Revealing human impact on natural ecosystems through soil bacterial DNA sampled from an archaeological site.

Human activities have affected the surrounding natural ecosystems, including belowground microorganisms, for millennia. Their short- and medium-term effects on the diversity and the composition of soil microbial communities are well-documented, but their lasting effects remain unknown. When unoccupied for centuries, archaeological sites are appropriate for studying the long-term effects of past human occupancy on natural ecosystems, including the soil compartment. In this work, the soil chemical and bacterial compositions were compared between the Roman fort of Hegra (Saudi Arabia) abandoned for 1500 years, and a preserved area located at 120 m of the southern wall of the Roman fort where no human occupancy was detected. We show that the four centuries of human occupancy have deeply and lastingly modified both the soil chemical and bacterial compositions inside the Roman fort. We also highlight different bacterial putative functions between the two areas, notably associated with human occupancy. Finally, this work shows that the use of soils from archaeological sites causes little disruption and can bring relevant information, at a large scale, during the initial surveys of archaeological sites.

The rhizobial type III effector ErnA confers the ability to form nodules in legumes.

Several Bradyrhizobium species nodulate the leguminous plant Aeschynomene indica in a type III secretion system-dependent manner, independently of Nod factors. To date, the underlying molecular determinants involved in this symbiotic process remain unknown. To identify the rhizobial effectors involved in nodulation, we mutated 23 out of the 27 effector genes predicted in Bradyrhizobium strain ORS3257. The mutation of nopAO increased nodulation and nitrogenase activity, whereas mutation of 5 other effector genes led to various symbiotic defects. The nopM1 and nopP1 mutants induced a reduced number of nodules, some of which displayed large necrotic zones. The nopT and nopAB mutants induced uninfected nodules, and a mutant in a yet-undescribed effector gene lost the capacity for nodule formation. This effector gene, widely conserved among bradyrhizobia, was named ernA for "effector required for nodulation-A." Remarkably, expressing ernA in a strain unable to nodulate A. indica conferred nodulation ability. Upon its delivery by Pseudomonas fluorescens into plant cells, ErnA was specifically targeted to the nucleus, and a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy approach supports the possibility that ErnA binds nucleic acids in the plant nuclei. Ectopic expression of ernA in A. indica roots activated organogenesis of root- and nodule-like structures. Collectively, this study unravels the symbiotic functions of rhizobial type III effectors playing distinct and complementary roles in suppression of host immune functions, infection, and nodule organogenesis, and suggests that ErnA triggers organ development in plants by a mechanism that remains to be elucidated.

Diversity and phenotypic analyses of salt- and heat-tolerant wild bean Phaseolus filiformis rhizobia native of a sand beach in Baja California and description of Ensifer aridi sp. nov.

In northern Mexico, aridity, salinity and high temperatures limit areas that can be cultivated. To investigate the nature of nitrogen-fixing symbionts of Phaseolus filiformis, an adapted wild bean species native to this region, their phylogenies were inferred by MLSA. Most rhizobia recovered belong to the proposed new species Ensifer aridi. Phylogenetic analyses of nodC and nifH show that Mexican isolates carry symbiotic genes acquired through horizontal gene transfer that are divergent from those previously characterized among bean symbionts. These strains are salt tolerant, able to grow in alkaline conditions, high temperatures, and capable of utilizing a wide range of carbohydrates and organic acids as carbon sources for growth. This study improves the knowledge on diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico and further enlarges the spectrum of microsymbiont with which Phaseolus species can interact with, including cultivated bean varieties, notably under stressed environments. Here, the species Ensifer aridi sp. nov. is proposed as strain type of the Moroccan isolate LMR001T (= LMG 31426T; = HAMBI 3707T) recovered from desert sand dune.

noeM, a New Nodulation Gene Involved in the Biosynthesis of Nod Factors with an Open-Chain Oxidized Terminal Residue and in the Symbiosis with Mimosa pudica.

The ß-rhizobium Cupriavidus taiwanensis is a nitrogen-fixing symbiont of Mimosa pudica. Nod factors produced by this species were previously found to be pentameric chitin-oligomers carrying common C18:1 or C16:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing terminal residue. Here, we report that, in addition, C. taiwanensis LMG19424 produces molecules where the reducing sugar is open and oxidized. We identified a novel nodulation gene located on the symbiotic plasmid pRalta, called noeM, which is involved in this atypical Nod factor structure. noeM encodes a transmembrane protein bearing a fatty acid hydroxylase domain. This gene is expressed during symbiosis with M. pudica and requires NodD and luteolin for optimal expression. The closest noeM homologs formed a separate phylogenetic clade containing rhizobial genes only, which are located on symbiosis plasmids downstream from a nod box. Corresponding proteins, referred to as NoeM, may have specialized in symbiosis via the connection to the nodulation pathway and the spread in rhizobia. noeM was mostly found in isolates of the Mimoseae tribe, and specifically detected in all tested strains able to nodulate M. pudica. A noeM deletion mutant of C. taiwanensis was affected for the nodulation of M. pudica, confirming the role of noeM in the symbiosis with this legume.

Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics.

BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the unique property of being root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions. RESULTS: A combination of phylogenetic analyses, comprising ITS and low-copy nuclear genes, along with cytogenetic experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance, two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem nodulation. CONCLUSION: A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some Aeschynomene species can be efficiently nodulated in a Nod-independent fashion.

A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses.

BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. RESULTS: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. CONCLUSIONS: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses.

nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth.

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity.

A Peptidoglycan-Remodeling Enzyme Is Critical for Bacteroid Differentiation in Bradyrhizobium spp. During Legume Symbiosis.

In response to the presence of compatible rhizobium bacteria, legumes form symbiotic organs called nodules on their roots. These nodules house nitrogen-fixing bacteroids that are a differentiated form of the rhizobium bacteria. In some legumes, the bacteroid differentiation comprises a dramatic cell enlargement, polyploidization, and other morphological changes. Here, we demonstrate that a peptidoglycan-modifying enzyme in Bradyrhizobium strains, a DD-carboxypeptidase that contains a peptidoglycan-binding SPOR domain, is essential for normal bacteroid differentiation in Aeschynomene species. The corresponding mutants formed bacteroids that are malformed and hypertrophied. However, in soybean, a plant that does not induce morphological differentiation of its symbiont, the mutation does not affect the bacteroids. Remarkably, the mutation also leads to necrosis in a large fraction of the Aeschynomene nodules, indicating that a normally formed peptidoglycan layer is essential for avoiding the induction of plant immune responses by the invading bacteria. In addition to exopolysaccharides, capsular polysaccharides, and lipopolysaccharides, whose role during symbiosis is well defined, our work demonstrates an essential role in symbiosis for yet another rhizobial envelope component, the peptidoglycan layer.

The evolutionary dynamics of ancient and recent polyploidy in the African semiaquatic species of the legume genus Aeschynomene.

The legume genus Aeschynomene is notable in the ability of certain semiaquatic species to develop nitrogen-fixing stem nodules. These species are distributed in two clades. In the first clade, all the species are characterized by the use of a unique Nod-independent symbiotic process. In the second clade, the species use a Nod-dependent symbiotic process and some of them display a profuse stem nodulation as exemplified in the African Aeschynomene afraspera. To facilitate the molecular analysis of the symbiotic characteristics of such legumes, we took an integrated molecular and cytogenetic approach to track occurrences of polyploidy events and to analyze their impact on the evolution of the African species of Aeschynomene. Our results revealed two rounds of polyploidy: a paleopolyploid event predating the African group and two neopolyploid speciations, along with significant chromosomal variations. Hence, we found that A. afraspera (8x) has inherited the contrasted genomic properties and the stem-nodulation habit of its parental lineages (4x). This study reveals a comprehensive picture of African Aeschynomene diversification. It notably evidences a history that is distinct from the diploid Nod-independent clade, providing clues for the identification of the specific determinants of the Nod-dependent and Nod-independent symbiotic processes, and for comparative analysis of stem nodulation.

Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides.

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

Ectomycorrhizal fungal communities of Coccoloba uvifera (L.) L. mature trees and seedlings in the neotropical coastal forests of Guadeloupe (Lesser Antilles).

We studied belowground and aboveground diversity and distribution of ectomycorrhizal (EM) fungal species colonizing Coccoloba uvifera (L.) L. (seagrape) mature trees and seedlings naturally regenerating in four littoral forests of the Guadeloupe island (Lesser Antilles). We collected 546 sporocarps, 49 sclerotia, and morphotyped 26,722 root tips from mature trees and seedlings. Seven EM fungal species only were recovered among sporocarps (Cantharellus cinnabarinus, Amanita arenicola, Russula cremeolilacina, Inocybe littoralis, Inocybe xerophytica, Melanogaster sp., and Scleroderma bermudense) and one EM fungal species from sclerotia (Cenococcum geophilum). After internal transcribed spacer (ITS) sequencing, the EM root tips fell into 15 EM fungal taxa including 14 basidiomycetes and 1 ascomycete identified. Sporocarp survey only weakly reflected belowground assessment of the EM fungal community, although 5 fruiting species were found on roots. Seagrape seedlings and mature trees had very similar communities of EM fungi, dominated by S. bermudense, R. cremeolilacina, and two Thelephoraceae: shared species represented 93 % of the taxonomic EM fungal diversity and 74 % of the sampled EM root tips. Furthermore, some significant differences were observed between the frequencies of EM fungal taxa on mature trees and seedlings. The EM fungal community composition also varied between the four investigated sites. We discuss the reasons for such a species-poor community and the possible role of common mycorrhizal networks linking seagrape seedlings and mature trees in regeneration of coastal forests.

