Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

Rhizobial nitrogen fixation efficiency shapes endosphere bacterial communities and Medicago truncatula host growth.

BACKGROUND: Despite the knowledge that the soil-plant-microbiome nexus is shaped by interactions amongst its members, very little is known about how individual symbioses regulate this shaping. Even less is known about how the agriculturally important symbiosis of nitrogen-fixing rhizobia with legumes is impacted according to soil type, yet this knowledge is crucial if we are to harness or improve it. We asked how the plant, soil and microbiome are modulated by symbiosis between the model legume Medicago truncatula and different strains of Sinorhizobium meliloti or Sinorhizobium medicae whose nitrogen-fixing efficiency varies, in three distinct soil types that differ in nutrient fertility, to examine the role of the soil environment upon the plant-microbe interaction during nodulation. RESULTS: The outcome of symbiosis results in installment of a potentially beneficial microbiome that leads to increased nutrient uptake that is not simply proportional to soil nutrient abundance. A number of soil edaphic factors including Zn and Mo, and not just the classical N/P/K nutrients, group with microbial community changes, and alterations in the microbiome can be seen across different soil fertility types. Root endosphere emerged as the plant microhabitat more affected by this rhizobial efficiency-driven community reshaping, manifested by the accumulation of members of the phylum Actinobacteria. The plant in turn plays an active role in regulating its root community, including sanctioning low nitrogen efficiency rhizobial strains, leading to nodule senescence in particular plant-soil-rhizobia strain combinations. CONCLUSIONS: The microbiome-soil-rhizobial dynamic strongly influences plant nutrient uptake and growth, with the endosphere and rhizosphere shaped differentially according to plant-rhizobial interactions with strains that vary in nitrogen-fixing efficiency levels. These results open up the possibility to select inoculation partners best suited for plant, soil type and microbial community. Video Abstract.

Soil Protists Can Actively Redistribute Beneficial Bacteria along Medicago truncatula Roots.

The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria: all play significant roles in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a bioavailable form. In soil, S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system, able to travel quickly along roots and water films, who prey on soil bacteria and have been known to egest undigested phagosomes. We show that a soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. Using model soil microcosms, we directly observed fluorescently labeled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal extended 52 mm farther down plant roots when Colpoda sp. was also present versus treatments that contained bacteria but not protists. Direct counts also showed protists are required for viable bacteria to reach the deeper sections of our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. IMPORTANCE Soil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here, we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen-fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

Insight into the control of nodule immunity and senescence during Medicago truncatula symbiosis.

Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix-). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix- nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix- nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

The legume-rhizobia symbiosis can be supported on Mars soil simulants.

Legumes (soybeans, peas, lentils, etc.) play important roles in agriculture on Earth because of their food value and their ability to form a mutualistic beneficial association with rhizobia bacteria. In this association, the host plant benefits from atmospheric nitrogen fixation by rhizobia. The presence of nitrogen in the Mars atmosphere offers the possibility to take advantage of this important plant-microbe association. While some studies have shown that Mars soil simulants can support plant growth, none have investigated if these soils can support the legume-rhizobia symbiosis. In this study, we investigated the establishment of the legume-rhizobia symbiosis on different Mars soil simulants (different grades of the Mojave Mars Simulant (MMS)-1: Coarse, Fine, Unsorted, Superfine, and the MMS-2 simulant). We used the model legume, Medicago truncatula, and its symbiotic partners, Sinorhizobium meliloti and Sinorhizobium medicae, in these experiments. Our results show that root nodules could develop on M. truncatula roots when grown on these Mars soil simulants and were comparable to those formed on plants that were grown on sand. We also detected nifH (a reporter gene for nitrogen fixation) expression inside these nodules. Our results indicate that the different Mars soil simulants used in this study can support legume-rhizobia symbiosis. While the average number of lateral roots and nodule numbers were comparable on plants grown on the different soil simulants, total plant mass was higher in plants grown on MMS-2 soil than on MMS-1 soil and its variants. Our results imply that the chemical composition of the simulants is more critical than their grain size for plant mass. Based on these results, we recommend that the MMS-2 Superfine soil simulant is a better fit than the MMS-1 soil and it's variants for future studies. Our findings can serve as an excellent resource for future studies investigating beneficial plant-microbe associations for sustainable agriculture on Mars.

Screening and Characterization of Two Extracellular Polysaccharide-Producing Bacteria from the Biocrust of the Mu Us Desert.

The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.

PHO1 family members transport phosphate from infected nodule cells to bacteroids in Medicago truncatula.

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.

Response of intercropped barley and fenugreek to mono- and co-inoculation with Sinorhizobium meliloti F42 and Variovorax paradoxus F310 under contrasting agroclimatic regions.

