The symbiotic relationship between the legume Medicago sativa and the soil bacteria Sinorhizobium meliloti results in the formation of nitrogen-fixing root nodules. Traditional destructive methods, including paraffin sectioning, vibratome sectioning, and cryosectioning, have been applied to visualize how bacteria occupy the nodule, making it extremely difficult to obtain reliable three-dimensional information. These approaches are often combined with fluorescent labeling or staining, which can introduce additional stress affecting plant growth and nodule formation. MicroCT has emerged as a relatively quick, easy, and robust tool for plant biology that can non-destructively visualize plant histological features in three dimensions (3D), thereby avoiding destructive artifacts during sample preparation and ensuring high-fidelity 3D reconstruction. While microCT has been applied to legume root nodules, a detailed established protocol that documents the process from plant harvest and sample preparation to scanning and software visualization is lacking. In this study, we show a step-by-step microCT workflow using Medicago sativa as a model. The protocol includes nodule excision from roots, fixation, contrast enhancement, mounting, scanning, and three-dimensional reconstruction. Critical parameters affecting elements such as image quality, tissue preservation, and contrast are highlighted. Using this approach, it is possible to visualize the overall tissue organization, bacteroid-infected cells, and vascular bundles in three dimensions without physically sectioning the nodules. The pipeline described here provides a reproducible method for non-destructive, high-resolution imaging of native root nodules and is likely adaptable to other legume species, offering researchers a practical tool for studying nodule structure and bacterial organization within nodules in 3D.

Symbiotic nitrogen fixation reduces reliance on synthetic fertilizers and is central to agriculture and ecosystem functioning. In legumes, this process occurs in root nodules where rhizobia differentiate into bacteroids that either remain viable or undergo terminal differentiation, a strategy that can enhance nitrogen fixation but limits nodule life span. This irreversible program suits annual legumes and has evolved convergently across multiple lineages, including the inverted repeat-lacking clade (IRLC), where it is enforced by nodule-specific cysteine-rich (NCR) peptides. By contrast, perennial legumes with indeterminate nodules must sustain symbiosis over extended periods, which is incompatible with terminally differentiated bacteroids. Here, we identify a family of nodule-specific proline-glycine-rich peptides (NPGs) in Robinia that are induced upon rhizobial infection. NPGs are highly expressed in nodules, encode intrinsically disordered peptides, and accumulate in infected cells within the fixation zone. Exposure of Mesorhizobium robiniae to recombinant NPGs induces transcriptional changes associated with a fixation-related physiological state while preserving bacterial viability. These findings identify NPGs as candidate host effectors at the plant-microbe interface and point to an alternative mode of symbiont modulation in perennial legumes.

In Medicago littoralis R-108, Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (MtSymCRK) is required to prevent defense reactions in symbiotic nodules during chronic infection. MtSymCRK represents a key legume symbiotic gene controlling nodule immunity and early bacteroid survival, but its mode of action in the cell signaling remains poorly understood. This study investigated proteins interacting with MtSymCRK by screening an Arabidopsis thaliana open reading frame (ORF) library using yeast-two-hybrid followed by translational genetics towards R-108. Finally, we conducted a reverse genetic approach in R-108 using Transposon of Nicotiana tabacum 1 (Tnt1) insertional mutants. We identified a 14-3-3 protein, AtGENERAL REGULATORY FACTOR 8 (AtGRF8) interacting with MtSymCRK kinase domain (MtSymCRK[KIN]) and confirmed the interaction between MtSymCRK[KIN] and MtGRF8, the closest homolog of AtGRF8 in R-108. Moreover, other members of the 14-3-3 family interact with MtSymCRK[KIN] but with a lower affinity than MtGRF8. We also demonstrated that the corresponding Tnt1 mutant line Mtgrf8 displays symbiotic dysfunctions in defense and senescence responses, resulting in a reduction of bacteroid differentiation. These dysfunctions compromise the intracellular survival of the rhizobia, thereby impairing the nitrogen fixation of rhizobia. Our findings show that MtGRF8 and MtSymCRK are required for effective functioning of symbiosis and prevention of bacteroid death.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease typically managed with broad-spectrum immunosuppressants that carry significant systemic side effects and often provide incomplete efficacy. While gut-microbiota-derived metabolites are known to influence AIH progression, the specific microbial drivers that maintain hepatic immune homeostasis remain poorly defined. Here, we show that Bacteroides acidifaciens (BA) and its metabolite 1-oleoyl-sn-glycero-3-phosphoethanolamine (O-LysoPE) are enriched in self-healing mouse models of hepatitis but markedly depleted in AIH patients. We demonstrate that O-LysoPE induces a 'hepatocyte-driven active immunosuppression' by targeting the Qa-1b (HLA-E): NKG2A immune checkpoint. Mechanistically, O-LysoPE selectively redirects the transcription factor Creb1 to the H2T23 promoter under inflammatory conditions, thereby upregulating hepatocytic Qa-1b expression. This elevation of Qa-1b engages the inhibitory receptor NKG2A on T cells, suppressing their overactivation and restoring a quiescent phenotype. Genetic disruption of H2T23 abrogates the hepatoprotective effects of O-LysoPE, confirming the central role of this metabolic-immune axis. Our findings reveal that the BA-O-LysoPE axis mobilizes the liver's intrinsic self-rescue mechanisms to restore immune quiescence. This study establishes a robust biological framework for liver-specific, targeted immunotherapy in AIH, offering a precision alternative to current systemic immunosuppression.

