Reprogramming, not silencing: How plant immunity is gated during legume nodulation 

Legume–Rhizobium symbiosis requires rhizobial entry and intracellular persistence without triggering host defense. Recent genetic and biochemical studies support an immune-gating view in which plant immunity pathways are not simply suppressed, but are dynamically regulated throughout rhizobial infection, nodule development and nodule senescence. Here, we review how LysM/LRR receptor complexes and co-receptor availability shape early discrimination of symbiotic signals and immune elicitors and how shared signaling modules (Ca2+, ROS and MAPKs) are differentially regulated to permit infection and nodule organogenesis. We then discuss rhizobial surface polysaccharides and type III-secreted effectors as modulators of these thresholds and highlight nodule-local regulators that prevent late immune reactivation and maintain bacteroid accommodation. We also discuss nodule-specific, regulator autophagy-associated processes and, in some legumes, NCR peptides that maintain bacteroids within symbiosomes while preventing plant overactivation of immunity.

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Use of translational fusions to express functional Klebsiella oxytoca dinitrogenase reductase in plant mitochondria

Engineering crop plants with the biological nitrogen fixation pathway is a longstanding goal of modern agriculture. Dinitrogenase reductase (NifH) is a critical component of the biological nitrogen fixation pathway, with multiple roles in metal cofactor assembly and catalysis. This enzyme must be folded correctly as a soluble homodimer and loaded with the [4Fe-4S] metallocluster for function. Previous studies have found that Klebsiella oxytoca (Ko) and Azotobacter vinelandii (Av) NifHs were mostly insoluble when targeted to plant mitochondria. Here we found that a translational fusion of two KoNifH or AvNifH monomers, forming KoNifHH or AvNifHH synthetic dimers, produced a soluble protein when targeted to plant mitochondria and co-expressed with the putative peptidyl-prolyl cis–trans isomerase NifM. KoNifHH isolated after expression in leaf mitochondria at ambient oxygen showed some acetylene reduction activity, which did not require co-expression of the nitrogenase-specific metallocluster machinery NifS and NifU. This activity increased after iron-sulfur cluster reconstitution in vitro with recombinant NifU. In a parallel study, we tested a translational fusion of a variant iron-only dinitrogenase reductase (AvAnfHv6) monomer that was soluble but not active as-isolated from plant mitochondria (AvAnfHHv6) (Gregg et al. 2025a). AvAnfHHv6 was abundant and fully soluble when isolated from plant mitochondria, like its monomer. AvAnfHHv6 was not active as-isolated but could be largely activated by iron-sulfur cluster reconstitution in vitro. This study demonstrates how translational fusions help improve solubility and have the potential to generate an active NifH enzyme within plant mitochondria.

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Microbial inoculation in green roofs: progress, challenges and ecological regulation strategies

As a core component of urban green infrastructure, green roofs play a crucial role in mitigating urban heat island effects, managing stormwater runoff and conserving biodiversity. However, substrates in green roofs are generally shallow, nutrient-poor, and subject to severe water stress, which significantly limits plant growth and the delivery of ecosystem functions. Soil microbes, especially arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR), play key roles in promoting plant growth, enhancing plant stress resistance, and improving substrate quality. This paper systematically reviews the research progress on microbial inoculation in green roofs, focusing on (1) the main types and mechanisms of AMF and PGPR, (2) the regulatory effects of environmental factors such as vegetation type, substrate characteristics, and roof age on plant-microbe interactions, and (3) the enhancement effects of microbial inoculation on plant growth, stormwater management, heat island mitigation, biodiversity conservation and biological carbon sequestration. Studies indicate that AMF significantly improve plant drought tolerance and nutrient uptake efficiency by expanding root absorption ranges and improving the rhizosphere environment; PGPR promote plant growth and stress resistance through multiple mechanisms including phytohormone production, nitrogen fixation, phosphate solubilization, and ACC deaminase activity. And the combined inoculation of appropriate AMF and PGPR can produce synergistic effects. However, the current research is confronted with challenges such as unstable inoculation effects, unclear long-term effects, inconsistent methodology, insufficient mechanistic understanding and absence of cost-benefit analyses. These efforts will facilitate the transformation of microbial inoculation technology from laboratory research to large-scale engineering applications, providing theoretical support and technical pathways for constructing resilient and self-sustaining green roof ecosystems.

