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.

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.

The nuclear pore complex controls the movement of proteins into and out of the nucleus, allowing cells to regulate protein localization and abundance. This process influences how organisms respond to environmental stimuli. Components of the nuclear pore complex, including the NUP107-160 sub-complex, NUP133, NUP85, and NENA, are required for root nodulation and arbuscular mycorrhization in Lotus japonicus. However, the specific role of these nucleoporins in symbiotic signaling was poorly understood. Through reverse genetics, we discovered that NUP133 is also required for symbiosis in Medicago truncatula, although the mutant phenotypes were less pronounced than in Lotus. Overexpression of the symbiotic ion channels Medicago DMI1 and Lotus Castor and Pollux in the Lotus Ljnup133, Ljnup85, and Ljnena mutants partially alleviated the nodulation defects. Notably, in NUP107-160 sub-complex mutants of Lotus and Medicago, the accumulation of GFP-labeled Pollux and DMI1 on the inner nuclear membrane was reduced, indicating the NUP107-160 sub-complex plays a key role in regulating the distribution of DMI1 and Pollux on the nuclear envelope. This highlights the extreme sensitivity of nodulation in Lotus to changes in the abundance of Pollux on the inner nuclear membrane. In contrast, Medicago appears to exhibit greater tolerance to alterations in the distribution of DMI1 on the nuclear envelope.

The fossil fuel–driven chemosynthesis of ammonia for nitrogen fertilizers is a major contributor to global greenhouse gas emissions, releasing approximately 1130 million metric tons (MMT) of CO2-equivalent annually. In addition, the extensive use of synthetic nitrogen fertilizers (≈108 MMT annually) results in substantial emissions of nitrous oxide (N2O), while nearly 50% of applied nitrogen is lost through leaching and runoff, leading to widespread nitrate pollution and ecosystem degradation. In contrast, solar-powered biological nitrogen fixation (BNF) by nitrogen-fixing plants offers a sustainable alternative that supports agricultural productivity and healthy ecosystem resilience.
BNF by leguminous and non-leguminous plants plays a critical role in reducing dependence on synthetic nitrogen fertilizers. Legumes alone contribute an estimated ∼32-149kg.hm-2 of fixed nitrogen annually. Beyond legumes, associative, endosymbiotic, and endophytic nitrogen fixation in non-legume plants further expands the ecological and taxonomic scope of BNF. Nitrogen-enriched cropping systems, such as legume–cereal rotations, can reduce greenhouse gas emissions by up to 88%, increase soil organic carbon stocks by approximately 8%, enhance soil microbiome activity by 45%, and provide farmers with 15–25% more stable revenues. Advances in biotechnology offer new opportunities to improve nitrogen fixation efficiency and develop novel nitrogen-fixing crops, including cereals and perennial fruit trees. By integrating nature’s evolutionary solutions (N2-fixing plants) with modern biotechnology, agriculture can transition from a fossil fuel–based chemo-nitrogen economy to a solar-powered bio-nitrogen economy.

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.

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.

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.

Drought is a major constraint on maize productivity, and its increasing frequency due to climate change necessitates improved stress adaptation strategies. Arbuscular mycorrhizal fungi (AMF) can enhance plant drought tolerance; however, the integrated mechanisms linking root development, host transcriptional regulation, and microbiome activity remain poorly understood. Here, we investigated these interactions in maize using an integrated phenotyping–transcriptomic–metatranscriptomic approach under controlled greenhouse conditions. Two inbred lines with contrasting drought tolerance (K1, tolerant; K2, sensitive) and their hybrid (KH) were grown under well-watered (60% soil moisture) and drought (30%) conditions, with or without Funneliformis mosseae inoculation. Mycorrhizal colonization reached 51.3–62.5% under drought, confirming effective symbiosis. RNA-seq analysis (FDR ≤ 0.05, |log2;FC| ≥ 1) revealed strong genotype-dependent transcriptional responses, with the drought-sensitive genotype showing the largest number of differentially expressed genes. Principal component analysis identified genotype as the primary driver of variation (PC1: 13%), followed by mycorrhizal status (PC2: 8%). AMF induced distinct, genotype-specific functional reprogramming. The drought-tolerant genotype showed moderated stress responses and maintained metabolic activity, whereas the drought-sensitive genotype exhibited sustained stress signaling and compensatory metabolic activation. The hybrid displayed a non-additive response associated with enhanced root remodeling and symbiosis-related functions. Metatranscriptomic analysis of the non-host root-associated transcript pool further revealed genotype-specific microbial functional activity patterns, ranging from activation to repression. These results demonstrate that AMF-mediated drought tolerance emerges from coordinated, genotype-dependent interactions among root development, host regulatory networks, and microbiome activity. This study provides a holobiont-level framework for understanding crop stress adaptation.

Endosymbiotic interactions in fungi are fundamental to ecological and evolutionary processes; however, despite their potential to elucidate key mechanisms of biological integration, they remain insufficiently explored. This review provides a comprehensive synthesis of the available evidence on these associations across major fungal lineages, encompassing their composition, functional attributes, and breadth across diverse model systems. Additionally, the current state of artificial endosymbiosis is examined, together with its applications in multiple areas of biotechnology, spanning both natural and engineered systems. Finally, a framework of progressive endosymbiosis is proposed to describe the continuum of integration states of endosymbionts within fungal hosts. Together, these insights highlight the central role of fungal endosymbiosis in biological integration and underscore its value as a model for understanding evolutionary transitions and developing innovative biotechnological applications.

