Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

The rhizosphere of a drought-tolerant plant species in Morocco: A refuge of high microbial diversity with no taxon preference.

Arid and semi-arid areas are facing increasingly severe water deficits that are being intensified by global climate changes. Microbes associated with plants native to arid regions provide valuable benefits to plants, especially in water-stressed environments. In this study, we used 16S rDNA metabarcoding analysis to examine the bacterial communities in the bulk soil, rhizosphere and root endosphere of the plant Malva sylvestris L. in Morocco, along a gradient of precipitation. We found that the rhizosphere of M. sylvestris did not show significant differences in beta-diversity compared to bulk soil, although, it did display an increased degree of alpha-diversity. The endosphere was largely dominated by the genus Rhizobium and displayed remarkable variation between plants, which could not be attributed to any of the variables observed in this study. Overall, the effects of precipitation level were relatively weak, which may be related to the intense drought in Morocco at the time of sampling. The dominance of Rhizobium in a non-leguminous plant is particularly noteworthy and may permit the utilization of this bacterial taxon to augment drought tolerance; additionally, the absence of any notable selection of the rhizosphere of M. sylvestris suggests that it is not significatively affecting its soil environment.

Guidelines for the description of rhizobial symbiovars.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

A Single Latent Plant Growth-Promoting Endophyte BH46 Enhances Houttuynia cordata Thunb. Yield and Quality.

Plant growth-promoting endophytes (PGPE) can effectively regulate plant growth and metabolism. The regulation is modulated by metabolic signals, and the resulting metabolites can have considerable effects on the plant yield and quality. Here, tissue culture Houttuynia cordata Thunb., was inoculated with Rhizobium sp. (BH46) to determine the effect of BH46 on H. cordata growth and metabolism, and elucidate associated regulatory mechanisms. The results revealed that BH46 metabolized indole-3-acetic acid and induced 1-aminocyclopropane-1-carboxylate deaminase to decrease ethylene metabolism. Host peroxidase synthesis MPK3/MPK6 genes were significantly downregulated, whereas eight genes associated with auxins, cytokinins, abscisic acid, jasmonic acid, and antioxidant enzymes were significantly upregulated. Eight genes associated with flavonoid biosynthesis were significantly upregulated, with the CPY75B1 gene regulating the production of rutin and quercitrin and the HCT gene directly regulating the production of chlorogenic acid. Therefore, BH46 influences metabolic signals in H. cordata to modulate its growth and metabolism, in turn, enhancing yield and quality of H. cordata.

Geographical and climatic distribution of lentil-nodulating rhizobia in Iran.

Lentil is one of the most important legumes cultivated in various provinces of Iran. However, there is limited information about the symbiotic rhizobia of lentils in this country. In this study, molecular identification of lentil-nodulating rhizobia was performed based on 16S-23S rRNA intergenic spacer (IGS) and recA, atpD, glnII, and nodC gene sequencing. Using PCR-RFLP analysis of 16S-23S rRNA IGS, a total of 116 rhizobia isolates were classified into 20 groups, leaving seven strains unclustered. Phylogenetic analysis of representative isolates revealed that the rhizobia strains belonged to Rhizobium leguminosarum and Rhizobium laguerreae, and the distribution of the species is partially related to geographical location. Rhizobium leguminosarum was the dominant species in North Khorasan and Zanjan, while R. laguerreae prevailed in Ardabil and East Azerbaijan. The distribution of the species was also influenced by agroecological climates; R. leguminosarum thrived in cold semiarid climates, whereas R. laguerreae adapted to humid continental climates. Both species exhibited equal dominance in the Mediterranean climate, characterized by warm, dry summers and mild, wet winters, in Lorestan and Kohgiluyeh-Boyer Ahmad provinces.

SeqCode facilitates naming of South African rhizobia left in limbo.

South Africa is well-known for the diversity of its legumes and their nitrogen-fixing bacterial symbionts. However, in contrast to their plant partners, remarkably few of these microbes (collectively referred to as rhizobia) from South Africa have been characterised and formally described. This is because the rules of the International Code of Nomenclature of Prokaryotes (ICNP) are at odds with South Africa's National Environmental Management: Biodiversity Act and its associated regulations. The ICNP requires that a culture of the proposed type strain for a novel bacterial species be deposited in two international culture collections and be made available upon request without restrictions, which is not possible under South Africa's current national regulations. Here, we describe seven new Mesorhizobium species obtained from root nodules of Vachellia karroo, an iconic tree legume distributed across various biomes in southern Africa. For this purpose, 18 rhizobial isolates were delineated into putative species using genealogical concordance, after which their plausibility was explored with phenotypic characters and average genome relatedness. For naming these new species, we employed the rules of the recently published Code of Nomenclature of Prokaryotes described from Sequence Data (SeqCode), which utilizes genome sequences as nomenclatural types. The work presented in this study thus provides an illustrative example of how the SeqCode allows for a standardised approach for naming cultivated organisms for which the deposition of a type strain in international culture collections is currently problematic.