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Metabolic modeling predicts synergistic growth benefits between arbuscular mycorrhizal fungi and theoretical N2-fixing rhizobia symbiosis in maize 

Engineering a novel N2-fixing rhizobia symbiosis in cereal crops is a strategy being pursued to improve agricultural sustainability. However, if such a symbiosis were introduced, it would have to be economically viable in the context of plants’ existing nutrient acquisition strategies, including existing symbioses with arbuscular mycorrhizal fungi (AMF) that most plants already engage in. It is important to understand how the metabolic costs and benefits from these symbioses with overlapping functions might impact plant growth when evaluating the potential benefits of this engineering strategy. To address this, we developed metabolic models describing how the relative growth rate of Zea mays is impacted by the AMF Rhizophagus irregularis and a hypothetical N2-fixing symbiosis with Bradyrhizobium diazoefficiens in isolation and in tandem. The metabolic models of the plant-AMF symbiosis and plant-AMF-rhizobia symbiosis are the first of their kind. To validate the AMF component of our model, we conducted a field evaluation comparing AMF-compatible and mutant AMF-incompatible maize hybrids. The empirically measured AMF-mediated growth benefit agreed well with model predictions. Our model of the rhizobium symbiosis predicted that the lower N content of cereal crops makes the growth penalty associated with acquiring nitrogen from rhizobia smaller than in legumes. Finally, the model of the plant-AMF-rhizobia symbiosis predicted positive synergies between rhizobia and AMF under nutrient-limited conditions but negative synergies under phosphorus-replete conditions. This indicates that these bioengineering strategies could improve cereal crop yields and may achieve greater gains in tandem, but soil nutrient levels and plant nitrogen requirements should be considered.

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Co-cultivation of diazotrophic bacteria enhances auxin-mediated root development in maize: implications for microbial inoculants

This study assessed the co-cultivation of Gluconacetobacter diazotrophicus and Paraburkholderia tropica, two diazotrophic species previously used in sugarcane, to evaluate their compatibility, phytohormone profiles, and effects on maize (Zea mays) root system architecture. Both strains were grown separately and together, followed by metabolomic analyses and plant bioassays under controlled and greenhouse conditions. The co-cultivation maintained stable populations and produced a wider range of phytohormones than individual cultures, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 4-Cl-IAA, gibberellins, cytokinins, and abscisic acid, with many of these compounds released into the culture medium. Short-term bioassays showed that auxins, particularly IAA, were the only phytohormones produced at concentrations high enough to cause quick and consistent changes in root length and surface area, with IAA levels increasing from 14.07 to 15.30 ng mL⁻¹ in monocultures to 30.68 ng mL⁻¹ under co-cultivation, indicating that auxin-driven signaling is the main mechanism behind early root responses. These findings suggest that co-cultivating compatible diazotrophic bacteria is a viable and practical strategy for developing advanced microbial inoculants to enhance maize root architecture and support sustainable crop production.

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Influence of climate and soil nutrients on the evolutionary abandonment of the arbuscular mycorrhizal symbiosis

The mutualistic symbiosis between plants and arbuscular mycorrhizal (AM) fungi is widespread in plants but has been abandoned several times. Abandonment is a two-step evolutionary process that proceeds from the AM state to full abandonment (a non-mycorrhizal (NM) state) via an intermediate AMNM state in which the presence of AM fungi in plants is dependent on ecological context. We tested the hypothesis that the evolution of AM fungal symbiosis abandonment is caused by colder and drier climates as well as elevated soil nutrients, which reduce the benefit-to-cost ratio of the symbiosis from the plant perspective. Using phylogenetic logistic multiple regression and georeferenced environmental data for more than 4000 species, we found that colder temperature was the strongest predictor of evolutionary transitions from the AM to AMNM states, but higher inorganic soil P was the strongest predictor of the evolution of full symbiosis abandonment from the AMNM to NM states. Our findings are consistent with observations that many NM clades evolved during the past 50 million years, as global temperature dropped during the Cenozoic. Furthermore, increased phosphorus deposition from anthropogenic sources could cause the evolution of symbiosis abandonment, particularly in lineages that are in the intermediate AMNM state.

