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Figure S3. Chromosome numbers in Aeschynomene species. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 135 kb)

Figure S2. Schematic representation of the different steps of the genotyping process from marker selection to data treatment. (PPTX 64 kb)

Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb)

Figure S1. Chromosome numbers in new Aeschynomene taxa. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 1008 kb)

Figure S8. Ancestral state reconstruction of adventive root primordia in the genus Aeschynomene and allied genera. Ancestral state reconstruction was estimated in SIMMAP software using the 50% majority-rule topology obtained by Bayesian analysis of the combined ITS + matK sequences. Data on the adventitious root primordia come from the present analysis and pertinent previously published data. Presence or not of adventitious root primordia is indicated by different colors. (PPTX 96 kb)

Figure S4. Chromosome numbers in species of Aeschynomene related genera. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome counts are indicated in brackets. Scale bars: 5 μm. (PPTX 57 kb)

Figure S5. Phylogenetic trees based on nuclear low-copy genes. Bayesian phylogenetic reconstructions obtained for the CYP1, eif1a, SuSy and TIP1;1 genes. Diploid species (2n = 20) are in blue, polyploid species (2n ≥ 28) in black excepted A. afraspera for which the A and B gene copies are distinguished in red and green respectively. -A, −A1, −A2, -B, -B1 and -B2 indicated the different copies found. Putative A and B subgenomes of the polyploid taxa are delineated by red and green boxes in dashed lines, respectively. Numbers at branches represent posterior probability. (PPTX 56 kb)

Table S3. GenBank numbers for the sequences used in the phylogenetic analyses. (PPTX 149 kb)

Table S2. Primers used for gene amplification and sequencing. (PPTX 134 kb)

Figure S6. Ancestral state reconstruction of ploidy levels in the genus Aeschynomene and allied genera. Ancestral state reconstruction was estimated in SIMMAP software using the 50% majority-rule topology obtained by Bayesian analysis of the combined ITS + matK sequences. Ploidy levels are indicated by different colors. Unknown ploidy levels are denoted by a dash. (PPTX 3568 kb)

Doc. S1. ITS sequences obtained for the Nod-independent Aeschynomene accessions. (DOCX 30 kb)

Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb)

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.

International audience; In rhizobium strains, the lipid A is modified by the addition of a very long-chain fatty acid (VLCFA) shown to play an important role in rigidification of the outer membrane, thereby facilitating their dual life cycle, outside and inside the plant. In Bradyrhizobium strains, the lipid A is more complex with the presence of at least two VLCFAs, one covalently linked to a hopanoid molecule, but the importance of these modifications is not well-understood. In this study, we identified a cluster of VLCFA genes in the photosynthetic Bradyrhizobium strain ORS278, which nodulates Aeschynomene plants in a Nod factor-independent process. We tried to mutate the different genes of the VLCFA gene cluster to prevent the synthesis of the VLCFAs, but only one mutant in the lpxXL gene encoding an acyltransferase was obtained. Structural analysis of the lipid A showed that LpxXL is involved in the transfer of the C-26:25OH VLCFA to the lipid A but not in the one of the C30:29OH VLCFA which harbors the hopanoid molecule. Despite maintaining the second VLCFA, the ability of the mutant to cope with various stresses (low pH, high temperature, high osmolarity, and antimicrobial peptides) and to establish an efficient nitrogen-fixing symbiosis was drastically reduced. In parallel, we investigated whether the BRADO0045 gene, which encodes a putative acyltransferase displaying a weak identity with the apo-lipoprotein N-acyltransferase Lnt, could be involved in the transfer of the C-30:29OH VLCFA to the lipid A. Although the mutant exhibited phenotypes similar to the lpxXL mutant, no difference in the lipid A structure was observed from that in the wild-type strain, indicating that this gene is not involved in the modification of lipid A. Our results advance our knowledge of the biosynthesis pathway and the role of VLCFAs-modified lipid A in free-living and symbiotic states of Bradyrhizobium strains.

Legumes harbor in their symbiotic nodule organs nitrogen fixing rhizobium bacteria called bacteroids. Some legumes produce Nodule-specific Cysteine-Rich (NCR) peptides in the nodule cells to control the intracellular bacterial population. NCR peptides have antimicrobial activity and drive bacteroids toward terminal differentiation. Other legumes do not produce NCR peptides and their bacteroids are not differentiated. Bradyrhizobia, infecting NCR-producing Aeschynomene plants, require the peptide uptake transporter BclA to cope with the NCR peptides as well as a specific peptidoglycan-modifying DD-carboxypeptidase, DD-CPase1. We show that Bradyrhizobium diazoefficiens strain USDA110 forms undifferentiated bacteroids in NCR-lacking soybean nodules. Unexpectedly, in Aeschynomene afraspera nodules the nitrogen fixing USDA110 bacteroids are hardly differentiated despite the fact that this host produces NCR peptides, suggesting that USDA110 is insensitive to the host peptide effectors and that nitrogen fixation can be uncoupled from differentiation. In agreement with the absence of bacteroid differentiation, USDA110 does not require its bclA gene for nitrogen fixing symbiosis with these two host plants. Furthermore, we show that the BclA and DD-CPase1 act independently in the NCR-induced morphological differentiation of bacteroids. Our results suggest that BclA is required to protect the rhizobia against the NCR stress but not to induce the terminal differentiation pathway.

