The research, led by Aura Aslan and Rumella Simarmata of Indonesia’s National Research and Innovation Agency (BRIN), with collaborators from IPB University, the University of Brawijaya, and PT. Pupuk Kalimantan Timur, focuses on an isolate designated B64. Using Illumina paired-end sequencing technology, the team assembled the whole genome of the strain and subjected it to the rigorous standards of modern prokaryotic taxonomy. Two complementary metrics were used to determine whether B64 belonged to an existing species: average nucleotide identity, or ANI, which measures the overall genetic similarity between two genomes, and digital DNA-DNA Hybridization, or dDDH, which estimates the degree of whole-genome relatedness in silico, replacing the laborious laboratory hybridization experiments of earlier decades.

The verdict was unambiguous. The highest ANI value recorded between B64 and its closest relatives was 94.4 percent, and the dDDH values ranged from 51.4 to 62.4 percent. Both figures fall below the internationally accepted thresholds for species membership, which are set at 95 percent for ANI and 70 percent for dDDH. In practical terms, these numbers mean that B64 is genetically distinct enough to be considered a novel species within the Bradyrhizobium genus, closely allied with Bradyrhizobium yuanmingense but clearly separated from it by a measurable genomic gulf. This kind of genome-based species delimitation has become the gold standard in bacterial taxonomy precisely because it removes the ambiguity that plagued older, phenotype-based classification systems.

But identifying a new species was only the opening act. The researchers also wanted to know whether B64 possessed the functional machinery that makes rhizobia so valuable to agriculture. Physiological assays painted an encouraging picture. The bacterium demonstrated nitrogen fixation activity measured at 1.97 parts per million, produced indole-3-acetic acid, a plant growth-promoting hormone better known as IAA, at 3.67 parts per million, and solubilized phosphate at 26.10 parts per million. Each of these traits contributes to plant growth through a different pathway: nitrogen fixation supplies the essential nutrient that most often limits crop productivity, IAA stimulates root development and branching, and phosphate solubilization unlocks a mineral that is abundant in many soils but chemically locked away from plant roots. An organism combining all three capabilities is a rare and valuable find.

The heart of the study, however, lies in its structural biology. The team zeroed in on NodD1, a transcriptional regulator of the LysR-type family that acts as the bacterial sensor for flavonoid molecules released by legume roots. When NodD1 binds the appropriate flavonoid, it switches on the nodulation genes, triggering the production of lipochitooligosaccharides, or Nod factors, which the plant perceives as the signal to begin building root nodules, the specialized organs that house the bacteria and provide the low-oxygen environment nitrogenase needs to function. In other words, NodD1 sits at the very gateway of symbiosis, deciding when the molecular dialogue between bacterium and plant begins.

Because no experimental structure of the B64 NodD1 protein was available, the researchers turned to AlphaFold3, the artificial intelligence system that has transformed structural biology by predicting protein conformations from amino acid sequences with remarkable accuracy. The resulting model was then subjected to Ramachandran plot analysis, a classical validation technique that examines whether the backbone dihedral angles of every amino acid residue fall within stereochemically permitted regions. The model passed with distinction: 100 percent of residues occupied allowed regions of the plot, a result that speaks to the reliability of the predicted fold and gives the team confidence that subsequent computational experiments were conducted on a structurally sound template.

With a validated protein structure in hand, the researchers performed molecular docking simulations to explore how NodD1 interacts with four flavonoid signaling molecules commonly exuded by legume roots: apigenin, daidzein, genistein, and naringenin. Docking is a computational method that predicts the preferred orientation and binding strength of a small molecule within a protein’s binding pocket, typically reporting the result as a binding free energy, where more negative values indicate more thermodynamically favorable interactions. The simulations, carried out using AutoDock Vina, demonstrated strong binding affinities across the entire flavonoid panel, with binding free energies ranging from −8.8 to −9.0 kilocalories per mole. These values fall comfortably within the range associated with biologically meaningful protein-ligand interactions.

Two ligands stood out for different reasons. Daidzein, an isoflavone characteristic of soybean root exudates, exhibited the highest thermodynamic stability of the four, with a binding free energy of −9.0 kilocalories per mole. Apigenin, by contrast, formed the most extensive network of residue-level interactions with the protein, suggesting that while its overall binding energy was marginally weaker, it engages a broader array of contact points within the binding pocket. Such differences matter: the constellation of hydrogen bonds, hydrophobic contacts, and aromatic stacking interactions that stabilize a ligand in its pocket determines not only how tightly the molecule binds but also how effectively it can allosterically activate the regulator and stimulate transcription of the nodulation genes. The finding provides a mechanistic rationale for why B64 nodulates its host plants efficiently and hints at which flavonoids might be most effective in priming the symbiosis under field conditions.