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Quantifying soybean biological nitrogen fixation using long-term isotope enrichment 

Background and aims Biological nitrogen fixation (BNF) supplies much of soybean (Glycine max (L.) Merr.) nitrogen (N) demand, but reported fixation rates vary widely, leading to uncertainty in the soybean N cycle. We quantified whole-plant soybean BNF and N allocation using long-term 15N labeling to assess whether BNF can offset grain N removal in a high-yielding system, and to quantify root contributions to fixed N. Methods Field mesocosms in Iowa, USA received three legacy fertilizer N rates as 2 atom% 15N-enriched urea for four prior seasons of maize (Zea mays L.) production. In 2024, N-fixing and non-N-fixing soybeans were grown to physiological maturity. Grain, aboveground biomass, and roots were analyzed to quantify BNF and to estimate whole-plant N budget. Results Soybean derived 81.8% ± 1% (SE) of total plant N from BNF. Legacy N rate had no effect on whole-plant N accumulation, BNF, or N partitioning among tissues (p > 0.05). Roots contained ~2% of total fixed N, with most BNF-derived N allocated to grain. High grain N removal was largely offset by BNF (174.7 ± 8.9 kg N ha−1), resulting in a near-neutral partial N balance (−5.7 ± 2 kg N ha⁻1). Conclusion From this one-season mesocosm study, our finding of negligible fixed N in soybean roots indicates that neglecting root BNF does not necessarily bias conclusions about soybean N cycle. However, the combination of long-term isotope labeling and non-nodulating isolines yielded much less variable and higher BNF rates than previous studies, suggesting the possibility of underestimation for soybean BNF in historical studies.

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Structural modelling uncovers diverse predicted transcriptional and post-transcriptional modulators among type III effectors of symbiotic Rhizobia | bioRxiv

Rhizobia are soil bacteria that establish nitrogen-fixing symbioses with legumes. While many rhizobia use a Type III Secretion System to deliver “Nodulation Outer Protein” (Nop) effectors, some uniquely use these proteins to initiate nodule organogenesis, bypassing classical signalling. The molecular functions of these effectors remain largely unknown due to extreme sequence divergence. Using AlphaFold2-mediated structural proteomics, we identified a modular architecture in rhizobial effectors composed of 22 distinct structural units. We reveal that many Nop effectors are cryptic transcriptional or post-transcriptional regulators, harbouring unrecognised nucleic acid–binding modules and RNA-dependent RNA polymerase domains. Crucially, these modules are conserved in specific plant pathogens, such as gall-inducing Pantoea, where our predicted structural units align with experimentally validated DNA-binding domains. Furthermore, we discovered the BPN (B3 and PUA-like nucleic acid binding) domain as a structural mimic of plant B3-domain transcription factors, pointing to a direct mechanism for hijacking legume development. Our findings strongly suggest that rhizobia employ a modular domain-fusion strategy to act as direct genetic modulators, uncovering a conserved mechanism used by both symbionts and pathogens to hijack host developmental programmes.

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Regulating arbuscular mycorrhizal permissiveness

Arbuscular mycorrhizal fungi (AMFs) have facilitated the colonization of land by plants some 470 million years ago. The vast majority of land plants have maintained a symbiotic association with these fungi to facilitate the uptake of mineral nutrients, such as phosphorus, at the cost of photosynthates delivered to the fungus in the form of lipids and sugars. Despite their importance for plant nutrient status, plants can refuse AMF if soil nutrient conditions are such that it is less costly for the plants to take up the nutrients by themselves or if environmental conditions are not appropriate. Recently, Hong et al. (2026) revealed how multiple signaling pathways in rice converge on a transcription factor complex involving the GRAS transcription factors NSP1 and NSP2 to control AM colonization. In this Spotlight, we highlight recent insights into the molecular mechanisms that control the permissiveness of plants to allow AMF entry into their roots (Figure 1).

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A rhizobial extracellular vesicle-conveyed auxin transporter mediates phytohormone mobilization and optimizes rhizobium-legume symbiosis | bioRxiv

Bacterial extracellular vesicles (bEVs) are emerging as key players in interkingdom communication, yet their role in delivering functional proteins to host cells during symbiosis remains unexplored. This study shows that Sinorhizobium fredii HH103 packages a PIN-like auxin transporter, AuxT, into bEVs that traffic within the peribacteroid space of soybean nodules. AuxT is chromosomally encoded and constitutively expressed, genetically uncoupled from the flavonoid-inducible auxin biosynthesis machinery located on the symbiotic plasmid. Structural prediction reveals that AuxT adopts an eight-transmembrane-helix architecture with striking homology to plant PIN auxin exporters, despite negligible sequence identity. Molecular docking demonstrates that AuxT binds indole-3-acetic acid within a central cavity, with dimerization inducing ligand-specific conformational changes consistent with transport activity. The auxT mutant exhibits significant symbiotic defects including reduced shoot biomass, nodule number, and nodule mass that are fully restored by complementation. Critically, AuxT-enriched bEVs contain elevated auxin levels, and nodules colonized by the complemented strain accumulate more auxin specifically within the bEVs peribacteroid space, while bacteroids themselves show no auxin retention. We postulate that bEV-associated AuxT mediates localized auxin export into the symbiosome, modulating the host hormonal environment to optimize symbiotic development. This work reveals a previously unrecognized mechanism of interdomain hormonal modulation, where a bacterium uses a structurally convergent transporter and vesicular delivery to actively shape host physiology and to improve the symbiotic performance.

