Nod factor receptors (NFRs) are essential for initiating symbiotic signaling in legumes, mediating rhizobial infection and nodule development. Tight regulation of NFR levels is crucial to prevent inappropriate immune responses and maintain cellular homeostasis. Co-expression of LjNFR1 and LjNFR5 triggers cell death in Nicotiana benthamiana, which is specifically inhibited by LjBAK1-mediated ubiquitination and subsequent degradation, suggesting the existence of a LjBAK1-E3 ligase complex for NFR protein turnover. Further analysis identified LjPUB7, a plant U-box E3 ubiquitin ligase in Lotus japonicus, as a regulator of early symbiotic interactions. LjPUB7 interacts with both LjBAK1 and NFRs, and directly ubiquitinates NFRs. Loss-of-function Ljpub7 mutants display increased infection thread formation, enhanced nodule development, and elevated expression of early nodulation genes. These findings reveal that LjPUB7 negatively regulates early rhizobial infection by targeting NFR1 and NFR5 for ubiquitination and degradation, thereby providing insights into the fine-tuned control of symbiotic signaling in legumes.

Root nodules are the only sites for symbiotic nitrogen fixation (SNF) in leguminous plants. The development and functioning of these nodules are governed by a cascade of gene expressions categorized as early and late nodulins. While early nodulins are rapidly induced by Nod factors and involved in infection and cortical cell division, late nodulins support mature nodule function. The regulation of these gene expressions involves several extra- and intracellular factors along withnon-coding RNAs (ncRNAs). Despite extensive studies on ncRNAsinSNF, the role of long ncRNAs (lncRNAs) in it remains largely unexplored excepting the well-characterized early nodulin lncRNA ENOD40 and its natural antisense transcript DONE40. Here, we report the identification and characterization of a novel lncRNA, Lotus japonicus PLP-IV Long non-coding RNA (LjPLR), discovered through in-silico transcriptome analysis followed by in-vivo validation. LjPLR is an antisense transcript complementary to the LjPLP-IV gene, which encodes a phosphatidylinositol transfer protein-like protein implicated in membrane biogenesis. We have identifiedLjPLP-IV as the only putative target of LjPLR. The negatively correlated temporal gene expression patterns of LjPLP-IV and LjPLR during nodule biogenesis providea new insight into the regulatory landscape of SNF in Lotus japonicus.

Arbuscular mycorrhizal (AM) fungi are essential members of the plant microbiome, exerting a profound influence on nutrient acquisition, and efficiency. While their role in facilitating phosphorus (P) uptake is well established, the broader contributions of AM fungi to nitrogen (N), P, and carbon (C), and the subsequent impacts on plant growth and development, remain comparatively underexplored. This review synthesizes recent advances in understanding AM symbiosis, highlighting its importance in regulating nutrient fluxes and plant physiological processes. AM fungi secrete glomalin-related soil proteins that improve soil aggregation and foster favorable conditions for microbial communities involved in nutrient transformations. In association with nitrogen-fixing bacteria, AM fungi contribute to improved N availability, providing up to 30% of the plant’s N demand. This supports amino acid biosynthesis and fundamental physiological processes, such as photosynthesis and production of metabolites, thereby promoting plant growth. Through the mycorrhizal pathway (MP), plants may acquire up to 80% of their P requirements, an important advantage during early plant’s developmental stages. In return, host plants allocate 20-40% of their photosynthetically derived C to sustain the fungal partner. Beyond individual nutrient exchanges, AM fungi play a pivotal role in regulating the stoichiometry of C:N:P in plant–fungal symbioses, with cascading effects on plant nutrition, soil fertility, and ecosystem functioning. By mediating these nutrient interactions, AM fungi not only support plant growth and development but also influence broader biogeochemical cycles. This review underscores the pivotal role of AM fungi in nutrient dynamics and provides insights to guide future research on mycorrhizal associations with nutrients and sustainable plant growth and development.

The majority of land plant species recruit fungi of the phylum Glomeromycotina to form one of the most widespread plant symbioses called arbuscular mycorrhiza (AM) for mutual nutritional benefit [1–3]. Arbuscular mycorrhiza fungi (AMF) take up mineral nutrients from the soil through an extended hyphal network that forms outside the root and transports the nutrients directly into the root cortex, where they are released to the plant from highly-branched fungal structures, the arbuscules [4]. In turn, the fungi obtain photosynthetically fixed organic carbon from their host plants in the form of sugars and lipids [5,6]. AM is often described as a strategy for plants to obtain inorganic phosphate, but it also improves uptake of other inorganic nutrients, such as nitrogen, potassium, and sulfur [7–9].

