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Summary: Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans‐Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes.Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP‐PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules.Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2.Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program. [ABSTRACT FROM AUTHOR]

Maize (Zea mays) is one of the most cultivated plants in the world. Due to the large area, the scale of its production, and the demand to increase the yield, there is a need for new environmentally friendly fertilizers. One group of such candidates is bacteria-produced nodulation (or nod) factors. Limited research has explored the impact of nodulation, factors on maize within field conditions, with most studies restricted to greenhouse settings and early developmental stages. Additionally, there is a scarcity of investigations that elucidate the metabolic alterations in the maize stem due to nod-factor exposure. It was therefore the aim of this study. Maize stem's metabolites and fibers were analyzed with various imaging analytical techniques: matrix assisted laser desorption ionization–mass spectrometry imaging (MALDI-MSI), Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR), and diffuse reflectance infrared Fourier transform spectroscopy. Moreover, the biochemical analyses were used to evaluate the proteins and soluble carbohydrates concentration and total phenolic content. These techniques were used to evaluate the influence of nod factor-based biofertilizer on the growth of a non-symbiotic plant, maize. The biofertilizer increased the grain yield and the stem mass. Moreover, the spectroscopic and biochemical investigation proved the appreciable biochemical changes in the stems of the maize in biofertilizer-treated plants. Noticeable changes were found in the spatial distribution and the increase in the concentration of flavonoids such as maysin, quercetin, and rutin. Moreover, the concentration of cell wall components (fibers) increased. Furthermore, it was shown that the use of untargeted analyses (such as Raman and ATR FT-IR, spectroscopic imaging, and MALDI-MSI) is useful for the investigation of the biochemical changes in plants. [ABSTRACT FROM AUTHOR]

SUMMARY: Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC‐deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC‐deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC‐deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3‐hydoxydecanoyl‐acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis. Significance Statement: The membrane lipid phosphatidylcholine (PC) is crucial for the bacterium Sinorhizobium meliloti when generating nitrogen‐fixing nodules on the roots of their host plant. Although PC‐deficient mutants of S. meliloti cannot form any nodules on their host plant, suppressor mutants were isolated which partially recover the ability to trigger nodule formation. Among these suppressor mutants, we detected a reduced function mutant of FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids thereby probably affecting lipid packing. [ABSTRACT FROM AUTHOR]

Plants perceive N-acetyl-d-glucosamine-containing oligosaccharides that play a role in the interaction with bacteria and fungi, through cell-surface receptors containing a tight bundle of three LysM domains in their extracellular region. However, the identification of LysM domains of receptor-like kinases (RLK)/receptor-like proteins (RLP) using sequence based methods has led to some ambiguity, as some proteins have been annotated with only one or two LysM domains. This missing annotation was likely produced by the failure of the LysM hidden Markov model (HMM) from the Pfam database to correctly identify some LysM domains in proteins of plant origin. In this work, we provide improved HMMs for LysM domain detection in plants, that were built from the structural alignment of manually curated LysM domain structures from the Protein Data Bank and AlphaFold Protein Structure Database. Furthermore, we evaluated different sets of ligand-specific HMMs that were able to correctly classify a limited set of fully characterised RLK/Ps by their ligand specificity. In contrast, the phylogenetic analysis of the extracellular region of RLK/Ps, or of their individual LysM domains, was unable to discriminate these proteins by their ligand specificity. The HMMs reported here will allow a more sensitive detection of plant proteins containing LysM domains and help improve their characterisation. [ABSTRACT FROM AUTHOR]

Ultisol is a type of soil with low organic matter, pH, and nutrient content, including molybdenum, leading to low productivity. This study aimed to investigate the use of dry land using molybdenum and CaCO3 inoculated with Rhizobium strain Nod+Fix+ to increase the soybean production of Willis and Baluran cultivars. This research was conducted from May to September 2021 in Pallangga Subdistrict, Gowa Regency, South Sulawesi, Indonesia. The study used a split-plot design with three replications for each treatment. The first factor was soybean varieties, consisting of Baluran and Willis cultivars. The second factor was the composition of the bacterial strain Nod+ Fix+, lime CaCO3 and NH4-molybdate, which consisted of without (Rhizobium strain Nod+ Fix+ + CaCO3 + NH4-molybdate); Rhizobium strain Nod+ Fix+ + CaCO3 1.0 ton/ha + NH4-molybdate 250 g/h); Rhizobium strain Nod+ Fix+ + CaCO3 1.5 tons/ha + NH4-molybdate 500 g/h); and Rhizobium strain Nod+ Fix+ + CaCO3 2.0 tons/ha + NH4-molybdate 750 g/h). The results showed that treating the bacterial strain Nod+ Fix+ + MoCo (1.0:0.6) kg/ha achieved the best results on growth, nutrient uptake (Nitrogen, Phosphorus and Potassium), and soybean yields, both for Willis and Baluran varieties on ultisol soils.