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Nanoplastics Pollution Threatens Sustainable Nitrogen Fixation in Agroecosystems by Disrupting Legume–Rhizobium Symbiosis

The rhizobium–legume symbiosis plays a vital role in the global nitrogen cycle. Although microplastics have been shown to affect this symbiotic system, the accumulation and impacts of nanoplastics (NPs) in rhizobia and their root nodules remain poorly understood, particularly regarding the interactive effects of NPs of different sizes on symbiotic nitrogen fixation. This study demonstrated that polystyrene (PS) NPs exhibited a significant size difference effect on rhizobia and their symbiotic nitrogen-fixing association with soybean (Glycine max). We found that both rhizobia and soybean nodules efficiently internalized PS NPs, with differently sized NPs showing mutual enhancement during the cellular uptake of rhizobia. 100 mg/kg of 20 nm PS NPs severely disrupted the symbiotic nitrogen fixation, reducing nitrogenase activity by 51.3% in single exposures and 28.6% in combined exposure to 200 nm PS NPs. This observed disruption caused by 20 nm PS NPs was associated with suppressed nodule formation (26.0% reduction in number, 50.4% decrease in fresh biomass), diminished leghemoglobin content (64.9% reduction), impaired nutrient acquisition (26.5% decrease in nodule Mo content), reduced rhizobia infection efficiency, impaired plant growth, and modified expression of nodulation- and nitrogen-fixation-related genes. These findings revealed that small-sized PS NPs posed a substantial threat to the rhizobium–legume symbiosis, underscoring the ecological risks of NP pollution in agricultural systems.

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Nitrate restricts nonsymbiotic leghemoglobin expression via inhibiting nodule inception proteins in nodules of Arachis hypogaea

An exquisite symbiotic relationship between legumes and rhizobia leads to the development of nitrogen-fixing specialized organs, known as nodules, in nitrate-deficient environments. By contrast, a high level of soil nitrate negatively regulates the pleiotropic phases of root nodule symbiosis (RNS), including rhizobial infection, nodule organogenesis, and leghemoglobin synthesis. Here, we identified a special group of nodule-specific nonsymbiotic leghemoglobin genes (AhLghs) in the crack-entry legume peanut and investigated their functional role and transcriptional regulation. A comparative transcriptomic analysis revealed that the downregulation of nodule inception (AhNIN) and nonsymbiotic leghemoglobin (AhLghs) genes plays a pivotal role in the nitrate-mediated inhibition of RNS in peanut. Knockdown of AhLghs and overexpression of AhLgh1 resulted in lower and higher leghemoglobin content, respectively, corroborating their roles as positive regulators of nitrogen fixation. Knockdown of AhNINs not only inhibited root nodulation but also decreased leghemoglobin content in peanut. Further, DNA-affinity purification sequencing (DAP-seq) analysis identified various nodulation genes, including AhLghs, as targets of AhNINs. Following the validation of DNA–protein interactions via electrophoretic mobility shift assay, transactivation assays revealed that AhNINs positively regulate AhLgh1 after binding to the NIN RESPONSIVE CIS ELEMENT (NRCE) of its promoter. Our work bridges a critical gap in understanding how nitrate influences nonsymbiotic leghemoglobin expression by targeting rhizobia-induced NINs in peanut and offers a potential model suggesting that the nitrate-NIN-Lgh module might represent a key evolutionary event in fine-tuning root nodulation.

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Bacteriology is Hiring!

Bioeconomy Assistant Professor The Department of Bacteriology is seeking applicants at the Assistant Professor level to launch a research program in the New Bioeconomy. Applicants may be interested in...

