Plant-Microbe Symbiosis

Bacteroid inorganic phosphorus (Pi) metabolism in the Rhizobium‐legume symbiosis differs between indeterminate and determinate legume nodules. In contrast to alfalfa bacteroids, bean ( Phaseolus vulgaris ) bacteroids exhibit high levels of alkaline phosphatase (AP), the native reporter enzyme for the bacterial Pi stress response. 14C and 32Pi whole plant labelling techniques were used in conjunction with diagnostic mutants (lacking AP or lacking high affinity Pi transport) to assess the relative importance of the Pi stress response in Rhizobium tropici bacteroids during symbiosis. The AP‐ mutant was not defective for symbiosis and did not differ from wildtype bacteroids for Pi acquisition. 14C‐CO2 feeding to host plants revealed 14C‐carbon uptake and accumulation in AP‐ mutant bacteroids, and their nodules were increased relative to wildtype bacteroids, implying that organo‐P compounds may account for meaningful levels of carbon exchange between symbionts. 32Pi tracer experiments implied that the high affinity transporter is important to bacteroid Pi acquisition and symbiotic performance in determinate nodules, but that the symbiosome Pi concentration does not meet the capacity of the high affinity transporter. 32P tracer work also illustrated that Pi taken up into the nodule does not remain in the nodule, but rather is redistributed to the host.

The CLAVATA signaling pathway regulates plant development and plant–environment interactions. CLAVATA signaling consists of mobile, cell-type or environment-specific CLAVATA3/ESR-related (CLE) peptides, which are perceived by a receptor complex consisting of leucine-rich repeat receptor-like kinases such as CLAVATA1 and receptor-like proteins such as CLAVATA2, which often functions with the pseudokinase CORYNE (CRN). CLAVATA signaling has been extensively studied in various plant species for its developmental role in meristem maintenance. In addition, CLAVATA signaling was implicated in plant–microbe interactions, including root nodule symbiosis and plant interactions with mutualistic arbuscular mycorrhizal (AM) fungi. However, knowledge on AM symbiosis regulation by CLAVATA signaling is limited. Here, we report a dual role for Medicago truncatula CRN in development and plant–microbe interactions. In shoots, MtCRN modulates inflorescence meristem branching. In roots, the MtCRN promoter is active in vascular tissues and meristematic regions. In addition, MtCRN expression is activated in cortex cells colonized by AM fungi and negatively regulates root interactions with these microbes in a nitrogen-dependent manner; negative AM symbiosis regulation by CRN was also observed in the monocot Zea mays, suggesting this function is conserved across plant clades. We further report that MtCRN functions partially independently of the AM autoregulation signal MtCLE53. Transcriptomic data revealed that M. truncatula crn roots display signs of perturbed nutrient, symbiosis, and stress signaling, suggesting that MtCRN plays various roles in plant development and interactions with the environment.

Crop domestication has revolutionized food production but increased agriculture’s reliance on fertilizers and pesticides. We investigate differences in the rhizosphere microbiome functions of wild and domesticated rice, focusing on nitrogen (N) cycling genes. Shotgun metagenomics and real-time PCR reveal a higher abundance of N-fixing genes in the wild rice rhizosphere microbiomes. Validation through transplanting rhizosphere microbiome suspensions shows the highest nitrogenase activity in soils with wild rice suspensions, regardless of planted rice type. Domesticated rice, however, exhibits an increased number of genes associated with nitrous oxide (N2O) production. Measurements of N2O emissions in soils with wild and domesticated rice are significantly higher in soil with domesticated rice compared to wild rice. Comparative root metabolomics between wild and domesticated rice further show that wild rice root exudates positively correlate with the frequency and abundance of microbial N-fixing genes, as indicated by metagenomic and qPCR, respectively. To confirm, we add wild and domesticated rice root metabolites to black soil, and qPCR shows that wild rice exudates maximize microbial N-fixing gene abundances and nitrogenase activity. Collectively, these findings suggest that rice domestication negatively impacts N-fixing bacteria and enriches bacteria that produce the greenhouse gas N2O, highlighting the environmental trade-offs associated with crop domestication.