Enhancing Pisum sativum growth and symbiosis under heat stress: the synergistic impact of co-inoculated bacterial consortia and ACC deaminase-lacking Rhizobium.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminase is a crucial bacterial trait, yet it is not widely distributed among rhizobia. Hence, employing a co-inoculation approach that combines selected plant growth-promoting bacteria with compatible rhizobial strains, especially those lacking ACC deaminase, presents a practical solution to alleviate the negative effects of diverse abiotic stresses on legume nodulation. Our objective was to explore the efficacy of three non-rhizobial endophytes, Phyllobacterium salinisoli (PH), Starkeya sp. (ST) and Pseudomonas turukhanskensis (PS), isolated from native legumes grown in Tunisian arid regions, in improving the growth of cool-season legume and fostering symbiosis with an ACC deaminase-lacking rhizobial strain under heat stress. Various combinations of these endophytes (ST + PS, ST + PH, PS + PH, and ST + PS + PH) were co-inoculated with Rhizobium leguminosarum 128C53 or its ΔacdS mutant derivative on Pisum sativum plants exposed to a two-week heat stress period.Our findings revealed that the absence of ACC deaminase activity negatively impacted both pea growth and symbiosis under heat stress. Nevertheless, these detrimental effects were successfully mitigated in plants co-inoculated with ΔacdS mutant strain and specific non-rhizobial endophytes consortia. Our results indicated that heat stress significantly altered the phenolic content of pea root exudates. Despite this, there was no impact on IAA production. Interestingly, these changes positively influenced biofilm formation in consortia containing the mutant strain, indicating synergistic bacteria-bacteria interactions. Additionally, no positive effects were observed when these endophytic consortia were combined with the wild-type strain. This study highlights the potential of non-rhizobial endophytes to improve symbiotic performance of rhizobial strains lacking genetic mechanisms to mitigate stress effects on their legume host, holding promising potential to enhance the growth and yield of targeted legumes by boosting symbiosis.

Effects of Soil Rhizobia Abundance on Interactions between a Vector, Pathogen, and Legume Plant Host.

Soil rhizobia promote nitrogen fixation in legume hosts, maximizing their tolerance to different biotic stressors, plant biomass, crop growth, and yield. While the presence of soil rhizobia is considered beneficial for plants, few studies have assessed whether variation in rhizobia abundance affects the tolerance of legumes to stressors. To address this, we assessed the effects of variable soil rhizobia inoculum concentrations on interactions between a legume host (Pisum sativum), a vector insect (Acyrthosiphon pisum), and a virus (Pea enation mosaic virus, PEMV). We showed that increased rhizobia abundance reduces the inhibitory effects of PEMV on the nodule formation and root growth in 2-week-old plants. However, these trends were reversed in 4-week-old plants. Rhizobia abundance did not affect shoot growth or virus prevalence in 2- or 4-week-old plants. Our results show that rhizobia abundance may indirectly affect legume tolerance to a virus, but effects varied based on plant age. To assess the mechanisms that mediated interactions between rhizobia, plants, aphids, and PEMV, we measured the relative expression of gene transcripts related to plant defense signaling. Rhizobia concentrations did not strongly affect the expression of defense genes associated with phytohormone signaling. Our study shows that an abundance of soil rhizobia may impact a plant's ability to tolerate stressors such as vector-borne pathogens, as well as aid in developing sustainable pest and pathogen management systems for legume crops. More broadly, understanding how variable rhizobia concentrations can optimize legume-rhizobia symbiosis may enhance the productivity of legume crops.

Hopanoid lipids promote soybean-Bradyrhizobium symbiosis.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200 million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

The evolutionary genomics of adaptation to stress in wild rhizobium bacteria.

Microbiota comprise the bulk of life's diversity, yet we know little about how populations of microbes accumulate adaptive diversity across natural landscapes. Adaptation to stressful soil conditions in plants provides seminal examples of adaptation in response to natural selection via allelic substitution. For microbes symbiotic with plants however, horizontal gene transfer allows for adaptation via gene gain and loss, which could generate fundamentally different evolutionary dynamics. We use comparative genomics and genetics to elucidate the evolutionary mechanisms of adaptation to physiologically stressful serpentine soils in rhizobial bacteria in western North American grasslands. In vitro experiments demonstrate that the presence of a locus of major effect, the nre operon, is necessary and sufficient to confer adaptation to nickel, a heavy metal enriched to toxic levels in serpentine soil, and a major axis of environmental soil chemistry variation. We find discordance between inferred evolutionary histories of the core genome and nreAXY genes, which often reside in putative genomic islands. This suggests that the evolutionary history of this adaptive variant is marked by frequent losses, and/or gains via horizontal acquisition across divergent rhizobium clades. However, different nre alleles confer distinct levels of nickel resistance, suggesting allelic substitution could also play a role in rhizobium adaptation to serpentine soil. These results illustrate that the interplay between evolution via gene gain and loss and evolution via allelic substitution may underlie adaptation in wild soil microbiota. Both processes are important to consider for understanding adaptive diversity in microbes and improving stress-adapted microbial inocula for human use.