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Structural and Functional Characterization of Heterologous Nitrogenase Complexes

Nitrogenase is the only known enzyme that catalyzes the reduction of dinitrogen to ammonia. The most prevalent isozyme, molybdenum nitrogenase, comprises the catalytic molybdenum–iron protein (MoFeP) and the ATP-dependent reductase iron protein (FeP). Although Mo-nitrogenases are widespread across bacteria and archaea and appear to share conserved mechanistic and structural features, FeP and MoFeP show considerable sequence variability across diazotrophs. This raises questions about the conservation of chemomechanical mechanisms coupling FeP-dependent ATP hydrolysis and electron transfer to MoFeP, and about the functional compatibility of nitrogenase components from divergent species. Previous studies showed that some heterologous FeP−MoFeP pairs can functionally complement each other, whereas other pairs lack catalytic activity, but the absence of structural information on such heterologous pairs has limited mechanistic understanding. To this end, we investigated the functional and structural compatibility of FeP and MoFeP from Azotobacter vinelandii (Av) and Gluconacetobacter diazotrophicus (Gd), two phylogenetically and ecologically distinct species. Building on our prior work with Gd-nitrogenase and recently developed cryogenic electron microscopy (cryoEM) protocols, we determined the ADP·BeFx-trapped structure of the homologous GdFeP–GdMoFeP complex and showed that it adopted the same geometry as its Av counterpart. Activity measurements showed that heterologous Gd/Av combinations retained 60–80% of homologous catalytic activities despite 30–50% sequence divergence in FeP and MoFeP. High-resolution cryoEM structures of GdFeP–AvMoFeP and AvFeP–GdMoFeP corroborated these activities and revealed that functional complementation tolerates substantial sequence variation when the core structural elements supporting ATP binding/hydrolysis, protein–protein interaction, electron transfer, and substrate reduction are conserved.

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From manual scoring to machine learning: recent developments in image-based arbuscular mycorrhizal fungi root quantification 

Selecting an appropriate method to quantify arbuscular mycorrhizal fungi (AMF) colonization in plants can be challenging, particularly for non-experts, due to the wide diversity of available techniques, some of which date back to the 1980s. Despite rapid methodological advances, comprehensive reviews focusing specifically on recent image analysis approaches for AMF root quantification are still scarce in the scientific literature. Given the growing potential of image-based methods to improve the accuracy, reproducibility, and high throughput of AMF root colonization assessment, this review synthesizes conceptual structure and thematic map of studies focusing on AMF colonization from 2001 to 2026. In addition, we examine widely used traditional approaches, such as the gridline intersect method, and emerging tools, including Visual Basic for Application–Arbuscular Mycorrhizal Fungi (VBA-AMF), MycoPatt, WinRHIZO, ImageJ-based workflows, Zeiss Intellesis, and deep learning approaches such as Automatic Mycorrhizal Finder (AMFinder), “Tool for Analysing root images to calculate the Infection rate of arbuscular Mycorrhizal fungi” (TAIM) and Mask Region-based Convolutional Neural Network (Mask R-CNN). By critically comparing their principles, capabilities, and limitations, this review aims to guide researchers in selecting the most appropriate image analysis methods for AMF quantification according to their experimental objectives and technical constraints.

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Arbuscular mycorrhizal plasticity in cereals reflects quantitative rather than taxonomically selective regulation

Grasses, including major cereal crops, associate with arbuscular mycorrhizal (AM) fungi to varying degrees depending on environmental conditions. Understanding mechanisms driving this environment-induced variation in mycorrhization, i.e. plant AM plasticity, is necessary to predict grass-mycorrhizal responses to global change factors, such as nutrient enrichment, and to resolve the role of AM symbiosis in cereal crop production. We compared AM plasticity in four cereal crops by testing the effect of nitrogen (N) fertilization on AM colonization and root PLFA 16:1ω5 concentration. To assess whether mycorrhization patterns reflect root functional traits, we compared specific root length among species. To determine whether plants regulate AM colonization qualitatively (by selectively associating with certain AM taxa) or quantitatively (by collectively suppressing colonization across taxa), we investigated directional shifts and variability in AM community structure in response to N fertilization. AM colonization varied between cereal species and was reduced by N fertilization, but we found limited evidence for interspecific differences in AM plasticity. Winter wheat appeared less AM responsive and associated more with uncultured AM fungi compared to the three spring-sown cereal species, oat, spring wheat and spring barley. Fertilization did not affect AM community composition, and within-species variation in AM community β-dispersion did not covary with variation in AM colonization or PLFA 16:1ω5 concentration. These results support the view that host plants regulate arbuscular mycorrhization quantitatively rather than through taxonomic selectivity. We propose AM plasticity as a plant-mycorrhizal trait to be used for more accurate predictions of plant environmental responses in eco-physiological and agroecological research.