Receptor signalling determines cellular responses and is crucial for defining specific biological outcomes. In legume root cells, highly similar and structurally conserved chitin and Nod factor receptor kinases activate immune or symbiotic pathways, respectively, when chitinous ligands are perceived1. Here we show that specific amino acid residues in the intracellular part of the Nod factor receptor NFR1 control signalling specificity and enable the distinction of immune and symbiotic responses. Functional investigation of CERK6, NFR1 and receptor variants thereof revealed a conserved motif that we term Symbiosis Determinant 1 in the juxtamembrane region of the kinase domain, which is key for symbiotic signalling. We show that two residues in Symbiosis Determinant 1 are indispensable hallmarks of NFR1-type receptors and are sufficient to convert Lotus CERK6 and barley RLK4 kinase outputs to enable symbiotic signalling in Lotus japonicus.

This study investigated the effects of different tillage practices (conventional tillage, no-tillage, stover dislocation, and deep tillage) on endophytic and rhizospheric microbial communities in a 9-year maize stover returning experiment in Inner Mongolia using Illumina MiSeq sequencing. Results showed that tillage practices significantly impacted microbial community composition, functional guilds, and interaction networks, with effects varying across plant growth stages. No-tillage (NT) increased denitrifying bacteria and plant pathogens, while stover dislocation (SD) and deep tillage (DT) enhanced nitrogen-fixing bacteria. Network analysis indicated that high-intensity tillage (SD and DT) improved microbial community stability, but DT also stabilized harmful microorganisms. Overall, SD was deemed the most suitable method. Future research should focus on long-term impacts and integrate more ecosystem health indicators for comprehensive evaluation.

The rhizosphere is a dynamic ecosystem that hosts diverse microbial communities, essential for nutrient cycling, and promoting plant health and resistance to environmental stresses and pathogens. Understanding the communication strategies between plant roots and these microbial communities is vital for sustainable agriculture, as these interactions can enhance crop resilience and productivity while reducing the need for chemical fertilizers. Extensive research has focused on how soybean plants shape the rhizosphere microbiota and the signaling processes that promote these interactions; however, many influencing factors, particularly environmental stresses, remain unexplored. Key elements, including soybean genetics, growth development stages, soil properties, agricultural practices, and environmental conditions, all play crucial roles in shaping microbial symbioses. This review examines the intricate interactions between soybean and their rhizospheric microbiota, emphasizing how various stresses affect these relationships. It also discusses the role of secondary metabolites from both microbes and soybean in facilitating communication, alongside other factors that significantly influence these microbial interactions and soybean productivity.

Aims
The role of strigolactones (SL) in molecular signalling with arbuscular mycorrhizal fungi (AMF) has been intensively investigated, but the impact of this interaction on defence mechanisms in soil microbiota and plant-pathogen systems is not fully understood. The aim of this study was to investigate the effects of SL and AMF treatments on molecular defence strategies and growth of wheat through soil–plant interactions during Fusarium culmorum (FC) infection.

Methods
Wheat varieties grown with and without AMF were inoculated with SL (rac-GR24; 15 µM) and FC (1 × 10⁶ spores ml−1) at the early stage of symbiosis. To evaluate symbiotic activity and infection effects in soil, expression levels of defence genes (PAL, PR2, PR3, PR4) and growth genes (TEF, Rubisco) were analysed by qRT-PCR before and after FC inoculation.

Results
SL was found to increase AMF activity in soil media and enhance symbiosis. This interaction improved both plant growth and defence responses. Increased expression of TEF and Rubisco genes favoured plant growth, while decreased expression of PR2 facilitated the entry of AMF hyphae into root tissues. Increased expression of PR3 enhanced the production of antifungal enzymes, while down-regulation of PR4 optimised energy utilisation through a ‘priming’ effect. Expression of the PAL gene showed cultivar-specific changes.

Conclusions
SL and AMF treatments significantly reduced disease severity during FC infection by optimising wheat defence and growth responses at the molecular level through the plant-soil system. These findings highlight the potential of SL and AMF for sustainable agricultural practices.

