BACKGROUND: The symbiosis among plants, rhizobia, and arbuscular mycorrhizal fungi (AMF) is one of the most well-known symbiotic relationships in nature. However, it is still unclear how bilateral/tripartite symbiosis works under resource-limited conditions and the diverse genetic backgrounds of the host.
RESULTS: Using a full factorial design, we manipulated mungbean accessions/subspecies, rhizobia, and AMF to test their effects on each other. Rhizobia functions as a typical facilitator by increasing plant nitrogen content, plant weight, chlorophyll content, and AMF colonization. In contrast, AMF resulted in a tradeoff in plants (reducing biomass for phosphorus acquisition) and behaved as a competitor in reducing rhizobia fitness (nodule weight). Plant genotype did not have a significant effect on AMF fitness, but different mungbean accessions had distinct rhizobia affinities. In contrast to previous studies, the positive relationship between plant and rhizobia fitness was attenuated in the presence of AMF, with wild mungbean being more responsive to the beneficial effect of rhizobia and attenuation by AMF.
CONCLUSIONS: We showed that this complex tripartite relationship does not unconditionally benefit all parties. Moreover, rhizobia species and host genetic background affect the symbiotic relationship significantly. This study provides a new opportunity to re-evaluate the relationships between legume plants and their symbiotic partners.

Nuclear Ca²⁺ signaling is crucial for symbiotic interactions between legumes and beneficial microbes, such as rhizobia and arbuscular mycorrhizal fungi. Key to generating repetitive nuclear Ca²⁺ oscillations are the ion channels DMI1 and CNGC15. Despite over 20 years of research on symbiotic nuclear Ca²⁺ spiking, important questions remain, including the exact function of the DMI1 channel. This review highlights recent developments that have filled knowledge gaps regarding the regulation of CNGC15 and its interplay with DMI1. We also explore new insights into the evolutionary conservation of DMI1-induced symbiotic nuclear Ca²⁺ oscillations and the roles of CNGC15 and DMI1 beyond symbiosis, such as in nitrate signaling, and discuss new questions this raises. As we delve deeper into the regulatory mechanisms and evolutionary history of these ion channels, we move closer to fully understanding the roles of nuclear Ca²⁺ signaling in plant life.

Biological nitrogen fixation (BNF) by symbiotic bacteria plays a vital role in sustainable agriculture. However, current quantification methods are often expensive and impractical. This study explores the potential of Raman spectroscopy, a non-invasive technique, for rapid assessment of BNF activity in soybeans. Raman spectra were obtained from soybean plants grown with and without rhizobia bacteria to identify spectral signatures associated with BNF. δN15 isotope ratio mass spectrometry (IRMS) was used to determine actual BNF percentages. Partial least squares regression (PLSR) was employed to develop a model for BNF quantification based on Raman spectra. The model explained 80% of the variation in BNF activity. To enhance the model\'s specificity for BNF detection regardless of nitrogen availability, a subsequent elastic net (Enet) regularisation strategy was implemented. This approach provided insights into key wavenumbers and biochemicals associated with BNF in soybeans.

Legume plants establish an endosymbiosis with nitrogen-fixing rhizobia bacteria, which are taken up from the environment anew by each host generation. This requires a dedicated genetic program on the host side to control microbe invasion, involving coordinated reprogramming of host cells to create infection structures that facilitate inward movement of the symbiont. Infection initiates in the epidermis, with different legumes utilizing distinct strategies for crossing this cell layer, either between cells (intercellular infection) or transcellularly (infection thread infection). Recent discoveries on the plant side using fluorescent-based imaging approaches have illuminated the spatiotemporal dynamics of infection, underscoring the importance of investigating this process at the dynamic single-cell level. Extending fluorescence-based live-dynamic approaches to the bacterial partner opens the exciting prospect of learning how individual rhizobia reprogram from rhizospheric to a host-confined state during early root infection.

The two-component regulatory system CenK-CenR has recently emerged as a regulator of cell envelope and cell division processes in the alpha-proteobacteria. In Sinorhizobium meliloti, CenK-CenR regulates the expression of SrlA, a thioredoxin-domain protein of unknown function. Deletion of srlA causes sensitivity to salt and oxidizing agents on solid growth medium. In this work, we report that the response regulator CenR, but not the histidine kinase CenK, is essential for cell viability in S. meliloti. We also demonstrate that phosphorylation of the target residue D55 is not required for viability, suggesting that the unphosphorylated transcription factor sufficiently regulates expression of one or more essential genes in the genome. Using transcription assays and phenotype testing we examine CenK-CenR-dependent activation of the srlA promoter and demonstrate its absolute dependence on phosphoryl-CenR for activity and that the CenR substitution D55E acts as a phosphomimetic that partially restores activity at the srlA promoter in the absence of phosphorylation by CenK. Finally, we report a mutational analysis of the CenR binding site in the srlA promoter required for transcriptional activation.

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii\'s capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga\'s fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide-a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.

Three strains of Gram-negative bacterium, Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson\'s correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility.

Herbarium specimens are increasingly being used as sources of information to understand the ecology and evolution of plants and their associated microbes. Most studies have used specimens as a source of genetic material using culture-independent approaches. We demonstrate that herbarium specimens can also be used to culture nodule-associated bacteria, opening the possibility of using specimens to understand plant-microbe interactions at new spatiotemporal scales. We used historic and contemporary nodules of a common legume, Medicago lupulina, to create a culture collection. We were able to recover historic bacteria in 15 genera from three specimens (collected in 1950, 2004, and 2015). This work is the first of its kind to isolate historic bacteria from herbarium specimens. Future work should include inoculating plants with historic strains to see if they produce nodules and if they affect plant phenotype and fitness. Although we were unable to recover any Ensifer, the main symbiont of Medicago lupulina, we recovered some other potential nodulating species, as well as many putative growth-promoting bacteria.

Plant-microbe symbioses require intense interaction and genetic coordination to successfully establish in specific cell types of the host and symbiont. Traditional RNA-seq methodologies lack the cellular resolution to fully capture these complexities, but single-cell and spatial transcriptomics (ST) are now allowing scientists to probe symbiotic interactions at an unprecedented level of detail. Here, we discuss the advantages that novel spatial and single-cell transcriptomic technologies provide in studying plant-microbe endosymbioses and highlight key recent studies. Finally, we consider the remaining limitations of applying these approaches to symbiosis research, which are mainly related to the simultaneous capture of both plant and microbial transcripts within the same cells.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg.
RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg.
CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.