Lotus japonicus alters in planta fitness of Mesorhizobium loti dependent on symbiotic nitrogen fixation

Symbiotic nitrogen fixation in legumes takes place in specialized organs called nodules,which become the main source of assimilated nitrogen for the whole plant. Symbiotic nitro‐gen fixation requires exquisite integration of plant and bacterial metabolism and involvesglobal changes in gene expression and metabolite accumulation in both rhizobia and thehost plant. In order to study the metabolic changes mediated by symbiotic nitrogen fixationon a whole‐plant level, metabolite levels were profiled by gas chromatography–mass spec‐trometry in nodules and non‐symbiotic organs of Lotus japonicus plants uninoculated or in‐oculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Furthermore, transcriptomic andbiochemical approaches were combined to study sulfur metabolism in nodules, its link tosymbiotic nitrogen fixation, and the effect of nodules on whole‐plant sulfur partitioning andmetabolism. It is well established that nitrogen and sulfur (S) metabolism are tightly en‐twined and sulfur is required for symbiotic nitrogen fixation, however, little is known aboutthe molecular and biochemical mechanisms governing sulfur uptake and assimilation duringsymbiotic nitrogen fixation. Transcript profiling in Lotus japonicus was combined with quan‐tification of S‐metabolite contents and APR activity in nodules and in non‐symbiotic organsof plants uninoculated or inoculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Moreover,sulfate uptake and its distribution into different plant organs were analyzed and 35S‐flux intodifferent S‐pools was monitored. Metabolite profiling revealed that symbiotic nitrogen fixa‐tion results in dramatic changes of many aspects of primary and secondary metabolism innodules which leads to global reprogramming of metabolism of the model legume on awhole‐plant level. Moreover, our data revealed that nitrogen fixing nodules represent athiol‐rich organ. Their high APR activity and 35S‐flux into cysteine and its metabolites in com‐bination with the transcriptional up‐regulation of several genes involved in sulfur assimila‐tion highlight the function of nodules as a new site of sulfur assimilation. The higher thiolcontent observed in non‐symbiotic organs of nitrogen fixing plants in comparison touninoculated plants cannot be attributed to local biosynthesis, indicating that nodules couldserve as a novel source of reduced sulfur for the plant, which triggers whole‐plant repro‐gramming of sulfur metabolism. Interestingly, the changes in metabolite profiling and theenhanced thiol biosynthesis in nodules and their impact on the whole‐plant sulfur, carbonand nitrogen economy are dampened in fix‐ plants, which in most respects metabolically re‐sembled uninoculated plants, indicating a strong interaction between nitrogen fixation andsulfur and carbon metabolism.

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The rhizobial autotransporter determines the symbiotic nitrogen fixation activity of Lotus japonicus in a host-specific manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913349117 ◽

2020 ◽

Vol 117 (3) ◽

pp. 1806-1815 ◽

Cited By ~ 2

Author(s):

Yoshikazu Shimoda ◽

Yuki Nishigaya ◽

Hiroko Yamaya-Ito ◽

Noritoshi Inagaki ◽

Yosuke Umehara ◽

...

Keyword(s):

Nitrogen Fixation ◽

Host Plant ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Lotus Japonicus ◽

Dependent Manner ◽

Strain Specificity ◽

Passenger Domain ◽

Aspartic Peptidase ◽

Specific Manner

Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix− mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix− phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix− nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1. The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix− nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.

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CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

New Phytologist ◽

10.1111/nph.16077 ◽

2019 ◽

Vol 224 (2) ◽

pp. 818-832 ◽

Cited By ~ 13

Author(s):

Longlong Wang ◽

Maria Carmen Rubio ◽

Xian Xin ◽

Baoli Zhang ◽

Qiuling Fan ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus

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The SNARE Protein SYP71 Expressed in Vascular Tissues Is Involved in Symbiotic Nitrogen Fixation in Lotus japonicus Nodules

PLANT PHYSIOLOGY ◽

10.1104/pp.112.200782 ◽

2012 ◽

Vol 160 (2) ◽

pp. 897-905 ◽

Cited By ~ 24

Author(s):

Tsuneo Hakoyama ◽

Ryo Oi ◽

Kazuya Hazuma ◽

Eri Suga ◽

Yuka Adachi ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus ◽

Snare Protein ◽

Vascular Tissues

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Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500777112 ◽

2015 ◽

Vol 112 (49) ◽

pp. 15232-15237 ◽

Cited By ~ 77

Author(s):

Beatrix Horváth ◽

Ágota Domonkos ◽

Attila Kereszt ◽

Attila Szűcs ◽

Edit Ábrahám ◽

...

