Experimental evolution can enhance benefits of rhizobia to novel legume hosts

Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host–symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host–symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts.

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Rhizobial Synthesized Cytokinins Contribute to But Are Not Essential for the Symbiotic Interaction Between Photosynthetic Bradyrhizobia and Aeschynomene Legumes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-13-0076-r ◽

2013 ◽

Vol 26 (10) ◽

pp. 1232-1238 ◽

Cited By ~ 29

Author(s):

Kateřina Podlešáková ◽

Joel Fardoux ◽

Delphine Patrel ◽

Katia Bonaldi ◽

Ondřej Novák ◽

...

Keyword(s):

Root Nodules ◽

Rna Degradation ◽

Nodule Development ◽

Nodule Formation ◽

Symbiotic Interaction ◽

Extracellular Medium ◽

Exogenous Addition ◽

Bradyrhizobium Sp ◽

Derivatives Of

Cytokinins (CK) play an important role in the formation of nitrogen-fixing root nodules. It has been known for years that rhizobia secrete CK in the extracellular medium but whether they play a role in nodule formation is not known. We have examined this question using the photosynthetic Bradyrhizobium sp. strain ORS285 which is able to nodulate Aeschynomene afraspera and A. indica using a Nod-dependent or Nod-independent symbiotic process, respectively. CK profiling showed that the most abundant CK secreted by Bradyrhizobium sp. strain ORS285 are the 2MeS (2-methylthiol) derivatives of trans-zeatin and isopentenyladenine. In their pure form, these CK can activate legume CK receptors in vitro, and their exogenous addition induced nodule-like structures on host plants. Deletion of the miaA gene showed that transfer RNA degradation is the source of CK production in Bradyrhizobium sp. strain ORS285. In nodulation studies performed with A. indica and A. afraspera, the miaA mutant had a 1-day delay in nodulation and nitrogen fixation. Moreover, A. indica plants formed considerably smaller but more abundant nodules when inoculated with the miaA mutant. These data show that CK produced by Bradyrhizobium sp. strain ORS285 are not the key signal triggering nodule formation during the Nod-independent symbiosis but they contribute positively to nodule development in Aeschynomene plants.

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A Convenient, Soil-Free Method for the Production of Root Nodules in Soybean to Study the Effects of Exogenous Additives

10.20944/preprints201809.0527.v1 ◽

2018 ◽

Author(s):

Swarup Roy Choudhury ◽

Sarah M. Johns ◽

Sona Pandey

Keyword(s):

Nitrogen Fixation ◽

Biological Nitrogen Fixation ◽

Root Nodules ◽

Growth Conditions ◽

Nodule Development ◽

Nodule Formation ◽

Legume Crop ◽

Simple Protocol ◽

Biological Nitrogen

Legumes develop root nodules that harbour endosymbiotic bacteria, rhizobia. These rhizobia convert nitrogen to ammonia by biological nitrogen fixation. A thorough understanding of the biological nitrogen fixation in legumes and its regulation is key to develop sustainable agriculture. It is well known that plant hormones affect nodule formation; however, most studies are limited to model legumes due to their suitability for in vitro, plate-based assays. Specifically, it is almost impossible to measure the effects of exogenous hormones or other additives during nodule development in crop legumes such as soybean as they have huge root system in soil. To circumvent this issue, the present research develops suitable media and growth conditions for efficient nodule development under in vitro, soil free conditions in an important legume crop, soybean. Moreover, we also evaluate the effects of all major phytohormones during soybean nodulation under identical conditions. This versatile, inexpensive, scalable and simple protocol provides several advantages over previously established methods. It is extremely time-and resource-efficient, does not require special training or equipment, and produces highly reproducible results. The approach is expandable to other large legumes as well as for other exogenous additives.

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Nitrate restricts nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus

Nature Communications ◽

10.1038/s41467-021-26820-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jieshun Lin ◽

Yuda Purwana Roswanjaya ◽

Wouter Kohlen ◽

Jens Stougaard ◽

Dugald Reid

Keyword(s):

Nitrogen Fixation ◽

Developmental Process ◽

Root Zone ◽

Root Nodules ◽

Lotus Japonicus ◽

Nodule Development ◽

Nodule Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cytokinin Biosynthesis

AbstractLegumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application rescues nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in species that do not form symbiotic root nodules. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.

