The Mesorhizobium huakuii opacity protein (MhOpa22) is essential for effective nodulation and nitrogen fixation in rhizobium–legume symbiosis. Astragalus sinicus AsGLP1 interacts with MhOpa22, but the molecular mechanisms underlying the ability of MhOpa22 to mediate symbiosis remain elusive. This study demonstrated that the Loop2–3 domains of MhOpa22 interact with AsGLP1 in planta. Inoculating A. sinicus with different M. huakuii loop deletion mutants revealed that the Loop2 mutant gives rise to fewer root nodules and decreased nodule nitrogenase activity relative to inoculation with the wild-type strain. The Loop2 mutation also significantly affects ROS production and the coordinated expression of defense and symbiosis genes in host plant roots during early symbiosis. Furthermore, MhOpa22 was confirmed to be a membrane protein that exerts its function via outer membrane vesicles (OMVs). Taken together, these results provide molecular insights into the function of the outer membrane protein MhOpa22 and its critical extracellular Loop2 domain during symbiosis. MhOpa22 inhibits host defense responses through OMVs during early symbiosis and plays an essential role in rhizobial infection and nodule formation.

Rhizobial symbiosis, a crucial source of nitrogen for legume hosts, is thought to have evolved from mycorrhizal symbiosis. Both symbioses share a common symbiotic signaling pathway (CSSP) in plants. One hypothesis is that the lack of nodulation-specific genes in the genome of mycorrhizal symbiotic plants limits their ability to establish rhizobial interactions. Here, we introduced nine key genes in nodulation pathway, including NFR1, NFR5, SYMRK, CCaMK, CYCLOPS, NSP1, NSP2, LHK1, and NIN) from Lotus japonicus, into rice (Oryza sativa ssp. japonica cv. Zhonghua 11) to create Nodulation Signaling Pathway Overexpression (NSPO) rice. Analysis of gene expression showed that NFR5 and CCaMK were robustly transcribed in transgenic rice roots determined by qPCR. SYMRK, CYCLOPS, NSP2, and NFR1 showed relatively low transcript abundance, while transcripts of NIN, NSP1, and LHK1 were not detected. NSPO rice did not exhibit enhanced rhizobial colonization at the roots but increased formation of 2,4-D-induced nodule-like structures at the ratoon roots compared to the wild type. Remarkably, field trials demonstrated higher grain yield in NSPO rice, despite a slight reduction in seed-setting rate. Additionally, the expression of immune-related transcription factor genes was downregulated in NSPO rice. These findings suggest that heterologous expression of nodulation-related genes can promote rhizobial interaction and improve agronomic traits, such as yield in non-leguminous crops.

The legume-rhizobia symbiosis is a cornerstone of sustainable agriculture due to its ability to facilitate biological nitrogen fixation. Still, real-time visualization and quantification of this interaction remain technically challenging, especially across different host backgrounds. In this study, we systematically evaluate the efficacy of the nitrogenase system nifH promoter (PnifH) in driving expression of distinct fluorescent reporters; superfolder yellow fluorescent protein (sfYFP), superfolder cyan fluorescent protein (sfCFP), and various red fluorescent proteins (RFPs) within root nodules of determinate (Lotus japonicus-Mesorhizobium japonicum) and indeterminate (Pisum sativum-Rhizobium leguminosarum) systems. We show that PnifH-driven sfYFP and sfCFP yield strong, uniform, and reproducible fluorescence in nodules of both systems, facilitating reliable quantification of nodulation traits and strain occupancy. In contrast, RFPs including monomeric (mScarlet-I, mRFP1, mARs1) and multimeric (Azami Red1.0) variants exhibited weak or inconsistent signals in pea. Notably, fluorescent labeling did not impair rhizobial competitiveness for root nodule occupancy, and PnifH-driven sfYFP and sfCFP reporters enabled robust multiplexed imaging in single-root and split-root assays. In the lotus, mScarlet-I worked robustly and facilitated a tripartite strain labeling system. Complementing our molecular toolkit, we established a deep learning-based analytical pipeline for high-throughput, automated quantification of nodulation traits, validated against standard ImageJ analysis. Altogether, our results identify PnifH-driven sfYFP and sfCFP as robust, broadly applicable reporters for legume-rhizobia symbiosis studies, while highlighting the need for optimized red fluorophores in some contexts. The integration of validated promoter-reporter constructs with state-of-the-art computational approaches provides a scalable framework for dissecting the spatial and competitive dynamics of plant-microbe mutualisms.