The geographical patterns of symbiont diversity in the invasive legume Mimosa pudica can be explained by the competitiveness of its symbionts and by the host genotype.

Variations in the patterns of diversity of symbionts have been described worldwide on Mimosa pudica, a pan-tropical invasive species that interacts with both α and ß-rhizobia. In this study, we investigated if symbiont competitiveness can explain these variations and the apparent prevalence of ß- over α-rhizobia. We developed an indirect method to measure the proportion of nodulation against a GFP reference strain and tested its reproducibility and efficiency. We estimated the competitiveness of 54 strains belonging to four species of ß-rhizobia and four of α-rhizobia, and the influence of the host genotype on their competitiveness. Our results were compared with biogeographical patterns of symbionts and host varieties. We found: (i) a strong strain effect on competitiveness largely explained by the rhizobial species, with Burkholderia phymatum being the most competitive species, followed by B. tuberum, whereas all other species shared similar and reduced levels of competitiveness; (ii) plant genotype can increase the competitiveness of Cupriavidus taiwanensis. The latter data support the likelihood of the strong adaptation of C. taiwanensis with the M. pudica var. unijuga and help explain its prevalence as a symbiont of this variety over Burkholderia species in some environments, most notably in Taiwan.

Radiation of the Nod-independent Aeschynomene relies on multiple allopolyploid speciation events.

• The semi-aquatic legumes belonging to the genus Aeschynomene constitute a premium system for investigating the origin and evolution of unusual symbiotic features such as stem nodulation and the presence of a Nod-independent infection process. This latter apparently arose in a single Aeschynomene lineage. But how this unique Nod-independent group then radiated is not yet known. • We have investigated the role of polyploidy in Aeschynomene speciation via a case study of the pantropical A. indica and then extended the analysis to the other Nod-independent species. For this, we combined SSR genotyping, genome characterization through flow cytometry, chromosome counting, FISH and GISH experiments, molecular phylogenies using ITS and single nuclear gene sequences, and artificial hybridizations. • These analyses demonstrate the existence of an A. indica polyploid species complex comprising A. evenia (C. Wright) (2n = 2x = 20), A. indica L. s.s. (2n = 4x = 40) and a new hexaploid form (2n = 6x = 60). This latter contains the two genomes present in the tetraploid (A. evenia and A. scabra) and another unidentified genome. Two other species, A. pratensis and A. virginica, are also shown to be of allopolyploid origin. • This work reveals multiple hybridization/polyploidization events, thus highlighting a prominent role of allopolyploidy in the radiation of the Nod-independent Aeschynomene.

A proteomic approach of bradyrhizobium/aeschynomene root and stem symbioses reveals the importance of the fixA locus for symbiosis.

Rhizobia are soil bacteria that are able to form symbiosis with plant hosts of the legume family. These associations result in the formation of organs, called nodules in which bacteria fix atmospheric nitrogen to the benefit of the plant. Most of our knowledge on the metabolism and the physiology of the bacteria during symbiosis derives from studying roots nodules of terrestrial plants. Here we used a proteomics approach to investigate the bacterial physiology of photosynthetic Bradyrhizobium sp. ORS278 during the symbiotic process with the semi aquatical plant Aeschynomene indica that forms root and stem nodules. We analyzed the proteomes of bacteria extracted from each type of nodule. First, we analyzed the bacteroid proteome at two different time points and found only minor variation between the bacterial proteomes of 2-week- and 3-week-old nodules. High conservation of the bacteroid proteome was also found when comparing stem nodules and root nodules. Among the stem nodule specific proteins were those related to the phototrophic ability of Bradyrhizobium sp. ORS278. Furthermore, we compared our data with those obtained during an extensive genetic screen previously published. The symbiotic role of four candidate genes which corresponding proteins were found massively produced in the nodules but not identified during this screening was examined. Mutant analysis suggested that in addition to the EtfAB system, the fixA locus is required for symbiotic efficiency.