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Strain distribution and in planta production of an extracellular polysaccharide depolymerase from Bradyrhizobium japonicum

Canadian Journal of Microbiology ◽

10.1139/m92-139 ◽

1992 ◽

Vol 38 (8) ◽

pp. 857-861 ◽

Cited By ~ 3

Author(s):

Michael F. Dunn ◽

Arthur L. Karr

Keyword(s):

Bradyrhizobium Japonicum ◽

Root Nodules ◽

Extracellular Polysaccharide ◽

Extracellular Polysaccharides ◽

Neutral Sugar ◽

In Planta ◽

In Vitro Production ◽

Polysaccharide Composition ◽

Vitro Production

Thirty-four strains of Bradyrhizobium japonicum were screened for the in vitro production of an extracellular polysaccharide depolymerase active against the B. japonicum acidic extracellular polysaccharide that contains mannose, glucose, galactose, and 4-O-methylgalactose as neutral sugar components. Over 90% of tested strains producing this type of extracellular polysaccharide also produced the extracellular polysaccharide depolymerase, whereas strains producing a compositionally different extracellular polysaccharide did not. In addition, representatives of species related to B. japonicum by extracellular polysaccharide composition or host range were also phenotypically depolymerase negative. Depolymerase was also present in soybean root nodules formed by B. japonicum strain 2143. In contrast to the cell-associated depolymerase activity found in free-living cells of this strain, most of the depolymerase activity present in nodules is free of the bacteroids. The widespread occurrence of the depolymerase among B. japonicum strains and the spatiotemporal distribution of its activity in planta are consistent with the enzyme playing a role in the removal of surface extracellular polysaccharide from the microorganism during the infection of nodulation process. Key words: Bradyrhizobium japonicum, soybean, extracellular polysaccharides, extracellular polysaccharide depolymerase, bacteroids.

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Characterization and Expression Analysis of Genes Encoding Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase of Lotus japonicus, a Model Legume

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.4.281 ◽

2003 ◽

Vol 16 (4) ◽

pp. 281-288 ◽

Cited By ~ 19

Author(s):

Tomomi Nakagawa ◽

Tomoko Izumi ◽

Mari Banba ◽

Yosuke Umehara ◽

Hiroshi Kouchi ◽

...

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Root Nodules ◽

Expression Patterns ◽

Phosphorylation Site ◽

Lotus Japonicus ◽

Specific Protein ◽

Mrna Levels ◽

Model Legume ◽

Putative Phosphorylation Site

Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.

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Expression Analysis of PIN Genes in Root Tips and Nodules of Lotus japonicus

International Journal of Molecular Sciences ◽

10.3390/ijms20020235 ◽

2019 ◽

Vol 20 (2) ◽

pp. 235 ◽

Cited By ~ 4

Author(s):

Izabela Sańko-Sawczenko ◽

Dominika Dmitruk ◽

Barbara Łotocka ◽

Elżbieta Różańska ◽

Weronika Czarnocka

Keyword(s):

Root Nodule ◽

Root Nodules ◽

Quantitative Polymerase Chain Reaction ◽

Lotus Japonicus ◽

Nodule Development ◽

Vascular Bundles ◽

Root Tips ◽

Gus Reporter ◽

Gene Assay ◽

Development And Differentiation

Auxins are postulated to be one of the pivotal factors in nodulation. However, their transporters in Lotus japonicus, the model species for the study of the development of determinate-type root nodules, have been scarcely described so far, and thus their role in nodulation has remained unknown. Our research is the first focusing on polar auxin transporters in L. japonicus. We analyzed and compared expression of PINs in 20 days post rhizobial inoculation (dpi) and 54 dpi root nodules of L. japonicus by real-time quantitative polymerase chain reaction (qPCR) along with the histochemical β-glucuronidase (GUS) reporter gene assay in transgenic hairy roots. The results indicate that LjPINs are essential during root nodule development since they are predominantly expressed in the primordia and young, developing nodules. However, along with differentiation, expression levels of several PINs decreased and occurred particularly in the nodule vascular bundles, especially in connection with the root’s stele. Moreover, our study demonstrated the importance of both polar auxin transport and auxin intracellular homeostasis during L. japonicus root nodule development and differentiation.