Ultisol is a type of soil with low organic matter, pH, and nutrient content, including molybdenum, leading to low productivity. This study aimed to investigate the use of dry land using molybdenum and lime (CaCO3) inoculated with Rhizobium strain Nod+Fix+ to increase the soybean production of Willis and Baluran cultivars. This research was conducted from May to September 2021 in Pallangga Subdistrict, Gowa Regency, South Sulawesi, Indonesia. The study used a split-plot design with three replications for each treatment. The first factor was soybean varieties, consisting of Baluran and Willis cultivars. The second factor was the composition of the bacterial strain Nod+ Fix+, lime CaCO3 and NH4-molybdate, which consisted of without (Rhizobium strain Nod+ Fix+ + CaCO3 + NH4-molybdate); Rhizobium strain Nod+ Fix+ + CaCO3 1.0 ton/ha + NH4-molybdate 250 g/h); Rhizobium strain Nod+ Fix+ + CaCO3 1.5 tons/ha + NH4-molybdate 500 g/h); and Rhizobium strain Nod+ Fix+ + CaCO3 2.0 tons/ha + NH4-molybdate 750 g/h). The results showed that treating the bacterial strain Nod+ Fix+ + MoCo (1.0:0.6) kg/ha achieved the best results on growth, nutrient uptake (Nitrogen, Phosphorus and Potassium), and soybean yields, both for Willis and Baluran varieties on ultisol soils. [ABSTRACT FROM AUTHOR]

Main conclusion: Lysin motif (LysM)-receptor-like kinase (RLK) and leucine-rich repeat (LRR)-RLK mediated signaling play important roles in the development and regulation of root nodule symbiosis in legumes. The availability of water and nutrients in the soil is a major limiting factor affecting crop productivity. Plants of the Leguminosae family form a symbiotic association with nitrogen-fixing Gram-negative soil bacteria, rhizobia for nitrogen fixation. This symbiotic relationship between legumes and rhizobia depends on the signal exchange between them. Plant receptor-like kinases (RLKs) containing lysin motif (LysM) and/or leucine-rich repeat (LRR) play an important role in the perception of chemical signals from rhizobia for initiation and establishment of root nodule symbiosis (RNS) that results in nitrogen fixation. This review highlights the diverse aspects of LysM-RLK and LRR receptors including their specificity, functions, interacting partners, regulation, and associated signaling in RNS. The activation of LysM-RLKs and LRR-RLKs is important for ensuring the successful interaction between legume roots and rhizobia. The intracellular regions of the receptors enable additional layers of signaling that help in the transduction of signals intracellularly. Additionally, symbiosis receptor-like kinase (SYMRK) containing the LRR motif acts as a co-receptor with Nod factors receptors (LysM-RLK). Cleavage of the malectin-like domain from the SYMRK ectodomain is a mechanism for controlling SYMRK stability. Overall, this review has discussed different aspects of legume receptors that are critical to the perception of signals from rhizobia and their subsequent role in creating the mutualistic relationship necessary for nitrogen fixation. Additionally, it has been discussed how crucial it is to extrapolate the knowledge gained from model legumes to crop legumes such as chickpea and common bean to better understand the mechanism underlying nodule formation in crop legumes. Future directions have also been proposed in this regard. [ABSTRACT FROM AUTHOR]