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Enhanced production of nitrogenase components in Nicotiana benthamiana through co-expression with Bacterioferritin A | bioRxiv

Engineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network To identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. BfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. Our results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems.

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Symbiotic peptides modulate rhizobial physiology without terminal differentiation

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.

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Nod factor signaling controlled genes in Medicago truncatula nodules | bioRxiv

Legume nodule formation is induced by rhizobia secreted Nod factors (NFs). It has been shown that NF receptors also accumulate in the apex of Medicago truncatula nodules. However, the NF signaling induced transcriptional changes in there have never been studied. Here, we studied this by using NF signaling mutant TE7, a weak allele of IPD3, blocked in rhizobial release. Nodule apices were isolated with laser microdissection and used for transcriptional analysis. We identified 1655 NF signaling controlled genes in nodule apex. By comparing this with the transcriptome data from VAMP721d&e RNAi nodule apices, we identified a subset of 445 genes whose expression depends on NF signaling and rhizobial release. Further, we compared the set of genes controlled by NF signaling in nodule apices with that controlled in root epidermis, and these showed only a small overlap. NIN is induced by NF signaling both in the root epidermis and in the nodule. By overexpression of NIN in TE7 and knock down of NIN in wildtype nodules we showed that NF signaling controlled rhizobial release depends on NIN. NF signaling controls a distinct set of genes in nodules, the function of which depends at least in part on NIN.

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Non-nodulating Paenirhizobium fix nitrogen in association with cereal roots

Cereal root microbiomes harbour diverse diazotrophic bacteria, yet the taxa capable of sustained nitrogen fixation in association with cereal roots remain poorly characterised. Here, two high-performing nitrogen-fixing strains (B6 and J2) were isolated from barley roots and identified as belonging to the family Rhizobiacae in the genus Paenirhizobium. Both strains possess plasmid-encoded canonical rhizobial nif and fix genes for nitrogen fixation but lack nodulation genes. Their genomes have a 5.7 Mb chromosome and four repABC plasmids. Unlike most nodulating rhizobia, strains B6 and J2 fixed nitrogen in laboratory culture on a range of carbon sources, achieving maximal activity on organic acids at low ammonium (<0.5 mM) and oxygen concentrations (1–3%). Both strains colonised the total root systems of barley plants, with population densities of 106 CFU g−1 fresh root weight. Strains fixed high levels of nitrogen on barley plants, similar to or greater than other known free-living diazotrophs. These findings expand the ecological context of rhizobial nitrogen fixation and identify cereal-associated Paenirhizobium as a previously unrecognised component of the diazotrophic cereal root microbiome.

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AI Models for Uncovering Plant–Microbiome Interactions

The plant microbiome (bacteria, fungi, archaea, and viruses) is critical for plant growth, health, and resilience to biotic and abiotic stresses. However, the complexity and dynamic nature of these i...

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My journey with fungi: the beauty and complexity of fungal natural products

Once viewed as niche curiosities, fungal natural products (NPs), or secondary metabolites, have become central to some of today's most dynamic areas of res

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Rhizobial auxin activates transcription factors to orchestrate YUC2-dependent auxin biosynthesis for soybean nodule development

Establishing the symbiosis between legumes and nitrogen-fixing rhizobia requires the precise modulation of auxin levels. However, our understanding of the regulatory roles of auxins, particularly rhizobia-derived auxins, remains limited. Our study reveals that the auxin biosynthesis gene YUC2a is essential for the spatiotemporal control of nodule development in soybean (Glycine max). This process is orchestrated by 3 transcription factors: Nuclear Factor-YA9 (NF-YA9), Lateral Organ Boundaries Domain 41 (LBD41), and Nodule Inception 1a (NIN1a). In the early stages of nodulation, rhizobial auxin stimulates NF-YA9 expression, NF-YA9 then activates YUC2a expression in the cortical cell layer, establishing optimal auxin levels for nodule initiation. In the middle stages, rhizobial auxin elevates LBD41 expression, and LBD41 suppresses YUC2a to control auxin levels, ensuring proper rhizobia colonization. In the late stages, rhizobial auxin inhibits NIN1a expression, which increases YUC2a expression in nitrogen-fixing symbiosomes, fine-tuning optimal auxin levels for nodule maturation. Disruption of YUC2a and its homologs impairs cell division in nodule primordia, reducing nodule density and nitrogen fixation capacity. Conversely, cortex-specific overexpression of YUC2a promotes nodule formation but inhibits rhizobia colonization. This dynamic auxin regulation optimizes nodule development in soybean, revealing rhizobia-derived auxin's critical role in nitrogen-fixing symbiosis.

