Eric Giraud

The evolutionary origin of nitrogen-fixing symbiosis has been a long-standing question. To address this, we focused on Bradyrhizobium , a globally abundant bacterial genus that includes classic symbiotic lineages, which rely on the common Nod factor signaling pathway to form nodules, and close relatives capable of fixing nitrogen in a free-living state. We isolated 88 strains carrying the key genes for nitrogen fixation ( nif ) from nonlegume environments and analyzed them alongside 586 public Bradyrhizobium genomes harboring these genes to reconstruct a robust phylogeny of nif genes. Analysis suggests that the earliest-diverging nif lineages are members capable of free-living nitrogen fixation, supporting the interpretation that this lifestyle is ancestral. The Nod factor-dependent symbiotic lineages are polyphyletic, with our data supporting at least three independent origins via horizontal acquisition of symbiosis islands. This evolutionary history is reflected in a genomic dichotomy: lineages capable of free-living nitrogen fixation possess a conserved nif island architecture that consistently includes the oxygen-protective gene glbO , whereas the symbiotic nif -associated regions are highly variable and universally lack glbO . Using both loss-of-function and gain-of-function genetic approaches, we show that glbO contributes significantly to nitrogenase activity under free-living conditions, whereas it is dispensable within the protected nodule environment. This work provides a framework for the evolution of nitrogen-fixing symbiosis, supporting the view that free-living nitrogen-fixing ancestors gave rise repeatedly and independently to symbiotic lineages in Bradyrhizobium .

The evolutionary origin of nitrogen-fixing symbiosis has been a long-standing question. To address this, we focused on Bradyrhizobium, a globally abundant bacterial genus that includes classic symbiotic lineages, which rely on the common Nod factor signaling pathway to form nodules, and close relatives capable of fixing nitrogen in a free-living state. We isolated 88 strains carrying the key genes for nitrogen fixation (nif) from non-legume environments and analyzed them alongside 586 public Bradyrhizobium genomes harboring these genes to reconstruct a robust phylogeny of nif genes. Analysis reveals that the earliest-diverging nif lineages are members capable of free-living nitrogen fixation, establishing this as the ancestral state. The Nod factor-dependent symbiotic lineages are polyphyletic, demonstrating at least three independent origins via horizontal acquisition of symbiosis islands. This evolutionary history is reflected in a genomic dichotomy: lineages capable of free-living nitrogen fixation possess a conserved nif island architecture that consistently includes the oxygen-protective gene glbO, whereas the symbiotic nif-associated regions are highly variable and universally lack glbO. Using both loss-of-function and gain-of-function genetic approaches, we show that glbO contributes significantly to nitrogenase activity under free-living conditions, whereas it is dispensable within the protected nodule environment. This work establishes a new framework for the evolution of symbiosis, identifying free-living ancestors as the source from which nitrogen-fixing symbiosis repeatedly and independently evolved in Bradyrhizobium.

In this study, we investigate two RpoN homologs in Bradyrhizobium sp. DOA9—chromosomal RpoNc and megaplasmid-borne RpoNp—and their roles in free-living conditions and nitrogen fixation. Phylogenetic analysis showed that RpoNc clusters with RpoN proteins from symbiotic nitrogen-fixing strains, whereas RpoNp forms a distinct clade, consistent with a function in stress responses. RpoNc proved essential for free-living conditions: ΔrpoNc mutants displayed severe growth defects that RpoNp could not compensate for. Transcriptomic comparisons between wild type and mutant RpoN identified 541 differentially expressed genes (DEGs) grouped into three clusters: 100 downregulated, 175 upregulated, and 254 moderately downregulated (with a fold change > 2, and a q-value (FDR, padj) < 0.05). Affected pathways involved nitrogen metabolism, motility, and environmental adaptation. RpoNc controlled major nitrogen fixation genes (nif and fix) along with core growth and stress response functions, while RpoNp mainly influenced stress-adaptation pathways. Genome-wide promoter motif analysis predicted 68 putative RpoNc targets, mainly associated with nitrogen fixation and metabolism, compared with only 22 predicted RpoNp targets, indicating a more restricted regulon. Electrophoretic mobility shift assays (EMSAs) further confirmed that both RpoN proteins directly bind σ⁵⁴-dependent promoters identified from transcriptomic data, supporting their regulatory roles under free-living conditions. Two mutants (ΔrpoNc and ΔrpoNp::ΩrpoNc) showed broad transcriptional disruption across nitrogen fixation, metabolism, and stress responses, underscoring complementary regulation. Overall, RpoNc is the dominant regulator of nitrogen fixation and core metabolism during free-living conditions, whereas RpoNp fine-tunes stress responses, revealing new regulatory insights for DOA9 adaptation. These results clarify how RpoN systems optimize survival across fluctuating conditions.