Aim: Rhizobium sp. IRBG74 (IRBG74), is a symbiont of Sesbania cannabina and a growth promoting endophyte of rice. Here, we compare the transcriptomic and physiological responses of IRBG74 in the rhizosphere of S. cannabina and rice. Methods: We used RNA sequencing to determine transcriptomic changes at 12 and 72 h post inoculation (hpi) in rhizosphere. Upregulation of key pathways was confirmed using β-glucuronidase (GUS) reporter strains and by histochemical and quantitative GUS activity. Results: Significant changes in transcriptome with S. cannabina were detected at 12hpi but most gene expression changes with rice were observed at 72hpi. Many pathways including Nod factor synthesis, two component systems, ABC transporters, and synthesis of indole acetic acid (IAA) were upregulated whereas translation, RNA degradation, protein export and sulfur metabolism were downregulated with both plants. In contrast, motility and chemotaxis genes were induced specifically with S. cannabina. The upregulation of nod and IAA genes was confirmed using reporter strains. Nod factor synthesis provides competitive advantage for colonization of rice and chemotaxis is required for colonization of S. cannabina but not of rice. Conclusions: IRBG74 responded to S. cannabina and rice using specific as well as common transcriptional changes. A mutant defective in Nod factor synthesis was outcompeted for rice colonization. Chemotaxis was required for colonization of S. cannabina but not for rice. IAA synthesis by IRBG74 could be a major mechanism of rice growth promotion. These results provide a foundation for further improvement of rhizobial interactions with rice and other cereals. [ABSTRACT FROM AUTHOR]

Soybean, which originated in China, is an important oil crop. The soybean–rhizobium symbiotic relationship provides soybean plants with abundant nitrogen. The Nod factor, which is an early signalling factor, affects the establishment of this symbiosis. In this study, the generated Sinorhizobium fredii HH103ΩNodB mutant exhibited defective Nod factor synthesis. The nodulation of 30 core collections, including 'Dongnong594' and 'Charleston', by the NodB mutant differed from the nodulation by the wild‐type strain. Soybean recombinant inbred lines derived from a 'Dongnong594' × 'Charleston' cross were used for mapping soybean genes related to the Nod factor signalling pathway. Eleven QTLs related to nodule number and eight QTLs related to nodule dry weight were revealed. On the basis of a qRT‐PCR analysis, Glyma.03G120700, Glyma.08G296000 and Glyma.15G21560 were identified as candidate genes influencing the NF interaction. These genes were differentially expressed following the inoculations with the wild‐type rhizobium and the NodB mutant. This study provides important insights into the mechanism mediating soybean responses to the NF after the soybean–rhizobium symbiosis is established. [ABSTRACT FROM AUTHOR]

Plasma membrane-bound receptor proteins play crucial roles in the perception and further transmission of regulatory signals to modulate numerous developmental and metabolic events. Precise functioning and fine-tuning of Nod factor receptor (NFR) mediated signalling is a critical requirement for root nodule symbiosis. Here, we have identified, cloned and phylogenetically characterized chickpea NFR1 and NFR5, which are showing significant homology with other legume NFR receptors. Homology modelling and molecular dynamics simulations highlight the molecular structure of ligand binding ectodomains [EDs] and cytosolic kinase domains [KDs] of NFRs in chickpea. Our detailed structural analysis also revealed that both NFR1 and NFR5 share resemblance as well as dissimilarity in sequence, structure and substrate-binding pocket. Further, molecular docking simulations provide us adequate insights into the active site of receptors where the Nod factor (NF) binds. The outcome of this work sheds light on the binding specificity of NFs towards NFRs and thus may significantly contribute to the design of new strategies in improving root-nodule symbiosis towards meeting the agricultural demands. • Several conserved and variable residues crucial for NF binding and phosphorylation remain in both CaNFR1 and CaNFR5. • CaNFR1 is an active kinase, whereas CaNFR5 is a pseudokinase, as it is not functional for catalysis. • CaNFR1 EDs show high conservation in the domain architecture with other model legumes EDs. • NFRs have strong binding efficacy towards their native NFs compared to other NFs. • CaNFR1-CaNFR5 ED can interact together to form a complex. [ABSTRACT FROM AUTHOR]

The difference in symbiotic specificity between peas of Afghanistan and European phenotypes was investigated using molecular modeling. Considering segregating amino acid polymorphism, we examined interactions of pea LykX-Sym10 receptor heterodimers with four forms of Nodulation factor (NF) that varied in natural decorations (acetylation and length of the glucosamine chain). First, we showed the stability of the LykX-Sym10 dimer during molecular dynamics (MD) in solvent and in the presence of a membrane. Then, four NFs were separately docked to one European and two Afghanistan dimers, and the results of these interactions were in line with corresponding pea symbiotic phenotypes. The European variant of the LykX-Sym10 dimer effectively interacts with both acetylated and non-acetylated forms of NF, while the Afghanistan variants successfully interact with the acetylated form only. We additionally demonstrated that the length of the NF glucosamine chain contributes to controlling the effectiveness of the symbiotic interaction. The obtained results support a recent hypothesis that the LykX gene is a suitable candidate for the unidentified Sym2 allele, the determinant of pea specificity toward Rhizobium leguminosarum bv. viciae strains producing NFs with or without an acetylation decoration. The developed modeling methodology demonstrated its power in multiple searches for genetic determinants, when experimental detection of such determinants has proven extremely difficult.