Tetracycline (TC), a widely used veterinary antibiotic, frequently accumulates in agricultural soils and disrupts nitrogen (N) cycling, thereby enhancing nitrous oxide (N2O) emissions. However, biologically based mitigation strategies and their underlying mechanisms remain poorly understood. In this study, a soybean pot experiment was conducted with four treatments: control, arbuscular mycorrhizal fungi (AMF) inoculation, TC addition, and AMF combined with TC. By integrating hyphosphere-specific sampling, potential N2O production rate measurements, 16S rRNA gene sequencing, quantitative PCR, metagenomics, and partial least squares path modeling, we systematically elucidated AMF-mediated regulation of N2O production under TC stress. TC significantly increased potential N2O production rate (+21.2%), primarily by selectively suppressing the terminal denitrification step, as evidenced by reduced nitrous oxide reductase (NOS) activity and decreased abundance of the nosZ gene, resulting in denitrification pathway disruption and N2O accumulation. In contrast, AMF inoculation under TC stress reduced potential N2O production rate by 29.5%, restoring it to control levels. Mechanistically, AMF improved hyphosphere soil properties (e.g., increased SOC and TN and enhanced TC dissipation) and selectively enriched functionally competent and TC-tolerant denitrifiers, particularly nosZ-harboring taxa such as Streptomyces, thereby repairing denitrification pathway completeness. Path modeling further demonstrated that AMF mitigated N2O production both directly by enhancing N-cycling microbial functional capacity and indirectly by optimizing soil physicochemical conditions. Our findings reveal the microbial and molecular mechanisms underlying antibiotic-enhanced N2O emissions and highlight AMF as a low-input, nature-based environmental biotechnology strategy to simultaneously remediate antibiotic-contaminated soils and mitigate agricultural greenhouse gas emissions.

Arbuscular mycorrhizal (AM) symbiosis is regulated by carotenoid-derived molecules, including strigolactones and other apocarotenoids. However, the role of root carotene availability remains poorly understood. Here we evaluated AM performance in tomato using two mutants showing contrasting root carotenoid profiles, i.e. cyc-b7, an EMS-derived TILLING mutant carrying a characterized mutation in Cyc-B, and 7458-Y, an uncharacterized mutant line showing high carotenoid accumulation), compared to their related wild type (Red Setter). Not-inoculated and AM fungal inoculated plants were grown under controlled conditions and root colonization parameters were assessed after two months. Root carotenoids were quantified by high-performance liquid chromatography (HPLC), and RNA-seq analysis was performed on root samples to understand the tomato response to the inoculation. Roots of cyc-b7 accumulated significantly lower total carotenoids than Red Setter, including reduced lutein and β-carotene, whereas 7458-Y showed increased β-carotene, together with higher arbuscule abundance than both Red Setter and cyc-b7. Transcriptome profiling revealed a genotype-dependent response to AMF inoculation, with symbiosis-related genes differentially regulated in the two mutant lines. In cyc-b7, AMF inoculation was associated with reduced expression of genes encoding nutrient transporters, as well as of a gene encoding a symbiosis receptor-like kinase (SYMRK), a component of the common symbiosis signaling pathway. By contrast, in 7458-Y, AMF inoculation was associated with up-regulation of a gene encoding a LysM receptor-like kinase involved in AM establishment, and of a gene, SlD27, related to strigolactone biosynthesis. Overall, our results support a link between root carotenoid metabolism and AMF colonization.

Rhizobial bacteria are known for their ability to fix nitrogen for leguminous plants and their essential function for sustainable agriculture. This study characterizes the taxonomic status and functional potential of the Bradyrhizobium B64 isolate using integrated genomic and molecular approaches. The whole genome of the B64 isolate was sequenced via Illumina paired-end technology. Species delimitation was performed using average nucleotide identity (ANI) and digital DNA-DNA Hybridization (dDDH). The NodD1 protein structure was modeled using AlphaFold3 and validated by Ramachandran plot analysis. Molecular docking was then conducted to evaluate interactions between NodD1 and four signaling flavonoids: Apigenin, Daidzein, Genistein, and Naringenin. Genomic analysis revealed a maximum ANI of 94.4% and dDDH values between 51.4 and 62.4%. Since these values fall below the standard prokaryotic thresholds (ANI < 95%; dDDH < 70%), the B64 isolate is identified as a novel species. Physiological assays confirmed nitrogen fixation (1.97 ppm), IAA production (3.67 ppm), and phosphate solubilization (26.10 ppm). Structural validation showed 100% of NodD1 residues in allowed regions, ensuring high model reliability. Docking simulations demonstrated strong binding affinities across all flavonoids, with binding free energies ranging from − 8.8 to − 9.0 kcal/mol. Daidzein exhibited the highest thermodynamic stability (− 9.0 kcal/mol), whereas apigenin showed the most extensive residue interaction network. The B64 isolate is a novel Bradyrhizobium species with a high symbiotic capacity. The stable NodD1-flavonoid interactions provide a molecular basis for efficient nodulation, positioning B64 as a promising candidate for developing lipo-chitooligosaccharide (LCO)-based biofertilizers.