Background and Aims
The establishment of bioenergy plantations as short-rotation coppice poplar systems has been proposed as a sustainable strategy to mitigate climate change through carbon capture. This study evaluates changes in soil organic carbon (SOC) after 12 years of poplar cultivation on former cropland and grassland in Belgium using repeated soil sampling to assess SOC stock changes and in-growth cores to identify carbon input pathways.

Methods
Using isotope tracing and in-growth cores with treatments excluding roots, mycorrhizae and above-ground inputs, we quantified the contributions of roots, mycorrhizae, and dissolved organic matter to new SOC formation and their interaction with the mineralization of native SOC.

Results
Results showed a significant increase in SOC in former croplands while grasslands experienced a slight SOC reduction, highlighting the influence of previous land use on SOC accrual potential. Root-derived inputs surpassed mycorrhizal contributions to SOC formation although both played a role in achieving a positive SOC balance.

Conclusion
This study underscores the critical role of roots in SOC accumulation and the importance of initial soil conditions when designing SOC accrual strategies through bioenergy plantations.

Plant cells constantly monitor both their surroundings and internal state, enabling dynamic responses to developmental and environmental cues. Central to this sensing capacity are membrane-bound receptor kinases (RKs), one of the largest and most functionally diverse protein families in plants. RKs detect a broad spectrum of signals, including microbial molecules, endogenous ligands, and changes in cellular status. They share a conserved modular architecture: an extracellular domain for signal perception, a single transmembrane domain, and a conserved cytoplasmic kinase domain that initiates intracellular signalling. Although structurally analogous to their animal counterparts, plant RKs evolved independently [1,2]. Their functional diversity stems primarily from their extracellular domains, with over 20 distinct structural classes described to date [1–3]. The repertoire of RKs expressed in a given cell type varies, as some RKs are ubiquitously expressed across tissues, while others are restricted to specific organs or developmental contexts [4].

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone.

Background
Legumes-rhizobia symbiosis has high specificity regulated by a specific class of genes, such as Rj4. Rj4 encodes a thaumatin-like protein belonging to the PR-5 family that restricts soybean from nodulation with many strains of Bradyrhizobium elkanii. How Rj4, a member of broad-spectrum resistance family, specifically regulates nodulation remains unclear. To uncover the molecular mechanism of Rj4, current study integrated transcriptome and proteome to analyze the downstream regulatory pathways and key genes mediated by Rj4, through investigating the gene expression and protein abundance in the roots of soybean BARC2 (Rj4/Rj4) after 0, 6 and 24 h post inoculation (hpi) with B. elkanii USDA61.

Results
The results showed that a total of 1660 differentially expressed genes (DEGs) and 2633 differentially abundance proteins (DAPs) were identified, in which resistance-related genes and symbiosis-related genes showed opposite expression trend. The key symbiosis-related genes, such as ENOD55, PUB1, and NFP, were up-regulated at 6 hpi but down-regulated at 24 hpi compared with control group. Conversely, the expression of most plant immunity related genes exhibited an initial decrease at 6 hpi followed by an increase at 24 hpi, suggested Rj4 restricted host plant from nodulation in early process through induction of plant immunity related genes and pathways. The key genes involved in plant immunity included pattern-triggered immunity (PTI)-related genes, such as pattern recognition receptors FLS2 and EFR, mitogen-activated protein kinases (MAPK) signaling pathway MPKs, calcium ion related genes CNGCs, CaMCML, and ROS related genes RBOH, as well as effector-triggered immunity (ETI) related resistance (R) genes RPM1 and Ptis, plant hormone signal transduction related genes JAZ and TGA, and flavonoid and isoflavonoid biosynthesis gene Cyps. We validated 8 DEGs via qRT-PCR, showing consistent trends with RNA-Seq (Spearman r > 0.9).

Conclusions
These findings provide new insights into how plant immunity inhibits legume-rhizobial symbiosis in the Rj4-mediated regulatory network, and provide key candidate genes to study legume-rhizobial nodulation specificity for future research.