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.

The difference in symbiotic specificity between peas of Afghanistan and European phenotypes was investigated using molecular modeling. Considering segregating amino acid polymorphism, we examined interactions of pea LykX-Sym10 receptor heterodimers with four forms of Nodulation factor (NF) that varied in natural decorations (acetylation and length of the glucosamine chain). First, we showed the stability of the LykX-Sym10 dimer during molecular dynamics (MD) in solvent and in the presence of a membrane. Then, four NFs were separately docked to one European and two Afghanistan dimers, and the results of these interactions were in line with corresponding pea symbiotic phenotypes. The European variant of the LykX-Sym10 dimer effectively interacts with both acetylated and non-acetylated forms of NF, while the Afghanistan variants successfully interact with the acetylated form only. We additionally demonstrated that the length of the NF glucosamine chain contributes to controlling the effectiveness of the symbiotic interaction. The obtained results support a recent hypothesis that the LykX gene is a suitable candidate for the unidentified Sym2 allele, the determinant of pea specificity toward Rhizobium leguminosarum bv. viciae strains producing NFs with or without an acetylation decoration. The developed modeling methodology demonstrated its power in multiple searches for genetic determinants, when experimental detection of such determinants has proven extremely difficult. [ABSTRACT FROM AUTHOR]

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2 , ARF3 , and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/ trans -acting small interference RNA 3 (TAS3) module through the action of TAS3 -derived trans - acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/ TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/ TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a , and MtARF4b , in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2 , MtARF3 , and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a , and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a , and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a , and MtARF4b , as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules. [ABSTRACT FROM AUTHOR]

Data on the physiological role of phytohormones (mainly cytokinin and auxin) in the initiation of cortical cell division of the root, in the formation of nodule primordium and in its further organogenesis are summarized. The necessity of high level of cytokinin and low level of auxin for this process is proved. The mechanism leading to an increase in the cytokinin / auxin ratio is associated with the inhibition of auxin transport from the aerial organs to the root with the involvement of cytokinin signaling. Reducing cytokinin / auxin ratio against the background of inhibition of cytokinin signaling initiates the formation of lateral roots. The role of other phytohormones as well as flavonoids which have a positive (gibberellins, brassinosteroids) or negative influence (ethylene, abscisic, jasmonic and salicylic acids) on the formation of the root nodule is discussed. The key role of the rhizobial Nod factor signaling in the organogenesis of the nodule is emphasized. The schemes of reactions and signaling processes involved in the initiation of nodule primordium and lateral roots formation are given.

Data on the role of the legume-rhizobial symbiosis (LRS) in national economic and a brief history of the fundamental study of this unique biological phenomenon are summarized. The features of the formation of root nodules of determinant and indeterminant types are described. The physiological role of the rhizobial Nod factor in suppressing the defense system of a legume plant and the role of the plant's immune systems (MTI and ETI) in the rhizobial infection and the formation of LRS are discussed. Signal systems of a legume plant (Ca2+, NO-synthase, NADPH oxidase) and their components (ROS, RNS) and other signaling molecules involved and interacting in LRS onset are described. The necessity of studying the local and systemic resistsnce of the legume plant to the rhizobial infection is emphasized.

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts. [ABSTRACT FROM AUTHOR]

Multimodal spectroscopic imaging methods such as Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI), Fourier Transform Infrared spectroscopy (FT-IR) and Raman spectroscopy were used to monitor the changes in distribution and to determine semi quantitatively selected metabolites involved in nitrogen fixation in pea root nodules. These approaches were used to evaluate the effectiveness of nitrogen fixation by pea plants treated with biofertilizer preparations containing Nod factors. To assess the effectiveness of biofertilizer, the fresh and dry masses of plants were determined. The biofertilizer was shown to be effective in enhancing the growth of the pea plants. In case of metabolic changes, the biofertilizer caused a change in the apparent distribution of the leghaemoglobin from the edges of the nodule to its centre (the active zone of nodule). Moreover, the enhanced nitrogen fixation and presumably the accelerated maturation form of the nodules were observed with the use of a biofertilizer.