Keyword(s):

Nitrogen Fixation ◽

Medicago Truncatula ◽

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Nitrogen Fixing ◽

In Planta ◽

Functional Roles ◽

Absolute Requirement ◽

Cell Layers

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

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

Journal of Bacteriology ◽

10.1128/jb.186.8.2439-2448.2004 ◽

2004 ◽

Vol 186 (8) ◽

pp. 2439-2448 ◽

Cited By ~ 162

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.

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Induction and Spatial Organization of Polyamine Biosynthesis During Nodule Development in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.12.1283 ◽

2004 ◽

Vol 17 (12) ◽

pp. 1283-1293 ◽

Cited By ~ 24

Author(s):

Emmanouil Flemetakis ◽

Rodica C. Efrose ◽

Guilhem Desbrosses ◽

Maria Dimou ◽

Costas Delis ◽

...

Keyword(s):

Nitrogen Fixation ◽

Symbiotic Nitrogen Fixation ◽

Expression Patterns ◽

Lotus Japonicus ◽

Cyclin D3 ◽

Nodule Development ◽

Cdna Clones ◽

Long Distance ◽

Polyamine Biosynthesis ◽

Transcript Levels

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L. japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an α-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

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Symbiotic Nitrogen Fixation at the Late Stage of Nodule Formation in Lotus japonicus and Other Legume Plants

Journal of Plant Research ◽

10.1007/pl00013957 ◽

2000 ◽

Vol 113 (4) ◽

pp. 467-473 ◽

Cited By ~ 7

Author(s):

Shigeyuki Tajima ◽

Kenichi Takane ◽

Mika Nomura ◽

Hiroshi Kouchi

Keyword(s):

Nitrogen Fixation ◽

Late Stage ◽

Symbiotic Nitrogen Fixation ◽

Lotus Japonicus ◽

Nodule Formation ◽

Legume Plants

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Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

Journal of Bacteriology ◽

10.1128/jb.01456-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2593-2600 ◽

Cited By ~ 13

Author(s):

Chrysanthi Kalloniati ◽

Daniela Tsikou ◽

Vasiliki Lampiri ◽

Mariangela N. Fotelli ◽

Heinz Rennenberg ◽

...

Keyword(s):

Nitrogen Fixation ◽

Carbonic Anhydrase ◽

Mutant Strain ◽

Symbiotic Nitrogen Fixation ◽

Plant Traits ◽

Nitrogenase Activity ◽

Nodule Development ◽

Free Living ◽

Model Legume ◽

Mesorhizobium Loti

ABSTRACT Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO2 to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an α-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active α-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and δ13C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH3 protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule δ13C abundance suggest the recycling of at least part of the HCO3 − produced by CAA1.

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Introduction of H2-Uptake Hydrogenase Genes Into Rhizobial Strains Improves Symbiotic Nitrogen Fixation in Vicia sativa and Lotus corniculatus Forage Legumes

Frontiers in Agronomy ◽

10.3389/fagro.2021.661534 ◽

2021 ◽

Vol 3 ◽

Author(s):

Mariana Sotelo ◽

Ana Claudia Ureta ◽

Socorro Muñoz ◽

Juan Sanjuán ◽

Jorge Monza ◽

...

Keyword(s):

Nitrogen Fixation ◽

Rhizobium Leguminosarum ◽

Symbiotic Nitrogen Fixation ◽

Nitrogenase Activity ◽

Root Nodules ◽

Lotus Corniculatus ◽

Vicia Sativa ◽

Uptake Hydrogenase ◽

Forage Crops ◽

Mesorhizobium Loti

Biological nitrogen fixation by the Rhizobium-legume symbiosis allows the conversion of atmospheric nitrogen into ammonia within root nodules mediated by the nitrogenase enzyme. Nitrogenase activity results in the evolution of hydrogen as a result of a side reaction intrinsic to the activity of this enzyme. Some rhizobia, and also other nitrogen fixers, induce a NiFe uptake hydrogenase (Hup) to recycle hydrogen produced by nitrogenase, thus improving the efficiency of the nitrogen fixation process. In this work we report the generation and symbiotic behavior of hydrogenase-positive Rhizobium leguminosarum and Mesorhizobium loti strains effective in vetch (Vicia sativa) and birsfoot trefoil (Lotus corniculatus) forage crops, respectively. The ability of hydrogen recycling was transferred to these strains through the incorporation of hup minitransposon TnHB100, thus leading to full recycling of hydrogen in nodules. Inoculation of Vicia and Lotus plants with these engineered strains led to significant increases in the levels of nitrogen incorporated into the host legumes. The level of improvement of symbiotic performance was dependent on the recipient strain and also on the legume host. These results indicate that hydrogen recycling has the potential to improve symbiotic nitrogen fixation in forage plants.

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