Legumes and rhizobia establish a nitrogen-fixing symbiosis that involves the formation of a lateral root organ, the nodule, and the infection process that allows intracellular accommodation of rhizobia within nodule cells. This process involves substantial gene expression changes regulated at the transcriptional and post-transcriptional levels. We have previously shown that a transcript encoding subunit 3 of the SUPERKILLER Complex (SKI), which guides mRNAs to the exosome for 3´-to-5´ degradation, is required for nodule formation and bacterial persistence within the nodule, as well as the induction of early nodulation genes, including early nodulin40 (MtENOD40), during the Medicago truncatula–Sinorhizobium meliloti symbiosis. Here, we reveal through transcript degradome and small RNA sequencing analysis that knockdown of MtSKI3 impairs the miR172-directed endonucleolytic cleavage of the mRNA encoding Nodule Number Control 1 (MtNNC1), an APETALA2 transcription factor that negatively modulates nodulation. Knockdown of MtNNC1 enhances nodule number, bacterial infection, and the induction of MtENOD40 upon inoculation with S. meliloti, whereas overexpression of an miR172-resistant form of MtNNC1 significantly reduces nodule formation. This work identifies miR172 cleavage of MtNNC1 and its control by MtSKI3, a component of the 3´-to-5´mRNA degradation pathway, as a regulatory hub controlling indeterminate nodulation.

Background: Pests and diseases can cause suboptimal tomato production. One alternative that can be used to control wilt disease in tomato plants is to use biological agents. Biological agents that have the potential to control wilt disease in tomato plants are mycorrhizae. This study aimed to determine whether mycorrhizal treatment was effective in the growth of tomato plants and the control of wilt.  Methods: The research design was a randomized block design (RBD) arranged factorially. This study used two factors and the first was the dose of mycorrhizal application (M) with three treatments consisting of M1 = mycorrhizal 25 spores/plant, M2 = mycorrhizal 50 spores/plant and M3 = mycorrhizal 75 spores/plant. The second factor was Mycorrhiza (T) application time using two treatments consisting of T1 = when the tomato seeds were sown and T2 = when transplanting. All treatment combinations were repeated 4 times. The varieties used are varieties that farmers usually plant, namely the F1-resistant variety, which was an introduced hybrid plant. The type of plant was a determinate plant. Result: The results showed that the mycorrhizal treatment with 75 spores significantly increased tomato plant growth on parameters such as plant height, number of leaves, fruit weight and reduced disease incidence.

Microbes are ubiquitous in the rhizosphere and play crucial roles in plant health, yet the metabolisms and physiologies of individual species in planta remain poorly understood. In this study, we examined microbial gene expression in response to the maize root environment for seven bacterial species originally isolated from maize roots. We grew each species individually, both in vitro in a minimal medium and in planta, and used differential metaproteomics to identify functions upregulated specifically when bacteria are grown on maize roots. We identified between 1,500 and 2,100 proteins from each species, with approximately 30-70% of these proteins being differentially abundant between the two conditions. While we found that transporter proteins were upregulated in all species in planta, all other differentially abundant functions varied greatly between species, suggesting niche specialization in root-associated microbes. Indeed, in vitro assays confirmed that Curtobacterium pusillum likely degrades plant hemicellulose, Enterobacter ludwigii may benefit the plant by phosphate solubilization, and Herbaspirillum robiniae colonizes maize roots more effectively when both of its Type VI Secretion Systems are functional. Together, our findings highlight both conserved and species-specific bacterial strategies for growth in the root environment and lay a foundation for future work investigating the mechanisms underlying plant-microbiota interactions.

Plastic pollution, particularly its breakdown into nanoplastics (NPs), poses a significant threat to ecosystem services, with notable effects on soil-plant-microbe interactions in agricultural systems. However, there is limited understanding of how NPs influence the soil microbiome and plant symbiotic functions. In this study, we applied polypropylene (PP) and polyethylene (PE) NPs, measuring 20 to 50 nm, to soybean growing conditions. We evaluated soil physicochemical properties, nodule counts, nitrogenase activity, and bacterial community composition in nodule, rhizosphere, and bulk soil under different concentrations of these NPs (200, 500, and 1000 mg/kg of soil w/w). Our results revealed that the impact of NPs on soil physicochemical properties was type-dependent, with PE-NPs exerting a more pronounced effect on soil enzyme activities than PP-NPs. Both NPs treatments accelerated nodulation and increased nitrogenase activity, with lower doses inducing more significant effects. Furthermore, PE and PP-NPs enriched bacterial species such as Ensifer and Arthrobacter, which positively interact with diazotrophs such as Bradyrhizobium, supporting symbiosis and biological nitrogen fixation. NPs treatments also significantly affected the bacteriome assembly process in the bulk soil, rhizosphere, and nodule, with an increased source ratio from the rhizosphere to the nodule and homogenous selection in the nodule bacteriome, likely benefiting bacteria involved in nodulation. Exposure to 500 mg/kg of both NPs caused alterations in the metabolic exudation profile of the plant rhizosphere, particularly influencing the biosynthesis pathways of flavonoids and isoflavonoids. Metabolites such as genistein and naringenin emerged as key mediators of plant-microbe interactions, further enhancing plant symbiotic processes under NPs exposure. This study demonstrates that NPs influence plants’ symbiotic potential both directly, by altering the composition of the soil bacteriome, and indirectly, by affecting exudation potential. It provides strong evidence that NPs, especially those smaller than a micrometer, can have long-term effects on the stability and functionality of agricultural ecosystems.

Plant responses to environmental stimuli are often shaped by a history of previous interactions, forming the foundation for stress memory and adaptive plasticity. Arbuscular mycorrhizal (AM) fungi establish a mutualistic relationship with most land plants, enhancing nutrient uptake and stress resilience, and are increasingly recognized as biological agents contributing to plant stress memory. However, quantifying AM colonization, especially in large-scale or time-course experiments investigating priming or memory effects, remains a technical bottleneck. Conventional staining methods are time-consuming, destructive, and incompatible with live imaging. This chapter presents a robust, nondestructive, and quantitative protocol to assess AM colonization in Medicago truncatula roots using a visible anthocyanin pigmentation marker. The method employs a synthetic construct expressing the R2R3 MYB transcription factor MtLAP1, driven by the AM-inducible Kunitz Protease Inhibitor 106 (KPI106) promoter, enabling visualization of arbuscule-containing root cells through purple/red pigmentation. The protocol encompasses Agrobacterium rhizogenes-mediated hairy root transformation, standardized mycorrhization assays, and anthocyanin pigment extraction and quantification. Anthocyanin accumulation correlates strongly with conventional staining-based colonization estimates, and the system enables early detection, live imaging, and high-throughput screening of mutants with altered AM phenotypes. This method offers a powerful tool for dissecting the functional role of mycorrhizal symbiosis in plant stress memory and is especially suited for forward genetic screens, stress priming experiments, and live-tracking of root–fungus interactions over time.

Background and Aims Legume root nodules host symbiotic rhizobia essential for nitrogen fixation but also harbor diverse non-rhizobial taxa that remain poorly characterized. Yellow pea (Pisum sativum) cultivars adapted to distinct seasonal growth (spring and winter) offer an opportunity to explore whether host genotype influences nodule microbiome composition and function. This study investigates the taxonomic and functional profiles of nodule-associated microbial communities in seasonal yellow pea varieties. Methods A field experiment with 6 field pea cultivars (spring and winter types) was conducted in South Dakota. Surface-sterilized root nodules were subjected to full-length 16S rRNA gene sequencing using Oxford Nanopore technology. Reads were quality filtered, organellar sequences removed, and taxonomic classification performed with the EMU pipeline. Microbial diversity, community structure, and core taxa were analyzed using R, with predicted functions inferred by FAPROTAX. Results The nodule microbiome was dominated by Rhizobium, accounting for up to 98% of classified reads. After excluding Rhizobium, non-rhizobial diversity revealed a conserved core microbiome shared across cultivars, including cyanobacteria with potential phototrophic and diazotrophic traits. Minor seasonal differences were observed, with winter cultivars exhibiting higher evenness and specific associations. Conclusion Yellow pea nodules harbor a stable, cyanobacteria-enriched core microbiome, largely consistent across seasonal cultivars. Season-specific microbial patterns suggest potential host-genotype influences, warranting further validation.

Arbuscular mycorrhizal (AM) symbiosis induces substantial metabolic rearrangement in host plants to facilitate nutrient exchange and symbiotic efficiency. While previous metabolomic studies have characterized metabolite shifts in AM symbiosis, the lipid-related metabolic rewiring underlying nutrient exchange in host plant roots remains poorly resolved. Here, we investigated the metabolic response in tomato roots colonized by AM fungi. A total of 219 differentially accumulated metabolites (DAMs) were identified by the ultra-high-performance liquid chromatography-tandem mass spectrometry analysis, with lipids and lipid-like molecules representing the predominant classes. The most significantly upregulated metabolite was 2-(14,15-epoxyeicosatrienoyl) glycerol, a 2-monoacylglycerols (2-MAGs) mapped to arachidonic acid metabolism. This compound represents a C20-based epoxy fatty acid-derived 2-MAG, distinct from the C16:0 2-MAG induced by AM symbiosis in legumes, thereby implying the possibility of transferring diverse lipid substrates from different host plants to AM fungi. Concurrently, enhanced accumulation of dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA) in AM fungi colonized roots underscored alterations of arachidonic acid metabolism and unsaturated fatty acid pathway. Gene set enrichment analysis based on the transcriptome data revealed significant transition of the glycerophospholipid metabolism pathway, primarily driven by multiple lysophosphatidylcholine (LPC) species that showed significant upregulation. Integrated transcriptomic and metabolomic analysis identified 31 overlapping KEGG pathways, emphasizing the importance of lipid and amino acid metabolism. In summary, our integrated analysis demonstrates that lipid-related metabolic reprogramming, represented by the induction of 2-MAGs and LPCs, is a feature of AM symbiosis that enables cross-kingdom nutrient exchange and host metabolic adaptation.

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Cell division and specification are crucial for plant development and coping with diverse environmental cues. Most land plants rely on symbiosis with arbuscular mycorrhizal (AM) fungi to cope with soil nutrient limitations by forming arbuscules in root inner cortex cells. What determines the AM susceptibility of these inner cortex cells is currently unknown. Here we show that DELLA transcriptional regulators control the number of inner cortex cells with an AM-susceptible identity at the root stem cell niche of Medicago truncatula in a dose-dependent manner. Genetic analyses suggest that this activity converges with the well-known mobile SHORT-ROOT transcription factor regulating ground tissue development. Furthermore, we show that MtDELLA1 protein moves from the stele/endodermis to the cortex in the mature part of the root to facilitate arbuscule formation. We propose that the formation of a root inner cortex cell identity controlled by mobile DELLA and SHORT-ROOT is a fundamental basis for AM symbiosis.

Plants interact with a plethora of organisms in the rhizosphere, with outcomes that range from detrimental to beneficial. Arbuscular mycorrhizal (AM) symbiosis is the most ubiquitous beneficial plant ...

When aiming to increase plants' nitrogen (N) budget, special attention is given to the microbial inoculum's capacity to perform biological N₂ fixation. However, we consider that other approaches can be explored. Here, we report initial results of plant growth promoting rhizobacteria (Azospirillum brasilense strains Sp245 and ARG2) capacity to scavenge atmospheric ammonia (NH₃). Using a bipartite Petri dish system, we grew the two A. brasilense strains with the appropriate controls, and with atmospheric NH₃ as a N source. By increasing the atmospheric NH₃ concentration, the growth rate of both A. brasilense strains increased almost 4 times in relation to the controls. By creating a gradient of atmospheric NH₃ concentrations we changed the growth rate of both A. brasilense strains, but its effect differed between the two bacterial strains, i.e., the Sp245 strain increased its growth rates up to pH 9.0, while the ARG2 strain reached maximum growth rates at pH 9.5. The fact that these two plant growth promoting rhizobacteria scavenge atmospheric NH₃, instead of fixing N₂, suggests that this overlooked microbial trait can be an interesting tool to mitigate atmospheric NH₃ concentrations, especially in farming environments.

•Bacteria strains altered roots, biomass, pigments, and K⁺ concentration •CJND1 and LN3BA improved root traits and foliar K⁺ in L. purpureus •Proline and Na⁺ blockage are the main salt tolerance mechanisms in L. purpureus •Active K⁺ transport helps L. purpureus retain leaf potassium under stress •L. purpureus is a promising climate-resilient crop for Mediterranean farmers

Glomalin, a substance produced by arbuscular mycorrhizal (AM) fungi, has well-documented benefits for plant and soil health, including water retention and soil aggregation. Glomalin quantification h...

Glomalin-related soil protein (GRSP) extracted from soil is considered crucial for the formation and stability of soil aggregates. However, due to limitations in extraction purity and interference from co-extracted products, the actual contribution of pure glomalin produced by arbuscular mycorrhizal fungi (AMF) to soil structure improvement and its specific mechanism of action remain elusive. Here, genetic engineering and cryo-electron microscopy (cryo-EM) are introduced to obtain purified glomalin and to determine its homo-tetradecamer structure. This allowed investigations of the effect of pure glomalin on soil aggregate stability and the specific glomalin-mineral interaction mechanism. The results showed that addition of glomalin significantly enhanced the formation of soil water-stable aggregates and soil macroaggregates. This enhancement was primarily attributed to the strong binding of glomalin to soil minerals, as evidenced by single molecule force spectroscopy (SMFS) and attenuated total reflectance-Fourier transform infrared spectrum (ATR-FTIR) experiments. Glomalin structural analysis, comparison of its amino sequence alignment with that of Escherichia coli heat shock protein 60 (E. coli Hsp60) and mineral binding experiments with several glomalin related mutants highlighted that the N-terminus disordered tail of glomalin composed of ∼39 amino acids were crucial for the glomalin super binding ability. These findings advance the understanding of glomalin's intrinsic mechanism for improving soil structure and open the opportunity for mass production of this ecologically important protein as a soil amendment.

Microalgal biomass is increasingly valued in industrial and agricultural sectors due to its bioactive compounds. However, large-scale production remains costly, mainly due to nitrogen fertilizer expenses. A promising sustainable alternative is co-cultivation with N2-fixing bacteria, capable of supplying biologically available nitrogen. In this study, Chlorella vulgaris was grown in synthetic medium with and without nitrogen, as well as in co-culture with three different N2-fixing bacteria in nitrogen-free medium. Microalgal growth was assessed by dry weight, Fv/Fm ratio, and flow cytometry, which also allowed evaluation of population dynamics and cell viability. Biomass composition (proteins, carbohydrates, lipids, chlorophyll, and carotenoids) was analyzed under all conditions. Co-cultures in nitrogen-free medium showed comparable biomass productivity to nitrogen-supplemented controls, although Fv/Fm values indicated physiological stress in some cases. Moreover, the agricultural potential of the resulting biomass and supernatants was evaluated through germination bioassays using lettuce seeds. All cultures tested at 0.2 g·L−1 significantly improved the germination index. Also, applying the culture supernatant (biomass removed) also yielded positive effects, with GI increases exceeding 40 %. These results suggest that co-cultivation with N2-fixing bacteria can support efficient microalgal production while generating biomass and supernatants with biostimulant potential, contributing to sustainable agriculture and circular bioeconomy strategies.

Soil phosphorus (P) deficiency can severely limit crop and forage productivity. With limited P resources, breeding programs to select high-P efficiency (HPE) genotypes have been developed, but the role of arbuscular mycorrhizal fungi (AMF) in altering root morphology and physiology to increase P use efficiency and production remains poorly understood. In this study, we compared mycorrhizal responsiveness, and plasticity of root morphological and physiological traits between two low-P efficiency (LPE) and two HPE alfalfa genotypes under low- and high-P treatments. Plants were grown either in soil with naturally occurring AMF or in sterilized soil with added AMF-free bacteria. The results indicated that the AMF symbiosis significantly increased alfalfa productivity and physiological P use efficiency by enhancing total root length and root surface area while reducing carboxylate release. Under low-P conditions, HPE genotypes with AMF symbiosis showed higher shoot DW, greater mycorrhizal responsiveness, thicker and more robust roots, as well as increased carboxylate release compared with LPE genotypes. We conclude that exploitation of the dominant species in indigenous AMF populations and breeding of crop genotypes with high mycorrhizal responsiveness show promising avenues with which to improve forage productivity and alleviate P limitation in modern agricultural ecosystems.

Plants form mycorrhizal symbioses to enhance nutrient acquisition, yet the biophysical principles governing carbon and nutrient exchange remain unclear. Here, we develop a theory of bi-directional carbon–nutrient transfer that integrates root anatomy, energetic costs, and mycorrhizal positioning. We show that nutrient uptake per unit carbon or energy investment declines with increasing root diameter due to higher carbon demands across thicker cortical tissues. Mycorrhizal fungi mitigate this constraint by enabling more carbon-efficient nutrient uptake, particularly when arbuscules are positioned in inner cortical layers. This spatial optimization minimizes the carbon cost of transporting nutrients to the stele. Our framework reconciles anatomical variation, symbiotic structure, and functional efficiency across root types and mycorrhizal strategies and offers a new lens for understanding the coevolution between roots and mycorrhizal fungi.