In the present research, we aimed to select efficient rhizobia and plant growth-promoting rhizobacteria (PGPR) from fenugreek nodules and assess their performance as bio-inoculum for intercropped fenugreek and barley. Inoculation effects with selected bacteria were investigated firstly on fenugreek plants under greenhouse experiment and secondly on intercropped fenugreek and barley under three different agro-environmental conditions for two consecutive years. Sinorhizobium meliloti F42 was selected due to its ability to nodulate fenugreek and effectively improve plant growth. Among non-nodulating endophytic bacteria, Variovorax paradoxus F310 strain was selected regarding its plant growth-promoting traits showed in vitro and confirmed in vivo under greenhouse experiment. Field inoculation trials revealed a significant improvement in fenugreek nodulation (up to + 97%) as well as in soil enzymes activities (up to + 209%), shoot N content (up to + 18%), shoot dry weight (up to + 40%), photosynthetic assimilation (up to + 34%) and chlorophyll content of both intercropped plants in response to the mono-inoculation with Sinorhizobium meliloti F42, compared to the un-inoculated treatment at the SBR and JBS sites. Variovorax paradoxus F310 inoculation significantly increased shoot P content of both intercropped plants at the three experimental sites compared to the un-inoculated treatment (up to + 48%). It was shown that bacterial inoculation was more efficient at the low-rainfall region than the high-rainfall region. The co-inoculation with Sinorhizobium meliloti F42 and Variovorax paradoxus F310 resulted in a significant reduction in fenugreek nodulation and shoot N content. This survey showed the benefits of rhizobial and PGPR inoculation as efficient bio-inoculums to promote the cereal-legume intercropping system and highlights the influence of site-specific environmental factors on Rhizobium-PGPR-plant interactions.

Specific tissue proteins 1 and 6 are involved in root biology during normal development and under symbiotic and pathogenic interactions in Medicago truncatula.

MAIN CONCLUSION: ST1 and ST6 are possibly involved in primary and lateral root and symbiotic nodule development, but only ST6 participates in the interaction with hemibiotrophic fungi. Specific tissue (ST) proteins have been shown to be involved in several processes related to plant nutritional status, development, and responses to biotic agents. In particular, ST1 and ST6 are mainly expressed in roots throughout plant development. Here, we analyze where and how the expression of the genes encoding both proteins are modulated in the legume model plant Medicago truncatula in response to the plant developmental program, nodulation induced by a beneficial nitrogen-fixing bacterium (Sinorhizobium meliloti) and the defense response triggered by a pathogenic hemibiotrophic fungus (Fusarium oxysporum). Gene expression results show that ST1 and ST6 participate in the vasculature development of both primary and lateral roots, although only ST6 is related to meristem activity. ST1 and ST6 clearly display different roles in the biotic interactions analyzed, where ST1 is activated in response to a N2-fixing bacterium and ST6 is up-regulated after inoculation with F. oxysporum. The role of ST1 and ST6 in the nodulation process may be related to nodule organogenesis rather than to the establishment of the interaction itself, and an increase in ST6 correlates with the activation of the salicylic acid signaling pathway during the infection and colonization processes. These results further support the role of ST6 in response to hemibiotrophic fungi. This research contributes to the understanding of the complex network that controls root biology and strengthens the idea that ST proteins are involved in several processes such as primary and lateral root development, nodule organogenesis, and the plant-microbe interaction.

MtNIP5;1, a novel Medicago truncatula boron diffusion facilitator induced under deficiency.

BACKGROUND: Legumes comprise important crops that offer major agronomic benefits, including the capacity of establishing symbiosis with rhizobia, fixing atmospheric N2. It has been proven that legumes are particularly susceptible to boron (B) stress, which leads to important yield penalties. Boron (B) deficiency or toxicity in plants causes the inhibition of growth and an altered development. Under such conditions, the participation of two distinct protein families (the major intrinsic protein family MIP and the Boron transporter family BOR) is required to minimize detrimental effects caused by B stress. However, in legumes, little is known about the transport mechanisms responsible for B uptake and distribution, especially under deficiency. RESULTS: A Medicago truncatula protein, MtNIP5;1 (Medtr1g097840) (homologous to the Arabidopsis thaliana AtNIP5;1) was identified as a novel legume B transporter involved in B uptake under deficiency. Further analyses revealed that this M. truncatula aquaporin expression was boron-regulated in roots, being induced under deficiency and repressed under toxicity. It localizes at the plasma membrane of root epidermal cells and in nodules, where B plays pivotal roles in symbiosis. Furthermore, the partial complementation of the nip5;1-1 A. thaliana mutant phenotype under B deficiency supports a functional role of MtNIP5;1 as a B transporter in this legume model plant. CONCLUSIONS: The results here presented support a functional role of MtNIP5;1 in B uptake under deficiency and provides new insights into B transport mechanisms in legume species.

Experimental evolution makes microbes more cooperative with their local host genotype.

Advances in microbiome science require a better understanding of how beneficial microbes adapt to hosts. We tested whether hosts select for more-cooperative microbial strains with a year-long evolution experiment and a cross-inoculation experiment designed to explore how nitrogen-fixing bacteria (rhizobia) adapt to legumes. We paired the bacterium Ensifer meliloti with one of five Medicago truncatula genotypes that vary in how strongly they "choose" bacterial symbionts. Independent of host choice, E. meliloti rapidly adapted to its local host genotype, and derived microbes were more beneficial when they shared evolutionary history with their host. This local adaptation was mostly limited to the symbiosis plasmids, with mutations in putative signaling genes. Thus, cooperation depends on the match between partner genotypes and increases as bacteria adapt to their local host.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level.

BACKGROUND: Leguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness. RESULTS: Rhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants. CONCLUSIONS: Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

Cellular Stoichiometry of Chemotaxis Proteins in Sinorhizobium meliloti.

Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems.IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in Escherichia coli and Bacillus subtilis In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.

A Cytokinin Signaling Type-B Response Regulator Transcription Factor Acting in Early Nodulation.

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (Mt NSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (Mt CCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.

Genome-scale metabolic reconstruction of the symbiosis between a leguminous plant and a nitrogen-fixing bacterium.

The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N2-fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N2-fixing bacteria, and N2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called 'Virtual Nodule Environment' (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation.

Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti.

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti, that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.