Rhizobium-legume Symbiosis Research Articles

CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions

Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

The Rpf107 gene, a homolog of LOR, is required for the symbiotic nodulation of Robinia pseudoacacia.

Rpf107 is involved in the infection process of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The LURP-one related (LOR) protein family plays a pivotal role in mediating plant defense responses against both biotic and abiotic stresses. However, our understanding of its function in the symbiotic interaction between legumes and rhizobia remains limited. Here, Rpf107, a homolog of LOR, was identified in Robinia pseudoacacia (black locust). The subcellular localization of Rpf107 was analyzed, and its function was investigated using RNA interference (RNAi) and overexpression techniques. The subcellular localization assay revealed that Rpf107 was mainly distributed in the plasma membrane and nucleus. Rpf107 silencing prevented rhizobial infection and hampered plant growth. The number of infected cells in the nitrogen fixation zone of the Rpf107-RNAi nodules was also noticeably lower than that in the control nodules. Notably, Rpf107 silencing resulted in bacteroid degradation and the premature aging of nodules. In contrast, the overexpression of Rpf107 delayed the senescence of nodules and prolonged the nitrogen-fixing ability of nodules. These results demonstrate that Rpf107 was involved in the infection of rhizobia and the maintenance of symbiotic nitrogen fixation in black locust root nodules. The findings reveal that a member of the LOR protein family plays a role in leguminous root nodule symbiosis, which is helpful to clarify the functions of plant LOR protein family and fully understand the molecular mechanisms underlying legume-rhizobium symbiosis.

Enhancing Rhizobium–Legume Symbiosis and Reducing Nitrogen Fertilizer Use Are Potential Options for Mitigating Climate Change

This review article explores the impact of nitrogen fertilizers on the symbiotic relationship between Rhizobium bacteria and legume plants. Nitrogen fixation has the potential to address the global protein shortage by increasing nitrogen supply in agriculture. However, the excessive use of synthetic fertilizers has led to environmental consequences and high energy consumption. To promote sustainable agriculture, alternative approaches such as biofertilizers that utilize biological nitrogen fixation have been introduced to minimize ecological impact. Understanding the process of biological nitrogen fixation, where certain bacteria convert atmospheric nitrogen into ammonia, is crucial for sustainable agriculture. This knowledge helps reduce reliance on synthetic fertilizers and maintain soil fertility. The symbiotic relationship between Rhizobium bacteria and leguminous plants plays a vital role in sustainable agriculture by facilitating access to atmospheric nitrogen, improving soil fertility, and reducing the need for chemical fertilizers. To achieve optimal nitrogen fixation and plant growth, it is important to effectively manage nitrogen availability, soil conditions, and environmental stressors. Excessive nitrogen fertilization can negatively affect the symbiotic association between plants and rhizobia, resulting in reduced soil health, altered mutualistic relationships, and environmental concerns. Various techniques can be employed to enhance symbiotic efficiency by manipulating chemotaxis, which is the ability of rhizobia to move towards plant roots. Plant-specific metabolites called (iso)flavonoids play a crucial role in signaling and communication between legume plants and rhizobia bacteria, initiating the symbiotic relationship and enhancing nitrogen fixation and plant growth. Excessive nitrogen fertilizer application can disrupt the communication between rhizobia and legumes, impacting chemotaxis, root exudation patterns, nodulation, and the symbiotic relationship. High levels of nitrogen fertilizers can inhibit nitrogenase, a critical enzyme for plant growth, leading to reduced nitrogenase activity. Additionally, excessive nitrogen can compromise the energy demands of nitrogen fixation, resulting in decreased nitrogenase activity. This review discusses the disadvantages of using nitrogenous fertilizers and the role of symbiotic biological nitrogen fixation in reducing the need for these fertilizers. By using effective rhizobial strains with compatible legume cultivars, not only can the amounts of nitrogenous fertilizers be reduced, but also the energy inputs and greenhouse gas emissions associated with their manufacturing and application. This approach offers benefits in terms of reducing greenhouse gas emissions and saving energy. In conclusion, this paper provides a comprehensive overview of the current understanding of the impact of nitrogen fertilizers on the symbiotic relationship between Rhizobium and legume plants. It also discusses potential strategies for sustainable agricultural practices. By managing nitrogen fertilizers carefully and improving our understanding of the symbiotic relationship, we can contribute to sustainable agriculture and minimize environmental impact.

How might bacteriophages shape biological invasions?