The symbiotic relationship between the nitrogen-fixing bacteria Frankia alni and the pioneer tree species Alnus glutinosa plays an important role for …

The senescence of alfalfa nodule severely affects symbiotic nitrogen fixation, whereas the detailed mechanism is yet to be revealed. Here, we report that the miR396b-MsGRF1c-MsGS2 pathway regulates a...

Les légumineuses forment des organes spécifiques sur leur système racinaire, les nodules fixateurs d'azote, grâce à une interaction symbiotique avec des bactéries du sol, collectivement appelées rhizobiums. Les rhizobiums induisent cependant non seulement la formation de ces nodules racinaires, mais modulent aussi l'architecture du système racinaire.     Dans une nouvelle étude publiée dans Current Biology par l'équipe SILEG de Florian Frugier, au sein de l’Institut des Sciences des Plantes de Paris-Saclay - IPS2 (CNRS/INRAE/UEVE/UPSaclay, Gif-sur-Yvette), les auteurs ont identifié chez la légumineuse modèle Medicago truncatula une nouvelle réponse symbiotique racinaire correspondant à une augmentation du diamètre du cylindre central en réponse à rhizobium. Cette réponse, observée également chez une autre légumineuse cultivée, le pois, se produit rapidement et localement après l'inoculation des rhizobiums, entraînant une augmentation du nombre de cellules vasculaires. De manière intéressante, cette réponse symbiotique d’augmentation du diamètre de la stèle racinaire implique les peptides de signalisation nommés « Tracheary Element Differentiation Inhibitory Factor » (TDIF), et notamment le gène MtCLE37 codant pour un peptide TDIF, dont l'expression est induite durant la nodulation, et qui a donc été nommé « TDIF lié à la nodulation symbiotique » (sTDIF). En effet, un mutant cle37/stdif ne répond pas aux rhizobiums en augmentant le diamètre du cylindre central racinaire, et présente un nombre réduit de nodules.     Des analyses transcriptomiques et métabolomiques combinées ont révélé que le mutant stdif présente un métabolisme primaire altéré, affectant notamment l'accumulation de glucides/sucres dans les racines et les nodules. De manière remarquable, un traitement exogène au saccharose ou au malate est capable de restaurer dans le mutant stdif la réponse symbiotique d’augmentation du diamètre du cylindre central induite par les rhizobiums. Ceci indique que cette dérégulation métabolique contribue donc à expliquer l'altération de la réponse symbiotique du mutant.     Au final, cette étude met en évidence une nouvelle fonction des peptides de signalisation TDIF chez les plantes légumineuses, qui, au-delà de la régulation du développement du cylindre central, modulent également l’adaptation du métabolisme primaire des racines nécessaire pour permettre le développement des nodules symbiotiques.   -> Contact: florian.frugier@cnrs.fr

Heat stress is a major limiting factor for soybean productivity worldwide. Recent studies have highlighted the critical role of the plant microbiome in enhancing plant resilience to heat stress. However, our understanding of the molecular and physiological mechanisms underlying root-microbiome interactions under heat stress remains limited. To elucidate the role of native soil microbes in the heat tolerance of soybean genotypes, we analyzed rhizosphere bacterial and fungal communities via 16S rRNA and ITS sequencing, and characterized root metabolites and anatomical traits in response to microbiome composition and heat stress. Soybean plants were grown under controlled conditions in either natural soil containing native microbiota or in microbiome-disturbed soil (via 3-hour autoclaving), under both optimal and elevated temperature regimes. Alpha and beta diversity analyses revealed significant microbial shifts between treatments. Distinct clustering of bacterial, fungal, and metabolite profiles was observed under high temperature and microbial disturbance. Nodule-forming bacteria such as Rhizobium and Janthinobacterium were markedly suppressed, and belowground traits exhibited sensitivity, with significantly reduced nodule numbers and nodulation efficiency under high temperature and soil microbial perturbation. Non-targeted root metabolomics identified 372 differentially accumulated metabolites. Integrative multi-omics analysis revealed associations between metagenomic profiles, metabolite levels, and nitrogen-fixation traits, implying a coordinated modulation of root physiological processes. These findings contribute to a growing understanding of how heat stress interacts with rhizosphere microbial communities and may support future efforts in breeding climate-resilient soybean cultivars.

