Fabienne Cartieaux

Legumes symbioses : Absence of Nod genes in photosynthetic bradyrhizobia

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

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

Several species from an ancient legume lineage independently evolved a novel class of cysteine-rich peptides to impose a differentiation process on their endosymbionts. 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.

Large-Scale Transposon Mutagenesis of Photosynthetic Bradyrhizobium Sp. Strain ORS278 Reveals New Genetic Loci Putatively Important for Nod-Independent Symbiosis with Aeschynomene indica

Photosynthetic Bradyrhizobium strains possess the unusual ability to form nitrogen-fixing nodules on a specific group of legumes in the absence of Nod factors. To obtain insight into the bacterial genes involved in this Nod-independent symbiosis, we screened 15,648 Tn5 mutants of Bradyrhizobium sp. strain ORS278 for clones affected in root symbiosis with Aeschynomene indica. From the 268 isolated mutants, 120 mutants were altered in nodule development (Ndv(-)) and 148 mutants were found to be deficient in nitrogen fixation (Fix(-)). More than 50% of the Ndv(-) mutants were found to be altered in purine biosynthesis, strengthening the previous hypothesis of a symbiotic role of a bacterial purine derivative during the Nod-independent symbiosis. The other Ndv(-) mutants were auxotrophic for pyrimidines and amino acids (leucine, glutamate, and lysine) or impaired in genes encoding proteins of unknown function. The Fix(-) mutants were found to be affected in a wide variety of cellular processes, including both novel (n = 56) and previously identified (n = 31) genes important in symbiosis. Among the novel genes identified, several were involved in the Calvin cycle, suggesting that CO(2) fixation could play an important role during this symbiosis.

The Nod Factor–Independent Symbiotic Signaling Pathway: Development of Agrobacterium rhizogenes–Mediated Transformation for the Legume Aeschynomene indica

The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes-mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.

Nod Factor-Independent Nodulation in Aeschynomene evenia Required the Common Plant-Microbe Symbiotic Toolkit

A tropical legume requires the common symbiotic pathway to interact with a nitrogen-fixing bacterium devoid of the canonical nodulation genes. Nitrogen fixation in the legume-rhizobium symbiosis is a crucial area of research for more sustainable agriculture. Our knowledge of the plant cascade in response to the perception of bacterial Nod factors has increased in recent years. However, the discovery that Nod factors are not involved in the Aeschynomene-Bradyrhizobium spp. interaction suggests that alternative molecular dialogues may exist in the legume family. We evaluated the conservation of the signaling pathway common to other endosymbioses using three candidate genes: Ca2+/Calmodulin-Dependent Kinase (CCaMK), which plays a central role in cross signaling between nodule organogenesis and infection processes; and Symbiosis Receptor Kinase (SYMRK) and Histidine Kinase1 (HK1), which act upstream and downstream of CCaMK, respectively. We showed that CCaMK, SYMRK, and HK1 are required for efficient nodulation in Aeschynomene evenia. Our results demonstrate that CCaMK and SYMRK are recruited in Nod factor-independent symbiosis and, hence, may be conserved in all vascular plant endosymbioses described so far.

Genotype Delimitation in the Nod-Independent Model Legume Aeschynomene evenia

Research on the nitrogen-fixing symbiosis has been so far focused 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 semi-aquatic Aeschynomene species present the distinctive feature to form nitrogen-fixing nodules on both roots and stems following elicitation by photosynthetic bradyrhizobia 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 molecular mechanisms of this unique Nod-independent nitrogen-fixing symbiosis, we previously identified A. evenia C. Wright as an appropriate model legume, because it displays all the requisites for molecular and genetic approaches. To advance the use of this new model legume species, here we characterized the intraspecific diversity found in A. evenia. For this, the accessions available in germplasm banks were collected and subjected to morphological investigations, genotyping with RAPD and SSR markers, molecular phylogenies using ITS and single nuclear gene sequences, and cross-compatibility tests. These combined analyses revealed an important intraspecific differentiation that led us to propose a new taxonomic classification for A. evenia comprising two subspecies and four varieties. The A. evenia ssp. evenia contains var. evenia and var. pauciciliata whereas A. evenia ssp. serrulata comprises var. serrulata and var. major. This study provides information to exploit efficiently the diversity encountered in A. evenia and proposes subsp. evenia as the most appropriate subspecies for future projects aimed at identifying plant determinants of the Nod-independent symbiotic process.