Many Bradyrhizobium strains are able to establish a Nod factor-independent symbiosis with the leguminous plant Aeschynomene indica by the use of a type III secretion system (T3SS). Recently, an important advance in the understanding of the molecular factors supporting this symbiosis has been achieved by the in silico identification and functional characterization of 27 putative T3SS effectors (T3Es) of Bradyrhizobium vignae ORS3257. In the present study, we experimentally extend this catalog of T3Es by using a multi-omics approach. Transcriptome analysis under non-inducing and inducing conditions in the ORS3257 wild-type strain and the ttsI mutant revealed that the expression of 18 out of the 27 putative effectors previously identified, is under the control of TtsI, the global transcriptional regulator of T3SS and T3Es. Quantitative shotgun proteome analysis of culture supernatant in the wild type and T3SS mutant strains confirmed that 15 of the previously determined candidate T3Es are secreted by the T3SS. Moreover, the combined approaches identified nine additional putative T3Es and one of them was experimentally validated as a novel effector. Our study underscores the power of combined proteome and transcriptome analyses to complement in silico predictions and produce nearly complete effector catalogs. The establishment of the ORS3257 effectome will form the basis for a full appraisal of the symbiotic properties of this strain during its interaction with various host legumes via different processes.

Lotus species develop infection threads to guide rhizobia into nodule cells. However, there is evidence that some species have a genetic repertoire to allow other modes of infection. By conducting confocal and electron microscopy, quantification of marker gene expression, and phenotypic analysis of transgenic roots infected with mutant rhizobia, we elucidated the infection mechanism used by Rhizobium leguminosarum Norway to colonize Lotus burttii. Rhizobium leguminosarum Norway induces a distinct host transcriptional response compared with Mesorhizobium loti. It infects L. burttii utilizing an epidermal and transcellular infection thread-independent mechanism at high frequency. The entry into plant cells occurs directly from the apoplast and is primarily mediated by 'peg'-like structures, the formation of which is dependent on the production of Nod factor by the rhizobia. These results demonstrate that Lotus species can exhibit duality in their infection mechanisms depending on the rhizobial strain that they encounter. This is especially relevant in the context of interactions in the rhizosphere where legumes do not encounter single strains, but complex rhizobial communities. Additionally, our findings support a perception mechanism at the nodule cell entry interface, reinforcing the idea that there are successive checkpoints during rhizobial infection. [ABSTRACT FROM AUTHOR]

Nitrogen is one of the essential plant nutrients and a major factor limiting crop productivity. To meet the requirements of sustainable agriculture, there is a need to maximize biological nitrogen fixation in different crop species. Legumes are able to establish root nodule symbiosis (RNS) with nitrogen-fixing soil bacteria which are collectively called rhizobia. This mutualistic association is highly specific, and each rhizobia species/strain interacts with only a specific group of legumes, and vice versa. Nodulation involves multiple phases of interactions ranging from initial bacterial attachment and infection establishment to late nodule development, characterized by a complex molecular signalling between plants and rhizobia. Characteristically, legumes like groundnut display a bacterial invasion strategy popularly known as “crack-entry’’ mechanism, which is reported approximately in 25% of all legumes. This article accommodates critical discussions on the bacterial infection mode, dynamics of nodulation, components of symbiotic signalling pathway, and also the effects of abiotic stresses and phytohormone homeostasis related to the root nodule symbiosis of groundnut and Bradyrhizobium. These parameters can help to understand how groundnut RNS is programmed to recognize and establish symbiotic relationships with rhizobia, adjusting gene expression in response to various regulations. This review further attempts to emphasize the current understanding of advancements regarding RNS research in the groundnut and speculates on prospective improvement possibilities in addition to ways for expanding it to other crops towards achieving sustainable agriculture and overcoming environmental challenges.

Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.

Recognition of Nod factors by LysM receptors is crucial for nitrogen-fixing symbiosis in most legumes. The large families of LysM receptors in legumes suggest concerted functions, yet only NFR1 and NFR5 and their closest homologs are known to be required. Here we show that an epidermal LysM receptor (NFRe), ensures robust signalling in L. japonicus. Mutants of Nfre react to Nod factors with increased calcium spiking interval, reduced transcriptional response and fewer nodules in the presence of rhizobia. NFRe has an active kinase capable of phosphorylating NFR5, which in turn, controls NFRe downstream signalling. Our findings provide evidence for a more complex Nod factor signalling mechanism than previously anticipated. The spatio-temporal interplay between Nfre and Nfr1, and their divergent signalling through distinct kinases suggests the presence of an NFRe-mediated idling state keeping the epidermal cells of the expanding root system attuned to rhizobia.