Invasions by eukaryotes dependent on environmentally acquired bacterial mutualists are often limited by the ability of bacterial partners to survive and establish free-living populations. Focusing on the model legume-rhizobium mutualism, we apply invasion biology hypotheses to explain how bacteriophages can impact the competitiveness of introduced bacterial mutualists. Predicting how phage-bacteria interactions affect invading eukaryotic hosts requires knowing the eco-evolutionary constraints of introduced and native microbial communities, as well as their differences in abundance and diversity. By synthesizing research from invasion biology, as well as bacterial, viral, and community ecology, we create a conceptual framework for understanding and predicting how phages can affect biological invasions through their effects on bacterial mutualists.

A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

Single-nucleus transcriptomes reveal spatiotemporal symbiotic perception and early response in Medicago.

Establishing legume-rhizobial symbiosis requires precise coordination of complex responses in a time- and cell type-specific manner. Encountering Rhizobium, rapid changes of gene expression levels in host plants occur in the first few hours, which prepare the plants to turn off defence and form a symbiotic relationship with the microbes. Here, we applied single-nucleus RNA sequencing to characterize the roots of Medicago truncatula at 30 min, 6 h and 24 h after nod factor treatment. We found drastic global gene expression reprogramming at 30 min in the epidermis and cortex and most of these changes were restored at 6 h. Moreover, plant defence response genes are activated at 30 min and subsequently suppressed at 6 h in non-meristem cells. Only in the cortical cells but not in other cell types, we found the flavonoid synthase genes required to recruit rhizobia are highly expressed 30 min after inoculation with nod factors. A gene module enriched for symbiotic nitrogen fixation genes showed that MtFER (MtFERONIA) and LYK3 (LysM domain receptor-like kinase 3) share similar responses to symbiotic signals. We further found that MtFER can be phosphorylated by LYK3 and it participates in rhizobial symbiosis. Our results expand our understanding of dynamic spatiotemporal symbiotic responses at the single-cell level.

Key role of phenol enzymes metabolism in the legume-rhizobial symbiosis under different water supply regimes

The legume-rhizobium interaction induces formation of specific reactions that take metabolism in the host plant up to a new functional level, increasing its tolerance to unfavourable cultivation conditions. Our objective was to study the participation of key enzymes – phenylalanine ammonia lyase, guaiacol peroxidase, and polyphenol oxidases – in the phenol-metabolism processes and synthesis of a broad spectrum of secondary metabolites in soybean plants that have established symbiotic interactions with rhizobia of varying effectiveness during optimal and insufficient water supplies. In our studies, we used symbiotic systems of soybean and rhizobia (Bradyrhizobium japonicum) that varied in efficiency and virulence. In the period of active nitrogen fixation by soybean, from the third-true-leaf stage until budding, we created different water-supply regimes for the plants, including optimal watering at the level of 60% of full field capacity (control) and insufficient, at the level of 30% (drought). When the soybean was flowering, we recovered the optimal level of water supply (resumed watering). In the studies, we employed microbiological, biochemical, and physiological approaches. We determined the specificity of how key enzymes of the phenol metabolism such as phenylalanine ammonia lyase, polyphenol oxidase and guaiacol peroxidase in the nodules, roots, and leaves of the soybean reacted to different levels of water supply, depending on the functional efficiency of the symbiotic system involving strains of B. japonicum, varying in effectiveness and virulence. In the effective soybean-rhizobium symbiosis, there occurred insignificant changes in the activity of phenol-metabolism enzymes in the nodules, roots, and leaves during drought and after action of the stress. This evidence is that in symbiosis with effective rhizobia B1-20, soybean could realize its own defensive systems that regulate optimal functioning of phenol metabolism in dehydration conditions. In the low-effective 107 and ineffective 604k symbiotic systems of soybean, there was observed unstable dynamics of the activity of enzymes in leaves and roots, manifested in intensification or inhibition of their activity levels during drought or post-stress period. This indicates malfunctioning of the processes associated with phenol metabolism in the soybean plants. We concluded that tolerance of legume-rhizobium symbiosis to water deprivation depends on mutual involvements of the both symbiotic partners – host plant and rhizobia, their ability to fully realize the defensive systems for activation of the key enzymatic complexes taking part in regulation of phenol metabolism in plants.

The Bax inhibitor GmBI-1α interacts with a Nod factor receptor and plays a dual role in the legume-rhizobia symbiosis.