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 gene-based map of the Nod factor-independent Aeschynomene evenia sheds new light on the evolution of nodulation and legume genomes

Aeschynomene evenia has emerged as a new model legume for the deciphering of the molecular mechanisms of an alternative symbiotic process that is independent of the Nod factors. Whereas most of the research on nitrogen-fixing symbiosis, legume genetics and genomics has so far focused on Galegoid and Phaseolid legumes, A. evenia falls in the more basal and understudied Dalbergioid clade along with peanut (Arachis hypogaea). To provide insights into the symbiotic genes content and the structure of the A. evenia genome, we established a gene-based genetic map for this species. Firstly, an RNAseq analysis was performed on the two parental lines selected to generate a F2 mapping population. The transcriptomic data were used to develop molecular markers and they allowed the identification of most symbiotic genes. The resulting map comprised 364 markers arranged in 10 linkage groups (2n = 20). A comparative analysis with the sequenced genomes of Arachis duranensis and A. ipaensis, the diploid ancestors of peanut, indicated blocks of conserved macrosynteny. Altogether, these results provided important clues regarding the evolution of symbiotic genes in a Nod factor-independent context. They provide a basis for a genome sequencing project and pave the way for forward genetic analysis of symbiosis in A. evenia.

The role of rhizobial (NifV) and plant (FEN1) homocitrate synthases in Aeschynomene /photosynthetic Bradyrhizobium symbiosis

In the most studied rhizobium-legume interactions, the host plant supplies the symbiont with homocitrate, an essential co-factor of the nitrogenase enzyme complex, via the expression of a nodule-specific homocitrate synthase FEN1. Photosynthetic bradyrhizobia interacting with Nod factor (NF) dependent and NF-independent Aeschynomene legumes are able to synthesize homocitrate themselves as they contain a nifV gene encoding a homocitrate synthase. Here, we show that in the model strain ORS285, nifV is required for free-living and symbiotic dinitrogen fixation with NF-independent Aeschynomene species. In contrast, in symbiosis with NF-dependent Aeschynomene species, the nifV requirement for efficient nitrogen fixation was found to be host plant dependent. Interestingly, orthologs of FEN1 were found in both NF-dependent and NF-independent Aeschynomene species. However, a high nodule specific induction of FEN1 expression was only observed in A. afraspera, a host plant in which nifV is not required for symbiotic dinitrogen fixation. These data indicate that efficient symbiotic nitrogen fixation in many of the tested Aeschynomene species requires rhizobial homocitrate synthesis. Considering that more than 10% of the fully sequenced rhizobium strains do contain a nifV gene, the Aeschynomene/photosynthetic Bradyrhizobium interaction is likely not the only rhizobium/legume symbiosis where rhizobial nifV expression is required.

Transcriptome Profiles of Nod Factor-independent Symbiosis in the Tropical Legume Aeschynomene evenia

Nod factors (NF) were assumed to be indispensable for the establishment of a rhizobium-legume symbiosis until the discovery that certain Bradyrhizobium strains interacting with certain Aeschynomene species lack the canonical nodABC genes required for their synthesis. So far, the molecular dialogue between Aeschynomene and its symbionts remains an open question. Here we report a time course transcriptional analysis of Aeschynomene evenia in response to inoculation with Bradyrhizobium ORS278. The NF-independent symbiotic process was monitored at five time points between bacterial infection and nodule maturity. The five time points correspond to three specific events, root infection by crack entry, nodule organogenesis, and the establishment of the nitrogen fixing process. During the third stage, about 80 NCR-like genes and eight symbiotic genes known to be involved in signaling, bacterial infection or nodulation regulation were highly expressed. Comparative gene expression analyses at the five time points also enabled the selection of genes with an expression profile that makes them promising markers to monitor early plant responses to bacteria. Such markers could be used in bioassays to identify the nature of the bacterial signal(s). Our data represent valuable resources for investigation of this Nod factor-independent symbiosis.