(s):  
In Vitro Regeneration ◽  
Lotus Japonicus ◽  
Shoot Formation ◽  
Direct Somatic Embryogenesis ◽  
Phenotypic Analysis ◽  
Indirect Organogenesis ◽  
Model Legume ◽  
Mesorhizobium Loti ◽  
Efficient Gene

As a prerequisite for the development of an efficient gene transfer methodology, the possibility of inducing direct somatic embryogenesis in Lotus japonicus (Regel) K. Larsen explants was investigated. Petiole bases, cotyledons, hypocotyls and stem segments were cultivated in the presence of different amounts of benzylaminopurine (BAP) and / or thidiazuron (TDZ). Regeneration was achieved differentially in the different explants and a higher efficiency of shoot formation was obtained with TDZ. By maintaining the same TDZ regime a second cycle of morphogenesis was achieved and the histological analysis of these structures indicated unambiguously their somatic embryogenic nature. Thidiazuron was also tested as an agent to improve the kinetics of shoot formation in a Lotus japonicus transformation–regeneration procedure based on indirect organogenesis. A very significant, highly reproducible, increase in the rate of the shoot formation was observed in independent transformation experiments. We also present an extensive analysis of the feasibility and reproducibility of an in vitro procedure, which can be very useful for the screening of symbiotic phenotypes in transgenic Lotus plants and for the analysis of the cascade of molecular and cytological events occurring soon after Mesorhizobium loti infection.

scholarly journals A Novel Fix¯ Symbiotic Mutant of Lotus japonicus, Ljsym105, Shows Impaired Development and Premature Deterioration of Nodule Infected Cells and Symbiosomes

2006 ◽  
Vol 19 (7) ◽  
pp. 780-788 ◽  
Author(s):  
Md. Shakhawat Hossain ◽  
Yosuke Umehara ◽  
Hiroshi Kouchi
Keyword(s):  
Nitrogen Deficiency ◽  
Lotus Japonicus ◽  
Nodule Development ◽  
Acetylene Reduction Activity ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Electron Microscopic ◽  
Model Legume ◽  
Mesorhizobium Loti ◽  
Infected Cells

Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix¯ mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.

scholarly journals Salicylic Acid Inhibits Indeterminate-Type Nodulation But Not Determinate-Type Nodulation

2003 ◽  
Vol 16 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Paulina C. van Spronsen ◽  
Teun Tak ◽  
Anita M. M. Rood ◽  
Anton A. N. van Brussel ◽  
Jan W. Kijne ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Rhizobium Leguminosarum ◽  
Acyl Chain ◽  
Lotus Japonicus ◽  
Biologically Active ◽  
Nodule Formation ◽  
Octadecanoid Pathway ◽  
Mesorhizobium Loti ◽  
Root Hair Deformation ◽  
Oxylipin Signaling

LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial symbiote of Vicia sativa subsp. nigra (vetch, an indeterminate-type nodulating plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an 18:1 acyl chain. This suggests that the 18:4 acyl chain specifically contributes to signaling in indeterminate-type nodulation. In a working hypothesis, we speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid (the octadecanoid pathway). Because salicylic acid (SA) is known to interfere with oxylipin signaling, we tested whether nodulation of vetch could be affected by addition of 10−4 M SA. This concentration completely blocked nodulation of vetch by Rhizobium leguminosarum bv. viciae and inhibited the mitogenic effect of 18:4 LCOs but did not affect LCO-induced root-hair deformation. SA did not act systemically, and only biologically active SA derivatives were capable of inhibiting nodule formation. SA also inhibited R. leguminosarum bv. viciae association with vetch roots. In contrast, addition of SA to Lotus japonicus (a determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit nodulation by Mesorhizobium loti. Other indeterminate-type nodulating plants showed the same inhibiting response toward SA, whereas SA did not inhibit the nodulation of other determinate-type nodulating plants. SA may be a useful tool for studying fundamental differences between signal transduction pathways of indeterminate- and determinate-type nodulating plants.

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