Symbiotic nitrogen fixation in legumes, driven by the interaction between rhizobia and host plants, provides essential nitrogen for plant growth but demands substantial energy. Sucrose, the principal product of photosynthesis, is critical in supporting this process. Despite its importance, the mechanisms underlying sucrose allocation following rhizobia inoculation remain poorly understood. Here, we identified and characterized GmSWEET3c, a rhizobia-induced sucrose transporter that is critical for sucrose allocation to the root susceptible zone. Functional analysis of the Gmsweet3c mutant revealed impaired sucrose allocation and a significant reduction in nodule formation, underscoring its critical role in symbiotic nodulation. Using a GmSWEET3c-GFP fusion protein, we found that the protein is located in both the plasma membrane of root cells and the membranes of infection threads, suggesting dual roles of GmSWEET3c in facilitating sucrose transport to the root susceptible zone and directing sucrose toward infection threads. Moreover, we demonstrated that GmNSP1, a key symbiotic transcription factor, directly binds to the promoter region of GmSWEET3c, activating its expression. Collectively, our findings highlight GmSWEET3c as a key mediator of sucrose distribution in soybean roots after rhizobia inoculation, enhancing our understanding of carbohydrate allocation in legume-rhizobia symbioses.

The functional divergence of GmBTSa in legumes supports iron availability through the activation of NSP–NIN, essential for nodulation.

Teyssendier de la Serve et al. identify a novel symbiotic response induced by nitrogen-fixing rhizobia on host plant roots, corresponding to a stele diameter enlargement. This response requires a TDIF...

Legume plants form specific organs on their root system, the nitrogen-fixing nodules, thanks to a symbiotic interaction with soil bacteria collectively named rhizobia. Rhizobia however do not only induce the formation of these nodule organs, but also modulate root system architecture. In a new study published in Current Biology by the F. Frugier SILEG team, we identified in the Medicago truncatula model legume a previously unnoticed increase of the root stele diameter occurring upon rhizobium inoculation. This symbiotic root response, similarly observed in another crop legume, pea, occurs rapidly and locally after rhizobium inoculation, leading to an increased number of vascular cells. Interestingly, this root stele diameter symbiotic response requires Tracheary Element Differentiation Inhibitory Factor (TDIF) signaling peptides, and notably the MtCLE37 TDIF-encoding gene which expression is increased during nodulation, thus being referred to as symbiotic nodulation TDIF (sTDIF). Indeed, a cle37/stdif mutant is not responsive to rhizobium regarding its root stele diameter increase, and has a reduced nodule number. Combined transcriptomic and metabolomic analyses revealed that stdif has a defective primary metabolism, notably affecting carbohydrate/sugar accumulation in both roots and nodules. Remarkably, a sucrose or a malate exogenous treatment is able to rescue the rhizobium-induced stele diameter symbiotic response in stdif. This metabolic deregulation is thus instrumental in explaining the altered symbiotic response of the mutant. Overall, this study highlights a novel function of TDIF signaling peptides in legumes plants, which beyond regulating stele development, also modulate the root primary metabolism adaptations required for symbiotic nodule development. Contact: florian.frugier@cnrs.fr

A gene identified more than 30 years ago has now revealed its role as a natural microRNA (miRNA) sponge to fine-tune the legume nodulation pathway, thanks to an international collaboration led by Wageningen University and the Sainsbury Laboratory, Cambridge University.

A gene identified more than 30 years ago has now revealed its role as a natural microRNA (miRNA) sponge to fine-tune the legume nodulation pathway. Researchers have discovered that ENOD40 sequesters a...