Identification and characterization of the TetR family transcriptional regulator NffT in Rhizobium johnstonii.

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF, nifA-fixA signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased fixA expression, was discovered through a fixA-promoter-ß-glucuronidase fusion assay performed with Rhizobium johnstonii. Real-time quantitative PCR analysis showed that nffT deletion increased the expression of symbiotic genes including nifA and fixA in nifA-fixA signaling, and fixL, fixK, fnrN, and fixN9 in fixL-fixN signaling. nffT overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091-93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located -32 to -99 bp upstream of RL0091-93 and NffT deletion significantly increased the expression of RL0091-93. nffT-promoter-ß-glucuronidase fusion assay indicated that nffT expression was regulated by the cobNTS genes and cobalamin. Mutations in cobNTS significantly decreased the expression of nffT, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of R. johnstonii-Pisum sativum.IMPORTANCESymbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of fixA in Rhizobium johnstonii. Furthermore, NffT was confirmed to play pleiotropic roles in R. johnstonii-Pisum sativum symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected R. johnstonii-P. sativum symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.

Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions.

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10-25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I-III) were the most frequent, whereas the other alleles (IV-XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

A comprehensive, improved protocol for generating common bean (Phaseolus vulgaris L.) transgenic hairy roots and their use in reverse-genetics studies.

Generating transgenic hairy roots has been the preferred strategy for molecular studies in common bean (Phaseolus vulgaris L.), since generating stable knockout lines in this species is challenging. However, the number of plants producing hairy roots following the original protocol published in 2007 is usually low, which has impeded progress. Since its initial publication, the original protocol has been extensively modified, but these modifications have not been adequately or systematically reported, making it difficult to assess the reproducibility of the method. The protocol presented here is an update and expansion of the original method. Importantly, it includes new, critical steps for generating transgenic hairy roots and using them in molecular analyses based on reverse-genetics approaches. Using this protocol, the expression of two different genes, used as an example, was significantly increased or decreased in approximately 30% of the transformed plants. In addition, the promoter activity of a given gene was observed, and the infection process of rhizobia in transgenic hairy roots was monitored successfully. Thus, this improved protocol can be used to upregulate, downregulate, and perform promoter activity analysis of various genes in common bean transgenic hairy roots as well as to track rhizobia infection.

Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants.

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.

An Integrated Affinity Chromatography-Based Approach to Unravel the sRNA Interactome in Nitrogen-Fixing Rhizobia.

The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography-based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5'-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively.

Impacts of Natural Selection on Evolution of Core and Symbiotically Specialized (sym) Genes in the Polytypic Species Neorhizobium galegae.

Nodule bacteria (rhizobia) represent a suitable model to address a range of fundamental genetic problems, including the impacts of natural selection on the evolution of symbiotic microorganisms. Rhizobia possess multipartite genomes in which symbiotically specialized (sym) genes differ from core genes in their natural histories. Diversification of sym genes is responsible for rhizobia microevolution, which depends on host-induced natural selection. By contrast, diversification of core genes is responsible for rhizobia speciation, which occurs under the impacts of still unknown selective factors. In this paper, we demonstrate that in goat's rue rhizobia (Neorhizobium galegae) populations collected at North Caucasus, representing two host-specific biovars orientalis and officianalis (N2-fixing symbionts of Galega orientalis and G. officinalis), the evolutionary mechanisms are different for core and sym genes. In both N. galegae biovars, core genes are more polymorphic than sym genes. In bv. orientalis, the evolution of core genes occurs under the impacts of driving selection (dN/dS > 1), while the evolution of sym genes is close to neutral (dN/dS ≈ 1). In bv. officinalis, the evolution of core genes is neutral, while for sym genes, it is dependent on purifying selection (dN/dS < 1). A marked phylogenetic congruence of core and sym genes revealed using ANI analysis may be due to a low intensity of gene transfer within and between N. galegae biovars. Polymorphism in both gene groups and the impacts of driving selection on core gene evolution are more pronounced in bv. orientalis than in bv. officianalis, reflecting the diversities of their respective host plant species. In bv. orientalis, a highly significant (P0 < 0.001) positive correlation is revealed between the p-distance and dN/dS values for core genes, while in bv. officinalis, this correlation is of low significance (0.05 < P0 < 0.10). For sym genes, the correlation between p-distance and dN/dS values is negative in bv. officinalis but is not revealed in bv. orientalis. These data, along with the functional annotation of core genes implemented using Gene Ontology tools, suggest that the evolution of bv. officinalis is based mostly on adaptation for in planta niches while in bv. orientalis, evolution presumably depends on adaptation for soil niches. New insights into the tradeoff between natural selection and genetic diversity are presented, suggesting that gene nucleotide polymorphism may be extended by driving selection only in ecologically versatile organisms capable of supporting a broad spectrum of gene alleles in their gene pools.