Sandwich-like docking configurations of the heterodimeric complex of NFR5 and K1 Vicia sativa receptor-like kinases together with the putative ligand, Nod factor (NF) of Rhizobium leguminosarum bv. viciae, were modeled and two of the most probable configurations were assessed through the analysis of the mutual polymorphisms and conservatism. We carried out this analysis based on the hypothesis that in a contact zone of two docked components (proteins or ligands) the population polymorphism or conservatism is mutual, i.e., the variation in one component has a reflected variation in the other component. The population material of 30 wild-growing V. sativa (leaf pieces) was collected from a large field (uncultivated for the past 25-years) and pooled; form this pool, 100 randomly selected cloned fragments of NFR5 gene and 100 of K1 gene were sequenced by the Sanger method. Congruence between population trees of NFR5 and K1 haplotypes allowed us to select two respective haplotypes, build their 3D structures, and perform protein–protein docking. In a separate simulation, the protein-ligand docking between NFR5 and NF was carried out. We merged the results of the two docking experiments and extracted NFR5–NF–K1 complexes, in which NF was located within the cavity between two receptors. Molecular dynamics simulations indicated two out of six complexes as stable. Regions of mutual polymorphism in the contact zone of one complex overlapped with known NF structural variations produced by R. leguminosarum bv. viciae. A total of 74% of the contact zone of another complex contained mutually polymorphic and conservative areas. Common traits of the obtained two stable structures allowed us to hypothesize the functional role of three-domain structure of plant LysM-RLKs in their heteromers.

Sandwich-like docking configurations of the heterodimeric complex of NFR5 and K1 Vicia sativa receptor-like kinases together with the putative ligand, Nod factor (NF) of Rhizobium leguminosarum bv. viciae, were modeled and two of the most probable configurations were assessed through the analysis of the mutual polymorphisms and conservatism. We carried out this analysis based on the hypothesis that in a contact zone of two docked components (proteins or ligands) the population polymorphism or conservatism is mutual, i.e., the variation in one component has a reflected variation in the other component. The population material of 30 wild-growing V. sativa (leaf pieces) was collected from a large field (uncultivated for the past 25-years) and pooled; form this pool, 100 randomly selected cloned fragments of NFR5 gene and 100 of K1 gene were sequenced by the Sanger method. Congruence between population trees of NFR5 and K1 haplotypes allowed us to select two respective haplotypes, build their 3D structures, and perform protein-protein docking. In a separate simulation, the protein-ligand docking between NFR5 and NF was carried out. We merged the results of the two docking experiments and extracted NFR5-NF-K1 complexes, in which NF was located within the cavity between two receptors. Molecular dynamics simulations indicated two out of six complexes as stable. Regions of mutual polymorphism in the contact zone of one complex overlapped with known NF structural variations produced by R. leguminosarum bv. viciae. A total of 74% of the contact zone of another complex contained mutually polymorphic and conservative areas. Common traits of the obtained two stable structures allowed us to hypothesize the functional role of three-domain structure of plant LysM-RLKs in their heteromers. [ABSTRACT FROM AUTHOR]

Plants are exposed to many different microbes in their habitat. These microbes may be benign or pathogenic, but in some cases they are beneficial. The rhizosphere provides an especially rich palette for colonization by beneficial (associative and symbiotic) microorganisms, which raises the question as to how roots can distinguish such ‘friends’ from possible ‘foes’ (i.e., pathogens). Plants possess an innate immunity system that can recognize pathogens, through an arsenal of protein receptors. These receptors include receptor-like kinases (RLK) and receptor-like proteins (RLP) located at the plasma membrane, as well as intracellular receptors (so called NBS-LRR proteins or R proteins) that recognize molecules released by microbes into the plant cell. The key rhizobial, symbiotic signaling molecule (called Nod factor) is perceived by the host legume plant using LysM-domain containing RLKs. Perception of the symbiotic Nod factor triggers signaling cascades leading to bacterial infection and accommodation of the symbiont in a newly formed root organ, the nodule, resulting in a nitrogen-fixing root nodule symbiosis (RNS). The net result of this symbiosis is the intracellular colonization of the plant with thousands of bacteria; a process that seems to occur in spite of the immune ability of plants to prevent pathogen infection. In this review, we discuss the potential of the invading rhizobial symbiont to actively avoid this innate immunity response, as well as specific examples of where the plant immune response may modulate rhizobial infection and host range.