Occurrence and diversity of stem nodulation in Aeschynomene and Sesbania legumes from wetlands of Madagascar.

Legumes have the ability to establish a nitrogen-fixing symbiosis with soil rhizobia that they house in specific organs, the nodules. In most rhizobium-legume interactions, nodulation occurs on the root. However, certain tropical legumes growing in wetlands possess a unique trait: the capacity to form rhizobia-harbouring nodules on the stem. Despite the originality of the stem nodulation process, its occurrence and diversity in waterlogging-tolerant legumes remains underexplored, impeding a comprehensive analysis of its genetics and biology. Here, we aimed at filling this gap by surveying stem nodulation in legume species-rich wetlands of Madagascar. Stem nodulation was readily observed in eight hydrophytic species of the legume genera, Aeschynomene and Sesbania, for which significant variations in stem nodule density and morphology was documented. Among these species, A. evenia, which is used as genetic model to study the rhizobial symbiosis, was found to be frequently stem-nodulated. Two other Aeschynomene species, A. cristata and A. uniflora, were evidenced to display a profuse stem-nodulation as occurs in S. rostrata. These findings extend our knowledge on legumes species that are endowed with stem nodulation and further indicate that A. evenia, A. cristata, A. uniflora and S. rostrata are of special interest for the study of stem nodulation. As such, these legume species represent opportunities to investigate different modalities of the nitrogen-fixing symbiosis and this knowledge could provide cues for the engineering of nitrogen-fixation in non-legume crops.

Photosynthetic Bradyrhizobium sp. strain ORS285 is capable of forming nitrogen-fixing root nodules on soybeans (Glycine max).

The ability of photosynthetic Bradyrhizobium strains ORS285 and ORS278 to nodulate soybeans was investigated. While the nod gene-deficient ORS278 strain induced bumps only on soybean roots, the nod gene-containing ORS285 strain formed nitrogen-fixing nodules. However, symbiotic efficiencies differed drastically depending on both the soybean genotype used and the culture conditions tested.

Evolution of symbiosis in the legume genus Aeschynomene.

Legumes in the genus Aeschynomene form nitrogen-fixing root nodules in association with Bradyrhizobium strains. Several aquatic and subaquatic species have the additional capacity to form stem nodules, and some of them can symbiotically interact with specific strains that do not produce the common Nod factors synthesized by all other rhizobia. The question of the emergence and evolution of these nodulation characters has been the subject of recent debate. We conducted a molecular phylogenetic analysis of 38 different Aeschynomene species. The phylogeny was reconstructed with both the chloroplast DNA trnL intron and the nuclear ribosomal DNA ITS/5.8S region. We also tested 28 Aeschynomene species for their capacity to form root and stem nodules by inoculating different rhizobial strains, including nodABC-containing strains (ORS285, USDA110) and a nodABC-lacking strain (ORS278). Maximum likelihood analyses resolved four distinct phylogenetic groups of Aeschynomene. We found that stem nodulation may have evolved several times in the genus, and that all Aeschynomene species using a Nod-independent symbiotic process clustered in the same clade. The phylogenetic approach suggested that Nod-independent nodulation has evolved once in this genus, and should be considered as a derived character, and this result is discussed with regard to previous experimental studies.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Aeschynomene evenia, a model plant for studying the molecular genetics of the nod-independent rhizobium-legume symbiosis.

Research on the nitrogen-fixing symbiosis has been focused, thus far, on two model legumes, Medicago truncatula and Lotus japonicus, which use a sophisticated infection process involving infection thread formation. However, in 25% of the legumes, the bacterial entry occurs more simply in an intercellular fashion. Among them, some Aeschynomene spp. are nodulated by photosynthetic Bradyrhizobium spp. that do not produce Nod factors. This interaction is believed to represent a living testimony of the ancestral state of the rhizobium-legume symbiosis. To decipher the mechanisms of this Nod-independent process, we propose Aeschynomene evenia as a model legume because it presents all the characteristics required for genetic and molecular analysis. It is a short-perennial and autogamous species, with a diploid and relatively small genome (2n=20; 460 Mb/1C). A. evenia 'IRFL6945' is nodulated by the well-characterized photosynthetic Bradyrhizobium sp. strain ORS278 and is efficiently transformed by Agrobacterium rhizogenes. Aeschynomene evenia is genetically homozygous but polymorphic accessions were found. A manual hybridization procedure has been set up, allowing directed crosses. Therefore, it should be relatively straightforward to unravel the molecular determinants of the Nod-independent process in A. evenia. This should shed new light on the evolution of rhizobium-legume symbiosis and could have important agronomic implications.

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species.

The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses.