Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs that rely on host-derived symbiotic carbohydrates. However, it remains unclear whether symbiotic AMF can access exogenous non-symbiotic carbon sources, complicating our understanding of their relationship with host plants. Here, we investigated the direct uptake of exogenous 13C1-labeled myristate by three symbiotic AMF species (Rhizophagus irregularis, R. intraradices, and R. diaphanous) and assessed their growth responses using AMF-carrot hairy root co-culture systems. Furthermore, we explored the environmental distribution of myristate, and evaluated the impact of exogenous myristate on the carbon-phosphorus exchange between R. irregularis and alfalfa or rice in a greenhouse experiment. Symbiotic AMF can absorb exogenous myristate, as evidenced by 13C enrichment and transcriptional activation of fatty acid transport and metabolism genes in AMF extraradical hyphae. Myristate is commonly present in various soil and plant environments, and its application increased both intraradical and extraradical fungal biomass, possibly linked to suppressed mycorrhizal-activated defense responses in host roots. Unexpectedly, exogenous myristate reduced the mycorrhizal phosphorus benefits for both alfalfa and rice and decreased their symbiotic carbon allocation to root-colonizing AMF, although these effects varied with soil phosphorus conditions. These findings provide new insights into understanding and manipulating the nutritional interactions between AMF and host plants.

Many plant endosymbionts are facultative, switching between host-associated and free-living stages. Extensive genomic and experimental studies suggest that adaptation during the saprophytic, off-host phase, rather than adaptation to hosts, primarily constrains the biogeographic distribution of these microbes. To test this hypothesis, we analyzed the growth capacities and genomic features of 38 Sinorhizobium and Ensifer strains isolated from the nodules of Medicago lupulina (black medic), collected from two regions with distinct thermal environments. The warmer region is predominantly inhabited by S. meliloti, while S. medicae and Ensifer strains are more common in the cooler region. Laboratory assays demonstrated that at 40 °C, the upper temperature limit of their region of origin, S. meliloti remained viable, albeit with reduced growth, whereas S. medicae and Ensifer strains failed to grow under heat stress. Comparative genomics revealed isolation-by-distance in both the core and accessory genomes, particularly in S. meliloti in the warmer region, which exhibits less within-region thermal variation. This is consistent with an isolation-by-distance model where population divergence is governed by restricted gene flow. These findings suggest that metabolic constraints shape the regional distribution of this facultative microbial symbiont, while limited gene flow influences local population structure.

Lateral roots (LR) and the root nodules (RN) of legumes are structurally related and the decision processes leading to RN formation involve signal exchange with the shoot. In order to disentangle these processes, we established a quantitative assay for LR formation in hairy root liquid cultures (HRLC) for the legume Lotus japonicus. In HRLC, ectopic expression of SymRK, or deregulated, auto-active versions of CCaMK and Cyclops stimulated LR formation in a NIN-dependent manner, but spontaneous RN were never observed. It appears that the previously described spontaneous RN formation induced by these versions requires the presence of the shoot. Interestingly, CCaMKT265D increased LR number in a cyclops mutant, revealing the presence of additional CCaMK targets mediating LR formation. Constitutive and ectopic expression of NIN under the ubiquitin promoter resulted in a significant increase in LR number. We compared the responsiveness of two Rosaceae that have either retained NIN (Dryas drummondii) or lost it (Fragaria vesca) to stimulation with the constitutively active variant CCaMK1−314. Intriguingly, CCaMK1−314 was able to increase LR formation in Dryas but not in Fragaria, pointing to consequences of the evolutionary loss of NIN on root architecture. Taken together our data provide evidence for NIN as a molecular link between symbiosis-signaling and LR formation. Non-inoculated nsp1 and nsp2 mutant plants as well as HRLC of these mutants exhibited increased LR densities that were no further increased by expression of CCaMK1-314. We propose a model in which LR density is balanced by the activation of NIN expression by SymRK and CCaMK and the LR suppressing activity of NSP1 and NSP2.

Phosphorus (P) is a primary mineral nutrient essential for the growth and productivity of many crop plants. Although abundant in nature, its bioavailability is limited due to the prevalence of insoluble forms. An alternative for meeting agricultural P demand is the application of P-solubilizing microorganisms (PSMs), which mobilize it. Although progress has been made in the study of PSMs, knowledge gaps still exist regarding their role in sustainable agriculture. Therefore, this review examines the barriers to P acquisition in low-solubility soils and highlights recent advances in understanding the mechanisms of P solubilization mediated by plant-associated bacteria and fungi. The molecular strategies involved in the uptake and transport of P from soil in plants are also analyzed. Bacteria from genera such as Bacillus, Pseudomonas, and Streptomyces, as well as fungi including arbuscular mycorrhizal fungi, Aspergillus spp., and Penicillium spp., employ various approaches to solubilize P, leading to improved plant nutrition. These mechanisms, which include the production of organic acids, cation chelation, proton exudation, and phosphatase activity, can be inferred from experimental approaches or genome mining strategies. The role of PSMs as plant growth promoters and enhancers of plant nutrition across diverse environmental conditions are also discussed. Finally, we propose the integration of PSM consortia as multifunctional bioinoculants to promote sustainable agricultural practices.

The advent of endosymbiosis underlies evolutionary innovation and ecosystem function. However, whether free-living partners tend to benefit or exploit each other during incipient endosymbiosis remains a dilemma. Rhizobia bacteria are plant endosymbionts capable of initiating root nodules and fixing nitrogen due to genes carried on mobile genetic elements (MGEs) such as the symbiosis island (SI). We conjugated marked SIs into the genomes of nonnodulating strains, which was sufficient to generate de novo root nodule-forming endosymbionts. Most novel endosymbionts originated as commensals that incurred no detectable costs to host plants, in contrast to predictions of exploitation. In fact, a third of endosymbionts originated as nitrogen fixing mutualists. Consistent with phylogenetic limits to transfer of MGE function, novel endosymbionts derived from more closely related SI donor and recipient strains showed greater nitrogen fixation. However, we did not detect phylogenetic limits to SI transmission, which could reflect selfish selection for generalized horizontal transfer of this MGE. In fact, the SI was able to displace other genomic elements residing at its characteristic tRNA gene insertion site. We thus provide genetic, genomic, and functional evidence of how MGEs can potentiate and constrain major evolutionary transitions to expand bacterial niches, with cascading effects on host organisms.

The demands for food are rising worldwide due to the planet’s finite resources and changing environment. The population of Earth is growing drastically every day, which may be in charge of ensuring global food security, which will ultimately have an impact on farming methods and human habitation. All living things require nitrogen, although it is the most restrictive in ecosystems. Despite the significant contribution of synthetic fertilizers, the amount of nitrogen needed for food production increases yearly, and the misuse of agrochemicals threatens the sustainability of agriculture and the health of the soil. Biological nitrogen fixation (BNF) is one solution to this issue. BNF accounts for about 60% of Earth’s fixed nitrogen. As a result, it is becoming more and more important to optimize BNF in agriculture to satisfy the expanding global demand for food production. BNF offers numerous practical advantages to agroecosystems. It is an essential method for enhancing soil N availability to enhance crop growth and refilling the reservoirs of soil organic N. A thorough understanding of the BNF mechanism would enable the transfer of this capacity to other non-fixing microbes or highly valuable non-leguminous plants. This chapter includes the history and contributions from both global and regional sources, gives the current understanding of BNF, the process of the global nitrogen fixation cycle, advantages of BNF, effectiveness in agriculture, commercialization of nitrogen as biofertilizers, limitations, and their prospects. It also addresses potential ways to increase BNF for potential benefit and draws attention to the opportunities and challenges on the adoption of BNFs.

Introduction: Microbial interactions in the rhizosphere are fundamental to soil health, plant growth, and ecosystem stability. Among these interactions, metabolic cross-feeding, the exchange of metabolites between microorganisms, plays a critical role in shaping microbial community structure and function. This study investigates the metabolic interplay between two PGPR (Priestia megaterium and Bacillus licheniformis), focusing on how metabolite exchange influences bacterial growth and metabolic reprogramming.

Methods: An integrative metabolomics approach was employed to examine metabolic exchanges between P. megaterium and B. licheniformis. Cultures were grown individually and in co-culture, followed by extraction of extracellular metabolites at distinct growth phases. Metabolomic profiling was conducted using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Data preprocessing and feature extraction were followed by molecular networking and multivariate statistical analysis to identify discriminant metabolites. Pathway enrichment and functional annotation were performed using KEGG and MetaboAnalyst to pinpoint key metabolic pathways altered during cross-feeding interactions.

Results and discussion: Metabolomic analysis revealed distinct metabolic shifts driven by reciprocal metabolite exchange between P. megaterium and B. licheniformis. Metabolites secreted by B. licheniformis exhibited a growth-inhibitory effect on P. megaterium, while those from P. megaterium stimulated the growth of B. licheniformis. Multivariate data analysis demonstrated significant variation in the production of amino acids, fatty acids, and cyclic lipopeptides across growth phases. Pathway enrichment identified the phenylalanine, tyrosine, and tryptophan biosynthesis (PTTB) pathway as a central metabolic hub mediating these interactions. The regulation of aromatic amino acid metabolism appeared critical in determining whether interactions were cooperative or competitive. The observed metabolic reprogramming reflects adaptive strategies employed by PGPR to thrive under nutrient-limited conditions, balancing cooperation and competition through selective metabolite secretion. These findings offer systems-level insight into the mechanistic basis of cross-feeding and highlight the potential of integrating metabolomics to guide microbial consortia design for agricultural applications. Understanding these metabolic determinants supports the development of tailored biofertilizer formulations that enhance soil fertility and plant resilience.

Conclusion: This study demonstrates that metabolite cross-feeding induces distinct metabolic reprogramming between Priestia megaterium and Bacillus licheniformis, underpinning adaptive interactions in nutrient-limited environments. These findings provide a mechanistic basis for microbial consortia design and biofertilizer optimization. Future multi-omics and systems-level investigations should elucidate the genetic and regulatory determinants of these metabolic exchanges, advancing sustainable biotechnological innovations aligned with SDGs 9, 12, and 13.

Legume plants have the capacity to incorporate atmospheric nitrogen by establishing an endosymbiotic interaction with soil bacteria resulting in the formation of nitrogen-fixing nodules. Bacteria are internalized through a tightly regulated process that requires membrane remodelling and vesicle trafficking, which are controlled by small GTPases. Members of the ARF family of GTPases mediate vesicle budding in a wide range of biological processes; however, the modulation of ARF members, their subcellular localization and the formation of complexes with other proteins during the root nodule symbiosis has not been investigated. Here, to identify proteins that physically interact with MtARFA1, a yeast two hybrid screening was performed using a cDNA library of Medicago truncatula roots inoculated with Sinorhizobium meliloti. One of the identified MtARF1 interactors is a protein that possesses a BTB/POZ domain. BTB/POZ domains are present in substrate-specific adaptors that form complexes with the Ubiquitin ligase E3 Cullin3 (CUL3), thus the interactor was designated as M. truncatula CUL3 substrate-adaptor protein 1 (MtCSP1). Physical interaction between MtARF1 and MtCSP1 was verified in planta by co-immunopurification assays and bimolecular fluorescence complementation, revealing that the interaction takes place in vesicles of the late endosome. The MtCSP1 promoter is active in lateral roots and in the meristem of indeterminate nodules. Phenotypic analysis of transgenic roots with altered mRNA levels of MtCSP1 evidenced the requirement of this gene for the progression of rhizobial infection and nodule organogenesis. This work establishes a link between small GTPases and protein degradation by the ubiquitin system in the context of the nitrogen-fixing symbiosis.

This thesis tests the hypothesis that phosphorus (P) fertiliser addition can optimise nitrogen (N2) fixation processes and increase soybean yield. Using a combination of controlled environment studies, field trial and global meta-analysis, this thesis aids in achieving sustainable soybean production through building improved understanding of the effects of P fertiliser addition to inform future fertiliser guidelines, crop models and management practices. Global meta-analysis showed an increase in soybean response to P fertiliser addition with seed yield increasing by 25%. This also highlighted the complexity of soybean yield response to P fertiliser, with a several key management and environmental conditions having a significant effect, including soil P concentration, pH, fertiliser type and rate of application and climatic conditions – indicating soybean yield cannot be increased by single P fertiliser applications alone. Controlled environment studies revealed P addition significantly increased nitrogen (N2) fixation. Key nodule traits significantly correlated with shoot N; however, further work should examine the mechanistic pathways driving the increase in nodule formation. Interestingly, controlled environment studies revealed nodule function was not influenced by P fertiliser addition. Instead, regulatory mechanisms such as maintenance of nodule P concentration and leghaemoglobin concentration under low P conditions maintained N2 fixation. Through combined analysis of multiple growth parameters and measures of plant physiology, seed yield was found to increase under P fertiliser addition. Seed P concentration also increased following P fertiliser addition. Results of this thesis contribute to our understanding of soybean response to P fertiliser addition, particularly the improvement to key nodule traits to improve N2 fixation and the partitioning and remobilisation of resources to improve yield. This now needs to be upscaled at differing environmental and management conditions and incorporated into crop models to ensure the sustainable use of P fertiliser in soybean production globally.

Arbuscular mycorrhizal symbiosis plays a pivotal role in nutrient acquisition and stress tolerance, making its regulation crucial for sustainable crop productivity. This review synthesizes current advances in understanding the molecular and physiological factors governing AM symbiosis, with emphasis on transcriptional, hormonal, and nutrient-mediated regulation. From pre-symbiotic signaling to root colonization and arbuscule development, AM formation is orchestrated by a complex network of molecular interactions. Transcription factors, including those with GRAS domains (e.g., NSP1, NSP2, RAM1, and DELLA), and other regulators such as MYB, SPX, WRKY, and CYCLOPS/IPD3, serve as central modulators of symbiosis-related gene expression. Phytohormones, including strigolactones, salicylic acid, and abscisic acid, generally promote symbiosis, whereas gibberellins and ethylene act as inhibitors; cytokinin exerts context-dependent effects. Nutrient status also modulates AM formation—low phosphorus and nitrogen promote, while high nutrient availability suppresses colonization. Collectively, these insights reveal the integrative regulatory networks driving AM symbiosis and offer new avenues to optimize symbiotic efficiency for enhanced plant growth and agricultural sustainability.