Biological nitrogen fixation converts atmospheric nitrogen into ammonia, essential to the global nitrogen cycle. While cyanobacterial diazotrophs are well characterized, recent studies have revealed a broad distribution of non-cyanobacterial diazotrophs (NCDs) in marine environments, although their study is limited by poor cultivability. Here we report a previously uncharacterized Bradyrhizobium isolated from the marine diatom Phaeodactylum tricornutum. Phylogenomic analysis places the strain within photosynthetic Bradyrhizobium, suggesting evolutionary adaptations to marine and terrestrial niches. Average nucleotide identity supports its classification as a previously undescribed species. Remarkably, inoculation experiments showed that the isolate induced nitrogen-fixing nodules in the Aeschynomene indica legume, pointing to symbiotic capabilities across ecological boundaries. Pangenome analysis and metabolic predictions indicate that this isolate shares more features with terrestrial photosynthetic Bradyrhizobium than with marine NCDs. Overall, these findings suggest that symbiotic interactions could evolve across different ecological niches, and raise questions about the evolution of nitrogen fixation and microbe–host interactions.

Many plants interact symbiotically with arbuscular mycorrhizal fungi to enhance inorganic phosphorus uptake, and legumes also develop a nodule symbiosis with rhizobia for nitrogen acquisition. The establishment and functioning of both symbioses rely on a common plant signaling pathway activated by structurally related Myc and Nod factors. Recently, a SPARK receptor-like kinase (RLK)/receptor-like cytoplasmic kinase (RLCK) complex was shown to be essential for arbuscular mycorrhiza formation in both monocot and dicot plants. Here, we show that in Aeschynomene legumes, the RLCK component of this receptor complex has undergone a gene duplication event and mediates a unique nodule symbiosis that is independent of rhizobial Nod factors. In Aeschynomene evenia, AeRLCK2 is crucial for nodule initiation but not for arbuscular mycorrhiza symbiosis. Additionally, AeRLCK2 physically interacts with and is phosphorylated by the cysteine-rich RLK, AeCRK, which is also required for nodulation. This finding uncovers an important molecular mechanism that controls the establishment of nodulation and is associated with Nod-independent symbiosis.

Summary: Rhizobial type III effectors (T3Es) play a crucial role in the symbiotic relationship between rhizobia and legumes by manipulating host cellular processes to promote nodule formation. Previously, we identified two T3Es, ErnA and Sup3, that trigger nodulation in Aeschynomene spp. in the absence of Nod factors. Here, we further investigate the mode of action of these T3Es during root nodule symbiosis. We employed protein interaction assays, in vitro binding and enzymatic activity assays, mutational analyses, and functional nodulation tests to dissect the roles of ErnA and Sup3 and their interactions with the host Small Ubiquitin‐like MOdifier (SUMO) pathway (SUMOylation). We demonstrate that ErnA contains a SUMO‐interacting motif (SIM) at its C terminus, which promotes its interaction with SUMO proteins in vitro and in plant nuclei. Additionally, we show that Sup3 possesses a C‐terminal SUMO protease domain, which not only interacts with SUMO proteins in vitro and in the nucleus but also exhibits SUMO protease activity. Deletion of the SIM in ErnA or mutation of the catalytic site in Sup3 abolished their ability to trigger nodulation in Aeschynomene indica. These findings suggest that type III secretion system‐dependent symbiosis is regulated by posttranslational modification through SUMOylation and that ErnA and Sup3 modulate this SUMOylation pathway to trigger nodulation.

Bradyrhizobium sp. DOA9 can nodulate a wide spectrum of legumes; however, unlike other bradyrhizobia, DOA9 carries a symbiotic plasmid harboring type III secretion system (T3SS) and several effector (T3E) genes, one of which encodes a putative type III effector SkP48. Here, we demonstrated the pivotal roles of SkP48 from Bradyrhizobium sp. DOA9 in inhibiting nodulation of various Vigna species and Crotalaria juncea and suppressing nodulation efficiency of Arachis hypogaea. By contrast, the nodulation efficiency of a SkP48 mutant did not differ significantly with the DOA9 wild-type strain on Macroptilium atropurpureum and Stylosanthes hamata. The SUMO domain of SkP48 is primarily responsible for the blocking nodulation phenotype V. radiata. An evolutionary analysis revealed that the SkP48 which contains a shikimate kinase and a SUMO protease (C48 cysteine peptidase) domain, SkP48 is distinct from other effectors previously reported in other bradyrhizobia and pathogenic bacteria. Our findings suggest that the putative T3E SkP48 is a key factor suppressing nodulation and nodule organogenesis in several legumes by activation of effector-triggered immunity through salicylic acid biosynthesis induction, which is deleterious to rhizobial infection. In addition, nodulation may be modulated by the function of defensins involved in jasmonic acid signalling in V. radiata SUT1.