The broader context of this work is the growing global effort to reduce agriculture’s reliance on industrially produced nitrogen fertilizer, the manufacture of which through the Haber-Bosch process consumes vast quantities of fossil fuel and contributes significantly to greenhouse gas emissions. Legume-rhizobium symbiosis offers a natural alternative: when the partnership functions well, the plant receives all the nitrogen it needs at essentially no energetic cost to the farmer. Biofertilizers based on rhizobial inoculants are already in commercial use, but their effectiveness is limited by strain selection, host specificity, and environmental factors. A novel species with strong nitrogen fixation, IAA production, and phosphate solubilization, combined with robust NodD1-flavonoid interactions, represents exactly the kind of candidate strain that inoculant developers are searching for. The authors specifically highlight the potential of B64 as a source material for lipochitooligosaccharide-based biofertilizers, products that deliver the Nod factor signal itself to stimulate nodulation even in the absence of live bacteria.

The study also exemplifies a methodological trend sweeping through microbiology: the seamless integration of genomics, structural prediction, and computational chemistry into a single research pipeline. What once would have required years of cloning, protein expression, crystallization, and X-ray diffraction work can now be initiated from a genome sequence, with AI-predicted structures validated by established stereochemical checks and interrogated by docking algorithms within weeks. This acceleration does not eliminate the need for experimental confirmation, and the authors themselves position their docking results as a molecular basis for further study rather than a definitive functional proof. Yet the approach dramatically lowers the barrier to screening large numbers of candidate strains and prioritizing the most promising symbionts for wet-lab validation and field trials.

For Indonesia, home to vast legume cultivation areas and a national push toward sustainable intensification, the identification of a locally isolated novel Bradyrhizobium species carries particular significance. Native strains adapted to local soils and climates often outperform imported commercial inoculants, and the work was supported by the country’s Research and Innovation Implementation Agency in partnership with the Indonesia Endowment Fund for Education. As the research team continues to characterize B64, including testing its nodulation performance across legume hosts and its resilience in diverse soil conditions, the bacterium may well find its way from genome databases and docking simulations into the seed coatings of farmers across the tropics, quietly fixing nitrogen and enriching soils one nodule at a time.

Arbuscular mycorrhizal fungi are among the oldest and most widespread partners of land plants. Fossil and molecular evidence suggests that this relationship began more than 400 million years ago, around the time plants were establishing themselves on land. Today, these fungi associate with the roots of most terrestrial plant species. The fungus extends a network of microscopic filaments, known as hyphae, through the surrounding soil, greatly expanding the plant’s reach beyond the physical limits of its roots. In return, the plant supplies the fungus with carbon-rich sugars and lipids produced through photosynthesis. The fungal network can improve the plant’s access to phosphate, nitrogen, and trace nutrients, while also influencing water uptake and stress tolerance. Yet the molecular details that allow a living fungus to enter root tissue without triggering a destructive immune response have remained difficult to resolve.

A major obstacle has been that colonization is spatially and temporally mixed. A single root may contain surface cells that have only just detected fungal signals, deeper cells preparing for invasion, cells actively building arbuscules, and cells already hosting mature exchange structures. Conventional genetic and biochemical methods generally average molecular signals across thousands or millions of cells, blurring these different stages together. “Despite decades of research, the molecular details of how plants accommodate fungal structures called arbuscules remained poorly understood,” said Prof. Sofie Goormachtig of VIB-UGent. Arbuscules are highly branched fungal structures formed inside root cells. They dramatically increase the membrane surface available for exchange, allowing nutrients to move from the fungus to the plant while carbon compounds move in the opposite direction.

To separate these overlapping stages, Goormachtig’s team worked with the VIB Single Cell Core to apply single-nucleus transcriptomics to tomato roots colonized by Rhizophagus irregularis, one of the best-studied arbuscular mycorrhizal fungi. Rather than measuring gene activity from a whole root, the technique profiles RNA molecules inside individual nuclei. Because messenger RNA reflects which genes are actively being transcribed, these profiles provide a molecular snapshot of each cell’s identity and condition. The researchers also used a fluorescent marker to identify root regions in which the fungus was actively present. This enrichment step allowed them to focus their sequencing effort on the most informative tissue instead of treating colonized and uncolonized regions as a single biological sample. In total, the dataset contained gene-activity profiles from nearly 66,000 individual root cells.