Legume–rhizobium symbiosis relies on the precise integration of receptor-mediated signaling with dynamic membrane trafficking to initiate infection threads (ITs) and accommodate bacterial infection via root hairs. Here, we identify the soybean Qa-SNARE GmSYNTAXIN111a (GmSYP111a), a close paralog of the cytokinesis-specific protein KNOLLE, as a critical regulator in symbiotic infection. A kinase client (KiC) assay revealed that GmSYP111a is a direct phosphorylation substrate of the receptor kinase GmSymRKβ (also known as DMI2 or NORK in other legumes), which modifies Ser-8 at its N-terminus. BiFC, co-immunoprecipitation, and in vitro kinase assays confirmed that GmSymRKβ phosphorylates GmSYP111a without disrupting their basal association, but phosphorylation alters the subcellular distribution of the complex. In soybean root hairs, GmSYP111a and GmSymRKβ co-localize to the plasma membrane, and Nod factor perception induces clathrin-mediated endocytosis accompanied by GmSYP111a re-localization into intracellular vesicles. A non-phosphorylatable mutant (GmSYP111aS8A) showed markedly reduced internalization and a corresponding decrease in infection events, resulting in impaired IT formation and a phenotype resembling GmSymRKβ-RNAi roots. In contrast, a phosphomimetic variant (GmSYP111aS8D) displayed partial vesicular recruitment upon infection. Functional analyses further demonstrated that GmSYP111a, but not its paralog SYP111b, is indispensable for nodule initiation in soybean, with this role conserved in Lotus japonicus. Collectively, our results show that GmSymRKβ-mediated phosphorylation of GmSYP111a functions as a molecular switch that links symbiotic signaling to clathrin-dependent endocytosis. This mechanism coordinates localized membrane remodeling at infection sites, parallels the role of KNOLLE in cytokinesis, and highlights how duplication and neo-functionalization of GmSYP111a contributed to the evolution of legume-specific symbiotic pathways.

Arbuscular mycorrhizal (AM) fungi enhance plant nutrient acquisition from soil; however, their ability to exploit organic nutrient forms in the absence of associated microbes capable of mineralization remains unclear. To test if the AM fungi carry their beneficial bacterial partners into nutrient-rich zones, we conducted three controlled experiments manipulating the microbial inputs, diversity and composition in plant–AM fungus–soil systems, ranging from open pots to semi-sterile mesocosms. We manipulated soil microbial diversity by imposing a microbial diversity gradient (complex communities fractionated by size, resulting in fractions passing through 1 µm to 1000 µm sieves) and cultivated Andropogon gerardii in previously sterilized substrate together with a bacterial-free Rhizophagus irregularis. In each experiment, 15N‐labeled chitin or mineral nitrogen (N) compartments were installed in the root‐free zone of each mesocosm. With decreasing microbial inputs into the root-free zone, the N uptake from chitin to plants, facilitated by the AM fungal hyphae, decreased. Upon complete absence of microbes in the root-free zone, AM hyphal foraging preferences assessed by quantitative PCR indicated that exploration of the mineral N compartments was more effective than that of the chitin compartments. AM fungal hyphae were ineffective in priming mineralization of organic N even if provided with complex soil microbiomes at a distance from the compartment. In summary, chitin-enriched compartments become attractive for the AM fungi only when previously mineralized by competent microbes. Such microbes, however, were not effectively transported to spatially restricted organic resources in soil via AM hyphal highways in our experiments.

Symbiotic mycorrhizal fungi are crucial drivers of ecosystem functioning through their associations with plants. Ecosystems dominated by different types of mycorrhizal fungi, such as ectomycorrhizal fungi (EM) and arbuscular mycorrhizal (AM) fungi, often exhibit variation in plant productivity. However, the mechanisms underlying these differences and their dependence on environmental context remain unclear. Furthermore, the lack of robust, fine-scale evidence linking plant productivity to measurable indicators of mycorrhizal colonization or dominance, together with limited information on environmental variables, constrains accurate global-scale modeling of mycorrhizal effects on ecosystem functioning. In this study, we synthesize existing knowledge on the competitive and complementary interactions between the two dominant mycorrhizal types. Building on this synthesis, we propose a new conceptual framework to describe the context-dependent and often idiosyncratic nature of these interactions. We then present case studies and a meta-analysis spanning local to global scales, examining how vegetation biomass is related to mycorrhizal colonization or dominance under different environmental conditions. Our findings indicate that mycorrhizal types and environmental variables interactively shape ecosystem productivity in a dynamic and resilient manner. This work offers a new foundation for spatially explicit, locally informed assessments of how mycorrhizal influence vegetation productivity across contrasting environmental constraints.