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Mycorrhizal associations in pteridophytes: current perspectives and emerging research gaps 

Pteridophytes occupy a key evolutionary position in land plant evolution and provide a framework for understanding the early establishment and diversification of plant-fungus symbioses. Early anatomical observations identified arbuscule- and vesicle-like structures in ferns and lycophytes, providing some of the earliest evidence for arbuscular mycorrhizal associations in this lineage. Throughout much of the twentieth century, however, research mainly remained descriptive, focusing on colonization patterns and morphological features. With the advent of molecular phylogenetics and high-throughput sequencing, this perspective has significantly expanded. Later studies revealed that pteridophytes associate not only with Glomeromycotina but also with Mucoromycotina, fine root endophytes, and dark septate endophytes, indicating a more diverse and complex symbiotic network than previously thought. Nevertheless, a comprehensive functional understanding of these interactions remains limited. Molecular insights into pteridophyte–mycorrhizal symbiosis remain underdeveloped compared with those in angiosperm model systems. Important aspects such as symbiotic signaling pathways, gene expression dynamics, nutrient exchange mechanisms, and regulatory networks are poorly understood. Furthermore, the extent of fungal specificity across different life stages of pteridophytes, such as gametophyte and sporophyte, and across various evolutionary lineages, remains unclear. Integrative approaches combining phylogenomics, transcriptomics, metabolomics, and microbiome profiling are scarce. Addressing these gaps will enhance our understanding of the origins and evolution of symbiotic mechanisms in early vascular plants and support the use of these associations in biodiversity conservation and ecosystem restoration.

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Evolutionary diversification of the SymRK receptor family in land plants

Plant receptor-like kinases (RLKs) are involved in diverse processes, ranging from growth and reproduction to interactions with microbes. Variation in the extracellular domains delineates several RLKs subfamilies, including the malectin-like domain leucine-rich repeat receptor-like kinases (MLD-LRR-RLKs). Symbiosis Receptor-like Kinase (SymRK) is the prototypical member of MLD-LRR-RLKs and is required for microbial accommodation in host roots during root endosymbiosis. Yet, comparative phylogenetic analysis of SymRK orthologs in the broader context of MLD-LRR-RLK subfamily evolution remains limited. In this study, we examined the inventory, phylogeny and clade-specific evolutionary and transcriptional characteristics of this receptor group. SymRK and its closest homologs are present in most land plant lineages and group into four major clades and six additional species-specific clades. These clades can be distinguished by their evolutionary characteristics as either conserved with reduced gene copy number changes (including SymRK) or expanded and diversified, as observed in clade IV. Clade IV dynamics are largely driven by tandem gene duplications, which often arise within gene clusters. We further analysed the evolutionary characteristics of MLD-LRR-RLKs at the population level in Arabidopsis thaliana accessions. We found that some genes are conserved across accessions and are therefore likely to be functionally important, whereas a subset of genes, often located within tandem clusters, are highly diverse and likely contribute to accession-specific adaptations. Finally, most MLD-LRR-RLKs in the A. thaliana Col-0 accession are expressed in roots and respond broadly to biotic stimuli at the transcriptional level. Notably, clustered genes frequently exhibited divergent expression profiles, suggesting transcriptional diversification. Together, we revealed two contrasting evolutionary characteristics among members of the MLD-LRR-RLK subfamily, potentially associated with their functions in plants.