Bradyrhizobium is a main rhizobial lineage of which most members nodulate legume plants using Nod factors synthetized by the nod genes. However, members of the Photosynthetic supergroup (phylogroup) within Bradyrhizobium are nod-free, but still capable of establishing nitrogen-fixing nodules with some tropical legumes of the Aeschynomene genus. These unusual findings are based on the genomic sequences of only 13 Photosynthetic Bradyrhizobium strains, and almost all were isolated from Aeschynomene nodules. Here, we report that Photosynthetic Bradyrhizobium supergroup members are more abundantly associated with rice root (endosphere and rhizosphere) compared to grassland, forest, and maize samples based on rpoB amplicon sequence analyses. We sequenced 263 new isolates of this supergroup mostly from two main subspecies of cultivated rice (Oryza sativa L. spp. indica and japonica). The extended supergroup comprises three major clades with their diversity broadly covering the natural community of this supergroup: a basal clade with significant expansion of its diversity, a clade composed by two phylogenetically diverse strains including one newly isolated, and a new clade exclusively represented by our new strains. Although this supergroup members universally lack the canonical nod genes, all 28 assayed strains covering the broad diversity induced nodules on Aeschynomene indica. The three clades displayed important differences in the efficiency of symbiosis, aligning well with their phylogenetic divergence. With this expanded ecological, phylogenetic, and functional diversity, we conclude that the nod factor-independent nodulation of Aeschynomene is a common trait of this supergroup, in contrast to the photosynthetic trait originally thought of as its unifying feature.

Effector proteins secreted via the type III secretion system (T3SS) of nitrogen-fixing rhizobia are key determinants of symbiotic compatibility in legumes. Previous report revealed that the T3SS of Bradyrhizobium sp. DOA9 plays negative effects on Arachis hypogaea symbiosis. In this study, we characterized the symbiotic role of 4 effector proteins (p0490, p0871, SkP48, and p0903) containing the small ubiquitin-like modifier (SUMO) protease domain identified in DOA9 during symbiosis. While the DOA9 strain and the two mutants of SUMO-proteases, p0490 and p0871, induced inefficient nodulation in A. hypogaea, the mutation of SUMO-proteases SkP48 or p0903 promoted efficient symbiosis comparable to the type strain Bradyrhizobium arachidis CCBAU051107. Complementation study of ∆p0903 with various mutated forms of p0903 highlighted importance of ubiquitin-like protein (ULP) domain in restriction of nodulation in A. hypogaea. We observed the accumulation of jasmonic acid (JA) and upregulation of several defence genes involved in the JA/ethylene (ET) signalling pathway at the early stage of infection in roots inoculated with DOA9 strain compared with those inoculated with the DOA9-∆p0903 strain. Our data highlight the importance of SUMO-protease effectors during the symbiotic interaction between bradyrhizobia and A. hypogaea, which could be useful for the development of high-performance inocula to improve its growth.

Summary: Some Bradyrhizobium strains nodulate certain Aeschynomene species independently of Nod factors, but thanks to their type III secretion system (T3SS). While different T3 effectors triggering nodulation (ErnA and Sup3) have been identified, the plant signalling pathways they activate remain unknown. Here, we explored the intraspecies variability in T3SS‐triggered nodulation within Aeschynomene evenia and investigated transcriptomic responses that occur during this symbiosis. Furthermore, Bradyrhizobium strains having different effector sets were tested on A. evenia mutants altered in various symbiotic signalling genes. We identified the A. evenia accession N21/PI 225551 as appropriate for deciphering the T3SS‐dependent process. Comparative transcriptomic analysis of A. evenia N21 roots inoculated with ORS3257 strain and its ∆ernA mutant revealed genes differentially expressed, including some involved in plant defences and auxin signalling. In the other A. evenia accession N76, all tested strains nodulated the AeCRK mutant but not the AeNIN and AeNSP2 mutants, indicating a differential requirement of these genes for T3SS‐dependent nodulation. Furthermore, the effects of AePOLLUX, AeCCaMK and AeCYCLOPS mutations differed between the strains. Notably, ORS86 nodulated these three mutant lines and required for this both ErnA and Sup3. Taken together, these results shed light on how the T3SS‐dependent nodulation process is achieved in legumes.