Pathogenic nematodes exploit Achilles' heel of plant symbioses.

Cyst nematode parasites disrupt beneficial associations of crops with rhizobia and mycorrhiza. Chen et al. discovered the mechanism and demonstrated that the soybean cyst nematode Heterodera glycines secretes a chitinase that destroys key symbiotic signals from the microbial symbionts. The authors further developed a chitinase inhibitor that alleviates symbiosis inhibition.

Nod Factor Lipopolysaccharide Purification to Study Nitrogen-Fixing Bacteria Symbiosis with Legumes.

Nod factors (NF) are lipochitooligosaccharides produced by nitrogen-fixing rhizobia bacteria. They are key components of the rhizobia-plant signaling exchange required for symbiosis. Thus, techniques to extract, detect, characterize, and purify NF are crucial for the identification of both rhizobial and plant mechanisms underlying nitrogen-fixing symbiosis. Here, we describe a method for NF detection using radiolabeling and thin-layer chromatography. Furthermore, we describe a technique for purifying NF for downstream analyses.

The T6SS-Dependent Effector Re78 of Rhizobium etli Mim1 Benefits Bacterial Competition.

The genes of the type VI secretion system (T6SS) from Rhizobium etli Mim1 (ReMim1) that contain possible effectors can be divided into three modules. The mutants in them indicated that they are not required for effective nodulation with beans. To analyze T6SS expression, a putative promoter region between the tssA and tssH genes was fused in both orientations to a reporter gene. Both fusions are expressed more in free living than in symbiosis. When the module-specific genes were studied using RT-qPCR, a low expression was observed in free living and in symbiosis, which was clearly lower than the structural genes. The secretion of Re78 protein from the T6SS gene cluster was dependent on the presence of an active T6SS. Furthermore, the expression of Re78 and Re79 proteins in E. coli without the ReMim1 nanosyringe revealed that these proteins behave as a toxic effector/immunity protein pair (E/I). The harmful action of Re78, whose mechanism is still unknown, would take place in the periplasmic space of the target cell. The deletion of this ReMim1 E/I pair resulted in reduced competitiveness for bean nodule occupancy and in lower survival in the presence of the wild-type strain.

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum bv. trifolii Requires a Complementary Function of Two Homologous Glycosyltransferases PssG and PssI.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction.

A complex regulatory network governs the expression of symbiotic genes in Sinorhizobium fredii HH103.

Introduction: The establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the nod regulon) of Sinorhizobium fredii HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, nodD2 and nolR. Both genes code for global regulators with a predominant repressor effect on the nod regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes. Methods: By combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a lacZ gene inserted into a regulatory gene, we have analysed the regulatory relations between the nodD1, nodD2, nolR, syrM, and ttsI genes, confirming previous data and discovering previously unknown relations. Results and discussion: Previously we showed that HH103 mutants in the nodD2, nolR, syrM, or ttsI genes gain effective nodulation with Lotus japonicus, a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the nodD2, nolR, and syrM single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with L. japonicus.

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

The Regulation of Pea (Pisum sativum L.) Symbiotic Nodule Infection and Defense Responses by Glutathione, Homoglutathione, and Their Ratio.

In this study, the roles of glutathione (GSH), homoglutathione (hGSH), and their ratio in symbiotic nodule development and functioning, as well as in defense responses accompanying ineffective nodulation in pea (Pisum sativum) were investigated. The expression of genes involved in (h)GSH biosynthesis, thiol content, and localization of the reduced form of GSH were analyzed in nodules of wild-type pea plants and mutants sym33-3 (weak allele, "locked" infection threads, occasional bacterial release, and defense reactions) and sym33-2 (strong allele, "locked" infection threads, defense reactions), and sym40-1 (abnormal bacteroids, oxidative stress, early senescence, and defense reactions). The effects of (h)GSH depletion and GSH treatment on nodule number and development were also examined. The GSH:hGSH ratio was found to be higher in nodules than in uninoculated roots in all genotypes analyzed, with the highest value being detected in wild-type nodules. Moreover, it was demonstrated, that a hGSHS-to-GSHS switch in gene expression in nodule tissue occurs only after bacterial release and leads to an increase in the GSH:hGSH ratio. Ineffective nodules showed variable GSH:hGSH ratios that correlated with the stage of nodule development. Changes in the levels of both thiols led to the activation of defense responses in nodules. The application of a (h)GSH biosynthesis inhibitor disrupted the nitrogen fixation zone in wild-type nodules, affected symbiosome formation in sym40-1 mutant nodules, and meristem functioning and infection thread growth in sym33-3 mutant nodules. An increase in the levels of both thiols following GSH treatment promoted both infection and extension of defense responses in sym33-3 nodules, whereas a similar increase in sym40-1 nodules led to the formation of infected cells resembling wild-type nitrogen-fixing cells and the disappearance of an early senescence zone in the base of the nodule. Meanwhile, an increase in hGSH levels in sym40-1 nodules resulting from GSH treatment manifested as a restriction of infection similar to that seen in untreated sym33-3 nodules. These findings indicated that a certain level of thiols is required for proper symbiotic nitrogen fixation and that changes in thiol content or the GSH:hGSH ratio are associated with different abnormalities and defense responses.