The gene networks surrounding Nod factor receptors that govern the symbiotic process between legumes and rhizobia remain largely unexplored. Here, we identify 13 novel GmNFR1α-associated proteins by yeast two-hybrid screening, and describe a potential interacting protein, GmBI-1α. GmBI-1α had the highest positive correlation with GmNFR1α in a co-expression network analysis, and its expression at the mRNA level in roots was enhanced by rhizobial infection. Moreover, GmBI-1α-GmNFR1α interaction was shown to occur in vitro and in vivo. The GmBI-1α protein was localized to multiple subcellular locations, including the endoplasmic reticulum and plasma membrane. Overexpression of GmBI-1α increased the nodule number in transgenic hairy roots or transgenic soybean, whereas down-regulation of GmBI-1α transcripts by RNA interference reduced the nodule number. In addition, the nodules in GmBI-1α-overexpressing plants became smaller in size and infected area with reduced nitrogenase activity. In GmBI-1α-overexpressing transgenic soybean, the elevated GmBI-1α also promoted plant growth and suppressed the expression of defense signaling-related genes. Infection thread analysis of GmBI-1α-overexpressing plants showed that GmBI-1α promoted rhizobial infection. Collectively, our findings support a GmNFR1α-associated protein in the Nod factor signaling pathway and shed new light on the regulatory mechanism of GmNFR1α in rhizobial symbiosis.

A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion

During legume-rhizobia symbiosis, differentiation of the symbiosome (engulfed intracellular rhizobia) is necessary for successful nitrogen fixation. To control symbiosome differentiation, host cell subcellular components, e.g., ER (endoplasmic reticulum), must adapt robustly to ensure large-scale host protein secretion to the new organelle. However, the key components controlling the adaption of ER in nodule cells remain elusive. We report that Medicago BID1, a nodule-specific signal peptide peptidase (SPP), is central to ER structural dynamics and host protein secretion. In bid1, symbiosome differentiation is blocked. BID1 localizes specifically to the ER membrane and expresses exclusively in nodule cells with symbiosomes. In the wild type ER forms proximal association structures with symbiosomes, but not in bid1. Consequently, in bid1 excessive ER stress responses are induced and ER-to-symbiosome protein secretion is impaired. In summary, a nodule-specific SPP is necessary for ER-symbiosome proximal association, host protein secretion, and symbiosome differentiation.

G-type receptor-like kinase AsNIP43 interacts with rhizobia effector nodulation outer protein P and is required for symbiosis.

In the Rhizobium-Legume symbiosis, the nodulation outer protein P (NopP) effector is one of the key regulators for rhizobial infection and nodule organogenesis. However, the molecular mechanism through which host legume plants sense NopP remains largely unknown. Here, we constructed an nopP deletion mutant of Mesorhizobium huakuii and found that nopP negatively regulates nodulation on Chinese milk vetch (Astragalus sinicus). Screening for NopP interacting proteins in host plants using the yeast 2-hybrid system identified NopP interacting protein 43 (AsNIP43), which encodes a G-type receptor-like kinase (LecRLK). The B-lectin domain at the N terminus of AsNIP43 was essential in mediating its interaction with NopP, which was confirmed in vitro and in vivo. Subcellular localization, co-localization, and gene expression analyses showed that AsNIP43 and NopP function tightly associated with earlier infection events. RNA interference (RNAi) knockdown of AsNIP43 expression by hairy root transformation led to decreased nodule formation. AsNIP43 plays a positive role in symbiosis, which was further verified in the model legume Medicago truncatula. Transcriptome analysis indicated that MtRLK (a homolog of AsNIP43 in M. truncatula) may function to affect defense gene expression and thus to regulate early nodulation. Taken together, we show that LecRLK AsNIP43 is a legume host target that interacts with rhizobia effector NopP is essential for rhizobial infection and nodulation.

VPT-like genes modulate Rhizobium-legume symbiosis and phosphorus adaptation.

Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role in Rhizobium-legume symbiosis is unclear. In this study, homologous genes of VPT1 (MtVPTs) were identified in Medicago truncatula to assess their roles in Rhizobium-legume symbiosis and phosphorus adaptation. MtVPT2 and MtVPT3 mainly positively responded to low and high phosphate, respectively. However, both mtvpt2 and mtvpt3 mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root-to-shoot phosphate transfer efficiency was significantly enhanced in mtvpt3 but weakened in mtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate induced MtVPT2 and MtVPT3 expressions in nodules. MtVPT2 and MtVPT3 mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower in mtvpt2 and mtvpt3 than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long-distance Pi transport and cytosolic Pi supply. Also, mtvpt2 and mtvpt3 could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show that MtVPT2 andMtVPT3 modulate phosphorus adaptation and rhizobia-legume symbiosis, possibly by regulating long-distance Pi transport.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

An emerging view of coevolution in the legume-rhizobium mutualism.

Mutualisms are often framed as 'delicately balanced antagonisms' (Bronstein, 1994), with the net fitness benefits to both partners potentially masking underlying conflicts of interest. How commonly symbionts evolve to 'cheat' their hosts and hosts evolve to 'sanction' or 'control' uncooperative symbionts is the subject of debate, especially in legume-rhizobium interactions (Frederickson, 2013; Kiers et al., 2003). This kind of antagonistic coevolution should result in either arms-race dynamics characterized by repeated selective sweeps or fluctuating selection dynamics that leave signatures of balancing selection in host and symbiont genomes (Frederickson, 2013; Kortright et al., 2022; O'Brien et al., 2021). In a From the Cover article in this issue of Molecular Ecology, Epstein et al. (2022) combine GWAS and population genomic analyses to assess the evidence for positive or balancing selection consistent with ongoing, antagonistic coevolution between legumes and rhizobia. They found few genomic signatures of fitness conflicts between mutualistic partners, suggesting that legume and rhizobium fitness interests are largely aligned and symbiotic traits are mostly under stabilizing selection. In combination with other recent work (e.g. Batstone et al., 2020), the results of Epstein et al. (2022) indicate that there is little ongoing fitness conflict between legumes and rhizobia that shapes host and symbiont genomes in this system. It may be time to move beyond symbiont 'cheating' and host 'control' as the dominant paradigm for understanding how partners in mutualism coevolve.

GFP labeling of a Bradyrhizobium strain and an attempt to track the crack entry process during symbiosis with peanuts.

Compared to the well-studied model legumes, where symbiosis is established via root hair entry, the peanut is infected by Bradyrhizobium through the crack entry, which is less common and not fully understood. Crack entry is, however, considered a primitive symbiotic infection pathway, which could be potentially utilized for engineering non-legume species with nitrogen fixation ability. We utilized a fluorescence-labeled Bradyrhizobium strain to help in understanding the crack entry process at the cellular level. A modified plasmid pRJPaph-bjGFP, harboring the codon-optimized GFP gene and tetracycline resistance gene, was created and conjugated into Bradyrhizobium strain Lb8, an isolate from peanut nodules, through tri-parental mating. Microscopic observation and peanut inoculation assays confirmed the successful GFP tagging of Lb8, which is capable of generating root nodules. A marking system for peanut root potential infection sites and an optimized sample preparation protocol for cryostat sectioning was developed. The feasibility of using the GFP-tagged Lb8 for observing crack entry was examined. GFP signal was detected at the nodule primordial stage and the following nodule developmental stages with robust GFP signals observed in infected cells in the mature nodules. Spherical bacteroids in the root tissue were visualized at the nodules' inner cortex under higher magnification, reflecting the trace along the rhizobial infection path. The GFP labeled Lb8 can serve as an essential tool for plant-microbe studies between the cultivated peanut and Bradyrhizobium, which could facilitate further study of the crack entry process during the legume-rhizobia symbiosis.

Legume plant yields using biopreparation and plant growth regulators

The effect of the biopreparation Rizotorfin and growth regulators Albit, Kornevin, Epin-Extra on symbiotic activity and yield of the bean plant varieties Geliada and Shokoladnitsa was studied under field conditions. The nitrogenase activity was increased in Heliada bean plants after treatment of seeds with Epin-Extra against the increase of bacteroides area, amount and area of volutin and decrease of area and amount of poly-ß-oxybutyric acid (POM). The protective effect of Rizotorfin was evident in the variety Shokoladnitsa. The relationship between symbiotic nitrogen fixation and yield of bean plants of varieties Heliada and Shokoladnitsa was detected. The varietal responses of these plants to the use of Risotorfin and growth regulators have been identified. To increase the efficiency of legume-rhizobium symbiosis and plant productivity, the pre-sowing treatment of seeds with a biopreparation based on nodule bacteria and growth regulators is recommended.

Evaluation of Legume-Rhizobial Symbiotic Interactions Beyond Nitrogen Fixation That Help the Host Survival and Diversification in Hostile Environments.