Symbioses have been fundamental to colonization of terrestrial ecosystems by plants and their evolution. Emergence of the ancient arbuscular mycorrhizal symbiosis was followed by the diversification of alternative intracellular symbioses, such as the ericoid mycorrhizae (ErM). We aimed at understanding how these diversifications occurred. We sequenced the genomes of ErM-forming liverworts, and reconstituted symbiosis under laboratory conditions. We demonstrated the existence of a nutrient-regulated symbiotic state that enables ErM and underlies intracellular colonization of plant tissues. Comparative transcriptomic analyses identified an ancestral gene module associated with intracellular symbiosis beyond ErM. Genetic manipulations in the liverwort Marchantia paleacea, phylogenetics and transactivation assays demonstrated its essential function for intracellular symbiosis. We conclude that plant have maintained, and convergently recruited, an ancestral gene module for intracellular symbioses.

Nitrogen is essential for plant growth, yet its availability often limits agricultural productivity. Some legumes have evolved a unique ability to form symbiotic relationships with nitrogen-fixing soil bacteria called rhizobia, enabling them to thrive in nitrogen-deficient soils. In five legume clades, an exploitive strategy has evolved in which rhizobia undergo Terminal Bacteroid Differentiation (TBD), where the bacteria become larger, polyploid, and have a permeabilized membrane. Terminally differentiated bacteria are associated with higher N2-fixation and, thus, a higher return on investment to the plant. In several members of the IRLC (Inverted Repeat-Lacking Clade) and the Dalbergioid clades of legumes, this differentiation process is triggered by a set of apparently unrelated plant antimicrobial peptides with membrane-damaging activity, known as Nodule-specific Cysteine-Rich (NCR) peptides. However, whether NCR peptides are also implicated in symbiotic TBD in other legume clades and whether they are evolutionarily related remains unknown. Here, to address the molecular identity of NCR peptides and their evolution in different legume clades, we performed inter- and intra-clade comparisons of NCR peptides in representative species of four TBD-inducing legume clades. First, we collected genomic and proteomic data of species for which NCR peptides are known (1523 NCR peptides). We then used sequence similarity-based clustering to regroup the NCR peptides, resulting in over 400 different NCR clusters, each clade-specific. We obtained Hidden Markov Models for each cluster and used them to predict NCR peptides in 21 legume genomes (6 clades), including newly generated deep-sequenced root and nodule RNA-seq data of Indigofera argentea (Indigoferoid clade) and newly assembled high-quality transcriptomes of Lupinus luteus and Lupinus mariae-josephae (Genistoid clade), using tailored gene prediction pipeline and transcriptome matching. This resulted in 3710 NCR peptides in species that induce TBD. To date, the rapid diversification of NCR peptides that reduces the sequence similarities has masked the origin of NCR peptide evolution. We obtained high-confidence structural models for one sequence of each cluster. We performed structure-based clustering and phylogenetics, which resulted in 23 superclusters (14 inter-clade and nine clade-specific) that we represent in a structural distance-based tree. Our study revealed that the evolution of NCR peptides is a mix of divergent and convergent processes within each clade. We further chose nine independently evolved NCR peptides to test in vitro whether they are functional analogs in symbiosis.

Drought alters the soil microbiota by selecting for functional traits that preserve fitness in dry conditions. Legacy effects or ecological memory refers to how past stress exposure influences microbiota responses to future environmental challenges. How precipitation legacy effects impact soil microorganisms and plants is unclear, especially in the context of subsequent drought. Here we characterized the metagenomes of six prairie soils spanning a precipitation gradient in Kansas, United States. A microbial precipitation legacy, which persisted over a 5-month-long experimental drought, mitigated the negative physiological effects of acute drought for a native wild grass species, but not for the domesticated crop species maize. RNA sequencing of roots revealed that soil microbiota with a low precipitation legacy altered expression of plant genes that mediate transpiration and intrinsic water-use efficiency during drought. Our results show how historical exposure to water stress alters soil microbiota, with consequences for future drought responses of some plant species.