Role of a LORELEI- like gene from Phaseolus vulgaris during a mutualistic interaction with Rhizobium tropici.

Reactive oxygen species (ROS), produced by NADPH oxidases known as RBOHs in plants, play a key role in plant development, biotic and abiotic stress responses, hormone signaling, and reproduction. Among the subfamily of receptor-like kinases referred to as CrRLK, there is FERONIA (FER), a regulator of RBOHs, and FER requires a GPI-modified membrane protein produced by LORELEI (LRE) or LORELEI-like proteins (LLG) to reach the plasma membrane and generate ROS. In Arabidopsis, AtLLG1 is involved in interactions with microbes as AtLLG1 interacts with the flagellin receptor (FLS2) to trigger the innate immune response, but the role of LLGs in mutualistic interactions has not been examined. In this study, two Phaseolus vulgaris LLG genes were identified, PvLLG2 that was expressed in floral tissue and PvLLG1 that was expressed in vegetative tissue. Transcripts of PvLLG1 increased during rhizobial nodule formation peaking during the early period of well-developed nodules. Also, P. vulgaris roots expressing pPvLLG1:GFP-GUS showed that this promoter was highly active during rhizobium infections, and very similar to the subcellular localization using a construct pLLG1::PvLLG1-Neon. Compared to control plants, PvLLG1 silenced plants had less superoxide (O2-) at the root tip and elongation zone, spotty hydrogen peroxide (H2O2) in the elongation root zone, and significantly reduced root hair length, nodule number and nitrogen fixation. Unlike control plants, PvLLG1 overexpressing plants showed superoxide beyond the nodule meristem, and significantly increased nodule number and nodule diameter. PvLLG1 appears to play a key role during this mutualistic interaction, possibly due to the regulation of the production and distribution of ROS in roots.

A Novel Rhizobium sp. Chiba-1 Strain Exhibits a Host Range for Nodule Symbiosis in Lotus Species.

Rhizobia are soil bacteria that induce the formation of nodules in the roots of leguminous plants for mutualistic establishment. Although the symbiotic mechanism between Lotus japonicus and its major symbiotic rhizobia, Mesorhizobium loti, has been extensively characterized, our understanding of symbiotic mechanisms, such as host specificity and host ranges, remains limited. In the present study, we isolated a novel Rhizobium strain capable of forming nodules on L. burttii from agricultural soil at Iwate prefecture in Japan. We conducted genomic and host range ana-lyses of various Lotus species. The results obtained revealed that the novel isolated Rhizobium sp. Chiba-1 was closely related to R. leguminosarum and had a wide host range that induced nodule development, including L. burttii and several L. japonicus wild-type accessions. However, L. japonicus Gifu exhibited an incompatible nodule phenotype. We also identified the formation of an epidermal infection threads that was dependent on the Lotus species and independent of nodule organ development. In conclusion, this newly isolated Rhizobium strain displays a distinct nodulation phenotype from Lotus species, and the results obtained herein provide novel insights into the functional mechanisms underlying host specificity and host ranges.

Effects of Elevated Temperature on Pisum sativum Nodule Development: I-Detailed Characteristic of Unusual Apical Senescence.

Despite global warming, the influence of heat on symbiotic nodules is scarcely studied. In this study, the effects of heat stress on the functioning of nodules formed by Rhizobium leguminosarum bv. viciae strain 3841 on pea (Pisum sativum) line SGE were analyzed. The influence of elevated temperature was analyzed at histological, ultrastructural, and transcriptional levels. As a result, an unusual apical pattern of nodule senescence was revealed. After five days of exposure, a senescence zone with degraded symbiotic structures was formed in place of the distal nitrogen fixation zone. There was downregulation of various genes, including those associated with the assimilation of fixed nitrogen and leghemoglobin. After nine days, the complete destruction of the nodules was demonstrated. It was shown that nodule recovery was possible after exposure to elevated temperature for 3 days but not after 5 days (which coincides with heat wave duration). At the same time, the exposure of plants to optimal temperature during the night leveled the negative effects. Thus, the study of the effects of elevated temperature on symbiotic nodules using a well-studied pea genotype and Rhizobium strain led to the discovery of a novel positional response of the nodule to heat stress.