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Effect of rhizobia and soil nitrate on the establishment and functioning of the soybean symbiosis in the field

Australian Journal of Agricultural Research ◽

10.1071/ar9840149 ◽

1984 ◽

Vol 35 (2) ◽

pp. 149 ◽

Cited By ~ 50

Author(s):

DF Herridge ◽

RJ Roughley ◽

J Brockwell

Keyword(s):

Soil Depth ◽

Root Nodules ◽

Xylem Sap ◽

Progressive Increase ◽

Rhizobium Japonicum ◽

Nodule Development ◽

Initial Increase ◽

Nodule Formation ◽

Mineral N ◽

Nodule Initiation

The symbiosis of the root-nodules of Bragg soybean [Glycine max (L.) Merrill] and the relative dependence of the plants on symbiotic and soil sources of N were evaluated in an experiment conducted on a vertisol which was high in organic- and mineral-N, free of Rhizobium japonicum, and where poor nodulation was characteristic of inoculated, new sowings. Effective inoculant containing R. japonicum strain CB 1809 was sprayed into the seed bed at three rates of application (10-fold intervals). Increasing rates of inoculant led to greater numbers of rhizobia in the rhizosphere and in the soil, and to improved nodulation. Uninoculated plants did not nodulate. High soil NO-3 (30 �g N/g, top 30 cm) did not prevent prompt, abundant colonization of rhizospheres by the bacteria from the inoculant, but nodule initiation was delayed and nodule development was retarded until 42 days after sowing. There was an acceleration in nodule formation and development between 42 and 62 days which coincided with a depletion of NO-3 from the top 60 cm of the soil profile. Nodulated and unnodulated soybeans took up NO-3 at similar times and rates to a soil depth of 90 cm; only unnodulated plants utilized soil NO-3 below 90 cm. Vacuum-extracted stem (xylem) exudate was sampled from plants throughout growth and analysed for nitrogenous solutes. The proportion of ureide-N relative to total-solutes-N in xylem sap was used as an index of symbiotic N2-fixation. The initial increase in concentrations of ureides coincided with the period of accelerated nodule formation and development between 42 and 62 days. Thereafter, there was a progressive increase in ureide concentrations in nodulated plants, and the levels were related to rate of inoculation, extent of nodulation, and to the decline in concentrations of soil NO-3. Ureide concentrations in unnodulated plants remained low throughout. The quantities of NO-3-N and �-NH2- N in xylem sap were not related to nodulation. The differences between treatments in terms of whole-plant N and grain N were less than predicted from the symbiotic parameters. This indicated that soybeans compensated for symbiotic deficiencies by more efficient exploitation of soil N and/or by more efficient redistribution of vegetative N into grain N, and that nodulation and soil NO-3 were interactive and complementary in meeting the N requirements of the crop.

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Differential Effects of Combined N Sources on Early Steps of the Nod Factor–Dependent Transduction Pathway in Lotus japonicus

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-8-0994 ◽

2007 ◽

Vol 20 (8) ◽

pp. 994-1003 ◽

Cited By ~ 50

Author(s):

Ani Barbulova ◽

Alessandra Rogato ◽

Enrica D'Apuzzo ◽

Selim Omrane ◽

Maurizio Chiurazzi

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Small Scale ◽

Nodule Formation ◽

Inhibitory Effects ◽

Nod Factor ◽

Mesorhizobium Loti ◽

N Sources ◽

N Starvation ◽

And Function

The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.

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Absence of Symbiotic Leghemoglobins Alters Bacteroid and Plant Cell Differentiation During Development of Lotus japonicus Root Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-7-0800 ◽

2009 ◽

Vol 22 (7) ◽

pp. 800-808 ◽

Cited By ~ 36

Author(s):

Thomas Ott ◽

John Sullivan ◽

Euan K. James ◽

Emmanouil Flemetakis ◽

Catrin Günther ◽

...

Keyword(s):

Gene Expression ◽

Nitrogen Fixation ◽

Cell Differentiation ◽

Root Nodules ◽

Lotus Japonicus ◽

Nodule Development ◽

Primary And Secondary Metabolism ◽

Bacterial Differentiation ◽

Plant Genes ◽

Bacterial Genes

During development of legume root nodules, rhizobia and their host plant cells undergo profound differentiation, which is underpinned by massive changes in gene expression in both symbiotic partners. Oxygen concentrations in infected and surrounding uninfected cells drop precipitously during nodule development. To assess what effects this has on plant and bacterial cell differentiation and gene expression, we used a leghemoglobin-RNA-interference (LbRNAi) line of Lotus japonicus, which is devoid of leghemoglobins and has elevated levels of free-oxygen in its nodules. Bacteroids in LbRNAi nodules showed altered ultrastructure indicating changes in bacterial differentiation. Transcript analysis of 189 plant and 192 bacterial genes uncovered many genes in both the plant and bacteria that were differentially regulated during nodulation of LbRNAi plants compared with the wild type (containing Lb and able to fix nitrogen). These included fix and nif genes of the bacteria, which are involved in microaerobic respiration and nitrogen fixation, respectively, and plant genes involved in primary and secondary metabolism. Metabolite analysis revealed decreased levels of many amino acids in nodules of LbRNAi plants, consistent with the defect in symbiotic nitrogen fixation of this line.

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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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