This article traces more than four decades of Eva Kondorosi's personal life and scientific journey in symbiotic nitrogen fixation, from early insights into nitrogenase structure to the molecular mechanisms governing root nodule development and symbiotic cell differentiation in the Medicago-Sinorhizobium meliloti symbiosis. Effective symbiosis depends on precise molecular communication between the partners, beginning in the soil and continuing through a highly coordinated, progressive differentiation program. A defining feature of symbiotic cell development is endoreduplication in both host plant cells and their Rhizobium partners. Host-induced bacterial endoreduplication results in the formation of large, polyploid, noncultivable nitrogen-fixing bacteroids. This terminal differentiation is orchestrated by plant-derived effector peptides, notably nodule-specific cysteine-rich (NCR) and nodulin glycine-rich (nodGRP) peptides, which act sequentially to reprogram bacterial physiology. Together, these findings establish symbiotic nitrogen fixation as a model for cross-kingdom cellular differentiation and highlight NCR and nodGRP peptides as a vast, largely unexplored resource with promising applications in agriculture and medicine.

Symbiotic nitrogen fixation (SNF) can provide a sustainable and self-sufficient nitrogen (N) source for plants. Since its discovery, SNF has remained a central focus of both breeders and fundamental researchers. For decades, extending the utility of SNF to broader agricultural systems has been considered a promising strategy to reduce reliance on synthetic N fertilizers, thereby lowering production costs and mitigating environmental pollution caused by N overuse. This review summarizes recent advances in understanding the molecular and regulatory mechanisms governing SNF in legume plants and highlights emerging strategies to optimize and extend its application in agricultural systems. Particular emphasis is placed on approaches that aim to achieve dominant, fine-tuned, and controllable regulation of N fixation to support sustainable crop production.

Legumes sanction root nodules containing rhizobial strains with low nitrogen fixation rates (less effectively fixing). Pea (Pisum sativum) nodules contain both undifferentiated bacteria and terminally differentiated nitrogen-fixing bacteroids. It is critical to understand how sanctions act on both bacteria and bacteroids, and how they differ. In addition, less effective strains could potentially evade sanctioning by entering the same nodule as an effective strain (i.e. piggybacking). Pisum sativum was co-inoculated with pairwise combinations of three strains of rhizobia with different effectiveness, to test whether ineffective strains can evade sanctions in this way. We assessed the effect of sanctions on nodule populations of bacteria and bacteroids using flow cytometry and the effects on nodule internal structure using confocal microscopy. We show that sanctioning lowered bacteroid populations and caused a reduction in the size of bacteria. Sanctions also precipitated an early change in nodule cell morphology. In nodules containing two strains that differed in their nitrogen-fixation ability, both were treated equally. Thus, peas sanction whole nodules based on their nitrogen output, but do not sanction at the cellular level. Our results demonstrate that peas conditionally sanction at the whole-nodule level, providing stability to the symbiosis by reducing the fitness of ineffective strains, but cannot target individual strains in a mixed nodule.

Bacterial ribonucleases (RNases) are central components of post-transcriptional networks underlying environmental adaptation. However, their contribution to the ecological specialization of bacteria with complex lifestyles, such as nitrogen-fixing legume symbionts, remains poorly understood. Here, we investigated the role of the double-stranded RNase III ortholog (SmRNase III) in Sinorhizobium meliloti, the symbiotic partner of alfalfa (Medicago sativa L.). Loss of SmRNase III function affected the expression of nearly 30% of protein-coding genes and 12% of annotated non-coding RNAs (sRNAs). Remarkably, more than 70% of these changes occurred under the microaerobic conditions typical of symbiotic nodules. Many SmRNase III-dependent transcripts encode pathways supporting microaerobic metabolism and nitrogen fixation in endosymbiotic bacteroids. Analysis of sequencing read coverage identified putative consensus cleavage signatures enriched in mRNA 5' untranslated regions, suggesting preferential processing at these sites. Altered expression of sRNAs and/or their predicted mRNA targets further supports a role for SmRNase III in sRNA-mediated silencing. Consistently, in vitro assays showed that base‑pairing between nifK (encoding the β‑subunit of the nitrogenase MoFe protein) and the antisense RNA asNifK promotes SmRNase III-mediated cleavage. In vivo assays further supported that silencing of nifK and dctA (encoding a major dicarboxylate transporter) requires SmRNase III, with dctA regulation involving a base‑pairing interaction between the trans‑sRNA AbcR1 and a predicted SmRNase III cleavage site within the mRNA. Our findings reveal a major impact of SmRNase III on shaping the symbiotic transcriptome of S. meliloti and provide a foundation for deeper investigation into RNase III-mediated regulation in rhizobia.