The resulting map revealed a sequence of four major cellular stages. The first involved root surface cells detecting the approaching fungus and initiating the earliest symbiotic responses. The second occurred in inner root cells that began preparing the tissue for fungal entry, changing their gene activity before mature fungal structures appeared. The third stage was characterized by cells constructing arbuscules, with extensive remodeling of cellular architecture and metabolism. The final stage involved cells containing fully developed arbuscules capable of sustained nutrient exchange. These stages were not merely anatomical categories. Each displayed a distinctive transcriptional signature, indicating that the plant progressively rewires its cells as the partnership develops. “Each stage has its own characteristic molecular signature,” said Dr. Naomi Stuer, first author of the study. “This helps us understand how the plant gradually rewires its cells as the partnership develops.”

The researchers next asked which molecular regulators coordinate these changes. They used MINI-EX, a computational framework designed to infer relationships between transcription factors and the genes they may control. Transcription factors are proteins that bind specific DNA sequences and influence whether target genes are activated or suppressed. By integrating the single-cell expression patterns with regulatory predictions, the team identified candidate transcription factors associated with each stage of colonization. The analysis recovered several regulators already known to participate in mycorrhizal symbiosis, providing an internal validation of the approach. It also identified new candidates whose roles had not previously been connected to the interaction. Three of these candidates were tested directly in living tomato roots, where they displayed the predicted stage-specific activity. This agreement between computational inference and experimental observation suggests that the candidates may function as genuine regulators rather than being passive markers of colonized cells.

One of the study’s most significant findings concerns a signaling pathway previously thought to operate mainly near the root surface. The new data indicate that the pathway remains active farther inside the root and continues functioning during the formation of arbuscules. Its activity may help prepare particular cortical cells for the demanding process of hosting fungal structures. Before an arbuscule can form, a plant cell must alter its metabolism, reorganize its internal membrane system, and establish a specialized interface around the invading fungal branches. These changes require precise coordination between developmental programs, nutrient signaling, and the plant’s immune system. The single-nucleus profiles suggest that some of this preparation begins before the cells show the obvious structural features associated with mature arbuscules. Because these prospective host cells initially look much like neighboring cortical cells, their early molecular state would have been almost impossible to recognize using microscopy alone.

The data also point to a sophisticated feedback system operating in mature arbuscule-containing cells. These cells appear to monitor information about the plant’s broader nutritional condition, including whether the plant is already receiving sufficient nutrients. Such sensing could allow the plant to adjust its investment in the fungus. Maintaining a symbiosis requires carbon and cellular resources, so the relationship must provide enough nutritional benefit to justify its cost. When phosphate or nitrogen is abundant, the plant may reduce colonization or limit the formation and lifespan of arbuscules. When nutrients are scarce, it may support a more extensive fungal network and intensify exchange. The findings suggest that mature host cells are not simply containers for fungal structures; they are active decision-making units that integrate local fungal signals with the plant’s systemic nutrient status.

“What excites me most about this dataset is that it does not just confirm what we suspected; it opens entirely new doors,” said Dr. Judith Van Dingenen of VIB-UGent, co-senior author. The resource gives researchers a way to ask precisely when and where symbiotic genes are activated, how long individual molecular programs persist, and what causes one root cell to become a fungal host while an adjacent cell follows a different developmental path. It may also enable comparisons between plant varieties that form highly efficient fungal partnerships and those that benefit less from colonization. Such comparisons could reveal whether improved symbiosis depends on stronger signaling, more effective nutrient transport, altered immune regulation, or a combination of several traits.

The practical implications extend beyond tomato biology. Modern agriculture often compensates for limited nutrient availability with industrial fertilizers, especially phosphate and nitrogen products whose manufacture, transport, and runoff carry substantial environmental costs. Engineering or breeding crops that make better use of arbuscular mycorrhizal fungi could offer another route to maintaining yields while reducing fertilizer inputs. The newly identified transcription factors provide possible entry points for that effort, although their agricultural value will require further testing in different crops, soils, climates, and microbial communities. The present study does not yet deliver a ready-made “super-symbiotic” crop, but it supplies the cellular atlas and regulatory hypotheses needed to pursue one. By revealing how a root changes cell by cell as it welcomes an ancient fungal partner, the researchers have transformed a hidden underground interaction into a process that can be measured, modeled, and potentially improved.

Article Title: Decoding stage-specific symbiotic programs in the Rhizophagus irregularis-tomato interaction using single-nucleus transcriptomics

News Publication Date: 18 August 2026

Web References: https://doi.org/10.1016/j.cub.2026.05.057

References: Current Biology, published 6 July 2026; DOI: 10.1016/j.cub.2026.05.057

Keywords: arbuscular mycorrhizal fungi, Rhizophagus irregularis, tomato roots, single-nucleus transcriptomics, single-cell biology, arbuscules, plant-fungus symbiosis, transcription factors, nutrient exchange, sustainable agriculture