Les Champignons mycorhiziens à arbuscules (CMA) hébergent dans leur cytoplasme sporal deux types d'endobactéries majeures : Candidatus Glomeribacter gigasporarum (CaGg) et des bactéries Mollicutes (Mre). Ces bactéries présentes dans les spores sont des endosymbioles obligatoires de Gigaspora margarita (Gim) en particulier, et auraient un rôle majeur dans la croissance du champignon dans les premières étapes de la symbiose avec la plante (Lum in i et al. 2007). Une étude fonctionnelle visant à comprendre celte relation symbiotique a été initiée afin d'évaluer l'impact de l'inoculation des spores contenant ces populations d'endobactéries sur la symbiose avec le riz. Pour cela, un recensement global de la communauté bactérienne (extérieur et intérieur de la spore) a été effectuée par étude du polymophisme par ARISA (Ribosomal Intergenic Spacer Analysis). Cette approche a été complétée par l'étude de la coexistence des deux endosymbiotes dans le cytoplasme sporal des CMA par différents approches (phylotypage moléculaire, microscopie électronique, hybridation ill situ Ouorescente (F ISH) ct PCR quantitative)(Desi ro ct al 2013). L'inoculation du riz à l'aide des spores provenant du Camero un et du japon a permis d'évaluer l'impact de la présence des bactéries sur les performances symbiotiques des CMA. Globalement, une communauté bactérienne dominée par les Actinobactéries, les Firmicutes et Burkholderia ont été recensés à l'extérieur et intérieur des spores. Dans l'espace cytoplasmique, une spore est susceptible d'héberger les deux types d'endobactéries à la fois (CaGg el Mre) avec une abondance variable de population de mollicutes el de CaGg en fonction de l'origine des CMA Ces découvertes révèlent ainsi pour la première fois que les champignons tolèrent un microbiome bactérien intracellula re dans lequel des types distincts d'endobactéries coexistent au se in d'une seule cellule. Une très faible abondance des CaGg observée dans les spores du Cameroun (1/20 à 11200 par rapport à l'espèce de référence provenant du japon) s'accompagne d'une forte présence des mollicutes (Mre). Une réponse plus importante a été notée sur la croissance et le développement du riz inoculé en serre avec les spores du Cameroun par rapport à celle du Japon, suggérant un effet positif de ces bactéries et un potentiel intéressant dans la tolérance des spores de CMA aux sols acides et la croissance du riz.

Two types of interactions have been described during the Aeschynomene-photosynthetic Bradyrhizobium symbioses-a classical one which is Nod factors (NF) dependent and an atypical one which is NF independent. It is the Aeschynomene sp. that determines the signaling modus. Some photosynthetic strains such as ORS285 that do contain the canonical nodABC genes are able to use bath strategies depending on the hast plant. Whereas the nod-Iacking strain such as BTAi 1 and ÛRS278 nodulate only some Aeschynomene species according a Nod-independent process. In this study, we investigate the role of lipopolysaccharides (LPS) in the establishment of the Aeschynomene-Bradyrhizobium symbiosis. Structural analysis of the LPS structure of photosynthetic Bradyrhizobium strains (BTAi1 and ORS278) reveals two original features : i) The only component of the 0- O-antigen region consists of a unique monosaccharide that had never been described before in nature. This new sugar was named Bradyrhizose (Alba S et al. 20 Il), il) the lipid A region displays the covalent attachment of an hopanoid molecule to a very long chain fatty acid. Screening of a mutant library of ûRS285 s train for clones showing an aberrant colony morphology has permitted to identify a mutant affected in a putative 0-antigen ligase that lacks the O-antigen part. Analysis of the symbiotic properties of this mutant shows that this region is important for the establishment of the Nod-dependent process but not for the Nod-independent one. To investigate the biological significance of the Hopanoid-lipid A, we cOl1structed a mutant in the squalene hopene cyclase (shc) gene that catalyzes a key step in hopanoid biosynthesis. This mutant was perfectly able to engage in an efficient symbiotic relationship with the plant but very rapidly the nodules lost their ability to fix nitrogen and senesce prematurely. Altogether these data indicate that Photosynthetic bradyrhizobia display a unique LPS structure that plays an important role in the establishment of the Nod-dependent symbiosis and in the chronic infection of the plant cells.