Plant roots form symbioses with beneficial microorganisms to enhance nutrient acquisition. Most terrestrial plants form arbuscular mycorrhizal symbiosis (AMS) with obligate biotrophic Glomeromycotina fungi, which supply hosts with mineral nutrients in exchange for carbon through specialized symbiotic hyphal structures (arbuscules) that develop within root cortex cells. Legumes form root nodule symbiosis (RNS) with nitrogen-fixing rhizobia, which are housed as differentiated bacteroids within specialized symbiotic organs (nodules) and provide plants with ammonia in return for carbon. RNS exhibits high partner specificity, occurring only between compatible hosts and microbes. Conversely, AMS is less specific, although symbiosis outcomes are context-dependent and influenced by host and fungal genotype, environmental conditions, and microbial competition. In both cases, plants favor high-performing microsymbionts by recognizing them during symbiosis initiation or by punishing low-performing symbionts through postcolonization sanctions. Microbes, in turn, employ strategies to manipulate plants for their own benefit. Here, we review the molecular mechanisms underlying partner preference in beneficial plant-microbe interactions and discuss how host partner selection strategies maintain mutualistic stability in AMS and RNS, alongside microbial strategies to evade host control. Understanding the dynamic interplay of functionally diverse plant-microbe symbioses provides a basis for improving mutualisms in both natural and agricultural systems.

With the aim to elucidate the interdependence between potassium transport by the host plant in nodule cells and potassium transport in bacteroids, a null mutant of rhizobial potassium ion transporter Smkup1 was created and investigated. The mutation, according to cytological analysis, has not caused specific aberrations in the root nodules' anatomy and ultrastructure, but a significant induction of the expression of host plant and rhizobial genes involved in the stress response was observed. At the same time, an opposite trend was observed for genes of the autophagy pathway that have shown a significant downregulation of expression. To identify the mechanisms of interplay between autophagy and senescence in the root nodule, an in silico analysis of protein-protein interactions of positive (Beclin 1) and negative (NAC1, BAK1) regulators of autophagy was performed. The resulting networks allowed the predictions of interacting proteins putatively linking symbiotic interactions, autophagy, stress, programmed cell death (PCD), and senescence. Based on these data, we hypothesized that modulation of the expression of these genes in the root nodule could be the way to extend the root nodule's lifespan and the duration of the nitrogen fixation process.

The root nodule symbiosis between legumes and nitrogen-fixing bacteria (NFB) acts as an important nitrogen source in terrestrial ecosystems. NFB in soil are affected by top-down predation in the food web. However, how protist predation affects abundant and rare sub-communities of NFB remains virtually unknown, limiting the exploitation of soil food webs to promote plant productivity. Here, a 10-yr field experiment combined with a glasshouse experiment was conducted to explore the effects of protist predation on abundant and rare NFB under organic material amendments. Our results revealed that organic material amendments increased the diversity of rare NFB and phagotrophic protists, but decreased the relative abundance of abundant NFB Correlation analysis combined with the glasshouse experiment suggested that protist predation decreased the relative abundance of NFB abundant taxa, but increased the diversity of rare taxa, which further promoted the cytokinin content and decreased the ethylene content in peanut (Arachis hypogaea L.) roots. Subsequent changes in plant hormones regulated the expression of genes involved in rhizobial infection, nodule organogenesis, and bacteroid differentiation, thereby promoting nodulation and increasing peanut yield. Overall, our findings provide unique insights into the interactions between phagotrophic protists and NFB, highlighting their links with plant productivity via predation-stimulated symbiotic nitrogen fixation.

Chronic alcohol consumption disrupts the gut microbiome, exacerbating alcohol-induced cognitive and social dysfunction (AICSD), which constitutes a primary etiology of early-onset dementia. Urolithin A (UA) has been well-reported as an effective intervention for neurodegenerative diseases. However, the protective efficacy of UA against AICSD, and its underlying mechanisms remain largely elusive. First, our study demonstrates that UA significantly enhances work memory (60.43%), short-term memory (12-fold), long-term memory (50.32%), social ability (10-fold), and social novelty (12-fold), while concurrently reducing synaptic impairments and neuroinflammation. Moreover, UA restores AICSD by upregulating the dopamine D2 receptor (DRD2) via RAP1 signaling. Furthermore, antibiotic treatment and fecal microbiota transplantation experiments confirm the causality between the host microbiota and behavioral alterations. Treatment with UA-enriched Bacteroids sartorii and Parabacteroids distasonis, or their derived endocannabinoid-anandamide (AEA), also ameliorates AICSD. Finally, AEA inhibits the Rap1 signaling through cannabinoid receptor 1 (CB1R) and DRD2 interaction, eventually ameliorating AICSD. Collectively, our study elucidates that microbiota-derived AEA mediates the therapeutic effects of UA on AICSD through the CB1R-DRD2-RAP1 signaling axis, providing valuable insights for UA and microbiome-targeted endocannabinoid interventions against AICSD.