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Structural basis of NSP1-NSP2 heterodimerization and its regulatory mechanism in legume nodulation

Legumes establish symbiotic relationships with rhizobia, leading to the development of nitrogen-fixing root nodules. Two GRAS transcription factors, nodulation signaling pathway (NSP) 1 and NSP2, are essential for Nod factor–induced transcription and subsequent nodulation in legumes. However, the structural basis of their interaction and functional mechanism remains poorly understood. Here, we report the crystal structure of the Medicago truncatula NSP1–NSP2 complex at 2.4 Å resolution. The structure reveals that NSP1 and NSP2 assemble into a heterodimer with a small, triangular interface exclusively composed of their leucine heptad repeat I motifs. This direct interaction is essential for nodulation, as NSP2 facilitates NSP1–DNA binding. Furthermore, we identified an HCCC-type zinc finger in NSP1 that modulates nodulation by influencing its DNA-binding activity. Together, our findings provide structural insights into NSP1–NSP2 heterodimerization and elucidate the regulatory mechanism underlying legume nodulation, offering a theoretical foundation for rationally engineering NSP1 and NSP2 to optimize plant–microbe relationships for agricultural applications.

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The GmCYCLOPS paralogs regulate soybean nodulation and exhibit signatures during domestication

CYCLOPS functions as a central regulator in legume-rhizobia symbiosis, but its role in cultivated soybean remains incompletely characterized. Through homology-based sequence analysis using Lotus japonicus LjCYCLOPS as a query, two soybean orthologs were identified, GmCYCLOPS1 and GmCYCLOPS2, sharing 98.1% amino acid identity. Both genes exhibited root- and nodule-enriched expression, with peak induction at 12 h post-rhizobial inoculation. Subcellular localization assays showed that both GmCYCLOPS1 and GmCYCLOPS2 proteins are enriched in the nucleus but also present in the cytoplasm. Functional analyses using Agrobacterium rhizogenes-mediated hairy root transformation demonstrated that RNA interference targeting either paralog reduced expression of both genes and significantly decreased nodulation. Consistently, CRISPR/Cas9-mediated disruption of both GmCYCLOPS1 and GmCYCLOPS2 generated double mutants with severe nodulation defects, whereas overexpression of these paralogs enhanced nodule formation. Population genomic analysis of 1504 accessions (34 wild, 423 landraces, and 1047 cultivars) revealed that the GmCYCLOPS1 haplotype Hap10, absent in wild accessions, increased to 91% in landraces and 99% in cultivars, showing strong selection signatures and enrichment in Northeast China. Meanwhile, GmCYCLOPS2 exhibited more moderate and regionally structured haplotype diversity. Together, these results reveal that GmCYCLOPS1 and GmCYCLOPS2 act as core regulators of soybean nodulation and have undergone divergent selection and geographic differentiation during soybean domestication. This study characterizes the functional and evolutionary features of the GmCYCLOPS paralogs, providing valuable gene and haplotype resources to improve soybean nodulation traits.

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Survey of scientific production on bio-inputs in Northern and Northeastern Brazil (2010–2025): A focus on plant growth-promoting microorganisms in legumes and grasses

Using bio-inputs, particularly those based on plant growth-promoting microorganisms (PGPM), has gained momentum in Brazil as a sustainable alternative to conventional agricultural inputs. This meta-analysis synthesizes data from 1,391 peer-reviewed studies published between 2010 and 2025, focusing on the spatial, temporal, and biological dimensions of PGPM research in Northern and Northeastern Brazil. An increase in the number of publications has been observed after 2018. Geographically, 61.2% of studies originated in the Northeast—primarily in Bahia, Pernambuco, and Ceará—while 38.8% were conducted in the North, especially in Pará, Acre, and Amazonas. Leguminous species accounted for 52% of studies, reflecting their nitrogen-fixing capacity and regional agronomic importance, followed by grasses (41%) and other species (7%). Diazotrophic bacteria such as Azospirillum, Bradyrhizobium, and Herbaspirillum were the most frequently studied microbial group, consistently enhancing plant growth metrics across crop types. Seed coating was the most widely used and effective inoculation method, followed by soil drenching and foliar application. PGPM treatments significantly improved aboveground biomass, root traits (notably nodulation and root density), and crop yield, especially in legumes. Subgroup analyses identified critical moderators influencing PGPM efficacy, including inoculant type, inoculation method, soil fertility, and environmental stressors. Despite these benefits, challenges were frequently reported. Approximately 31% of studies cited inconsistent field performance, and 28% highlighted microbial viability and soil compatibility constraints. Regulatory gaps, quality control deficiencies, and biosafety concerns regarding releasing of non-native strains were also noted. These findings highlight the potential of PGPM to support regional food systems and contribute to national sustainability goals, underscoring the urgent need for improved standardization, targeted funding, and stronger policy frameworks. The study provides evidence-based insights to guide research, regulation, and investment in bio-inputs for a more equitable and resilient Brazilian agriculture.

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