Plants often experience unfavorable conditions during their life cycle that impact their growth and sometimes their survival. A temporary phase of such stress, which can result from heavy metals, drought, salinity, or extremes of temperature or pH, can cause mild to enormous damage to the plant depending on its duration and intensity. Besides environmental stress, plants are the target of many microbial pathogens, causing diseases of varying severity. In plants that harbor mutualistic bacteria, stress can affect the symbiotic interaction and its outcome. To achieve the full potential of a symbiotic relationship between the host and rhizobia, it is important that the host plant maintains good growth characteristics and stay healthy under challenging environmental conditions. The host plant cannot provide good accommodation for the symbiont if it is infested with diseases and prone to other predators. Because the bacterium relies on metabolites for survival and multiplication, it is in its best interests to keep the host plant as stress-free as possible and to keep the supply stable. Although plants have developed many mitigation strategies to cope with stress, the symbiotic bacterium has developed the capability to augment the plant's defense mechanisms against environmental stress. They also provide the host with protection against certain diseases. The protective features of rhizobial-host interaction along with nitrogen fixation appear to have played a significant role in legume diversification. When considering a legume-rhizobial symbiosis, extra benefits to the host are sometimes overlooked in favor of the symbionts' nitrogen fixation efficiency. This review examines all of those additional considerations of a symbiotic interaction that enable the host to withstand a wide range of stresses, enabling plant survival under hostile regimes. In addition, the review focuses on the rhizosphere microbiome, which has emerged as a strong pillar of evolutionary reserve to equip the symbiotic interaction in the interests of both the rhizobia and host. The evaluation would draw the researchers' attention to the symbiotic relationship as being advantageous to the host plant as a whole and the role it plays in the plant's adaptation to unfavorable environmental conditions.

Rhizobium nodule diversity and composition are influenced by clover host selection and local growth conditions.

While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.

GmBES1-1 dampens the activity of GmNSP1/2 to mediate brassinosteroid inhibition of nodulation in soybean

Soybean (Glycine max) forms root nodules to house rhizobial bacteria for biological nitrogen fixation. The development of root nodules is intricately regulated by endogenous and exogenous cues. The phytohormones brassinosteroids (BRs) have been shown to negatively regulate nodulation in soybean, but the underlying genetic and molecular mechanisms remain largely unknown. Here, we performed transcriptomic analyses and revealed that BR signaling negatively regulates nodulation factor (NF) signaling. We found that BR signaling inhibits nodulation through its signaling component GmBES1-1 by dampening NF signaling and nodule formation. In addition, GmBES1-1 can directly interact with both GmNSP1 and GmNSP2 to inhibit their interaction and the DNA-binding activity of GmNSP1. Furthermore, BR-induced nuclear accumulation of GmBES1-1 is essential for inhibiting nodulation. Taken together, our results demonstrate that regulation of GmBES1-1 subcellular localization by BRs plays a key role in the legume–rhizobium symbiosis and plant development, indicating a crosstalk mechanism between phytohormone and symbiosis signaling pathways.

Effect of Triazole Fungicides Titul Duo and Vintage on the Development of Pea (Pisum sativum L.) Symbiotic Nodules.

Triazole fungicides are widely used in agricultural production for plant protection, including pea (Pisum sativum L.). The use of fungicides can negatively affect the legume-Rhizobium symbiosis. In this study, the effects of triazole fungicides Vintage and Titul Duo on nodule formation and, in particular, on nodule morphology, were studied. Both fungicides at the highest concentration decreased the nodule number and dry weight of the roots 20 days after inoculation. Transmission electron microscopy revealed the following ultrastructural changes in nodules: modifications in the cell walls (their clearing and thinning), thickening of the infection thread walls with the formation of outgrowths, accumulation of poly-β-hydroxybutyrates in bacteroids, expansion of the peribacteroid space, and fusion of symbiosomes. Fungicides Vintage and Titul Duo negatively affect the composition of cell walls, leading to a decrease in the activity of synthesis of cellulose microfibrils and an increase in the number of matrix polysaccharides of cell walls. The results obtained coincide well with the data of transcriptomic analysis, which revealed an increase in the expression levels of genes that control cell wall modification and defense reactions. The data obtained indicate the need for further research on the effects of pesticides on the legume-Rhizobium symbiosis in order to optimize their use.

Plant-Environment Response Pathway Regulation Uncovered by Investigating Non-Typical Legume Symbiosis and Nodulation.

Nitrogen is an essential element needed for plants to survive, and legumes are well known to recruit rhizobia to fix atmospheric nitrogen. In this widely studied symbiosis, legumes develop specific structures on the roots to host specific symbionts. This review explores alternate nodule structures and their functions outside of the more widely studied legume-rhizobial symbiosis, as well as discussing other unusual aspects of nodulation. This includes actinorhizal-Frankia, cycad-cyanobacteria, and the non-legume Parasponia andersonii-rhizobia symbioses. Nodules are also not restricted to the roots, either, with examples found within stems and leaves. Recent research has shown that legume-rhizobia nodulation brings a great many other benefits, some direct and some indirect. Rhizobial symbiosis can lead to modifications in other pathways, including the priming of defence responses, and to modulated or enhanced resistance to biotic and abiotic stress. With so many avenues to explore, this review discusses recent discoveries and highlights future directions in the study of nodulation.