A legume-specific novel type of phytosulfokine, PSK-δ, promotes nodulation by enhancing nodule organogenesis.

Phytosulfokine-α (PSK-α), a tyrosine-sulfated pentapeptide with the sequence YSO3IYSO3TQ, is widely distributed across the plant kingdom and plays multiple roles in plant growth, development, and immune response. Here, we report a novel type of phytosulfokine, PSK-δ, and its precursor proteins (MtPSKδ, LjPSKδ, and GmPSKδ1), specifically from legume species. The sequence YSO3IYSO3TN of sulfated PSK-δ peptide is different from PSK-α at the last amino acid. Expression pattern analysis revealed PSK-δ-encoding precursor genes to be expressed primarily in legume root nodules. Specifically, in Medicago truncatula, MtPSKδ expression was detected in root cortical cells undergoing nodule organogenesis, in nodule primordia and young nodules, and in the apical region of mature nodules. Accumulation of sulfated PSK-δ peptide in M. truncatula nodules was detected by LC/MS. Application of synthetic PSK-δ peptide significantly increased nodule number in legumes. Similarly, overexpression of MtPSKδ in transgenic M. truncatula markedly promoted symbiotic nodulation. This increase in nodule number was attributed to enhanced nodule organogenesis induced by PSK-δ. Additional genetic evidence from the MtPSKδ mutant and RNA interference assays suggested that the PSK-δ and PSK-α peptides function redundantly in regulating nodule organogenesis. These results suggest that PSK-δ, a legume-specific novel type of phytosulfokine, promotes symbiotic nodulation by enhancing nodule organogenesis.

The Medicago truncatula IEF Gene Is Crucial for the Progression of Bacterial Infection During Symbiosis.

Legumes are able to meet their nitrogen need by establishing nitrogen-fixing symbiosis with rhizobia. Nitrogen fixation is performed by rhizobia, which has been converted to bacteroids, in newly formed organs, the root nodules. In the model legume Medicago truncatula, nodule cells are invaded by rhizobia through transcellular tubular structures called infection threads (ITs) that are initiated at the root hairs. Here, we describe a novel M. truncatula early symbiotic mutant identified as infection-related epidermal factor (ief), in which the formation of ITs is blocked in the root hair cells and only nodule primordia are formed. We show that the function of MtIEF is crucial for the bacterial infection in the root epidermis but not required for the nodule organogenesis. The IEF gene that appears to have been recruited for a symbiotic function after the duplication of a flower-specific gene is activated by the ERN1-branch of the Nod factor signal transduction pathway and independent of the NIN activity. The expression of MtIEF is induced transiently in the root epidermal cells by the rhizobium partner or Nod factors. Although its expression was not detectable at later stages of symbiosis, complementation experiments indicate that MtIEF is also required for the proper invasion of the nodule cells by rhizobia. The gene encodes an intracellular protein of unknown function possessing a coiled-coil motif and a plant-specific DUF761 domain. The IEF protein interacts with RPG, another symbiotic protein essential for normal IT development, suggesting that combined action of these proteins plays a role in nodule infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

NIN-Like Proteins: Interesting Players in Rhizobia-Induced Nitrate Signaling Response During Interaction with Non-Legume Host Arabidopsis thaliana.

Nitrogen is an essential macronutrient and a key cellular messenger. Plants have evolved refined molecular systems to sense the cellular nitrogen status. This is exemplified by the root nodule symbiosis between legumes and symbiotic rhizobia, where nitrate availability inhibits this mutualistic interaction. Additionally, nitrate also functions as a metabolic messenger, resulting in nitrate signaling cascades which intensively crosstalk with other physiological pathways. Nodule inception-like proteins (NLPs) are key players in nitrate signaling and regulate nitrate-dependent transcription during legume-rhizobia interactions. Nevertheless, the coordinated interplay between nitrate signaling pathways and rhizobacteria-induced responses remains to be elucidated. In our study, we investigated rhizobia-induced changes in the root system architecture of the non-legume host arabidopsis under different nitrate conditions. We demonstrate that rhizobium-induced lateral root growth and increased root hair length and density are regulated by a nitrate-related signaling pathway. Key players in this process are AtNLP4 and AtNLP5, because the corresponding mutants failed to respond to rhizobia. At the cellular level, AtNLP4 and AtNLP5 control a rhizobia-induced decrease in cell elongation rates, while additional cell divisions occurred independently of AtNLP4. In summary, our data suggest that root morphological responses to rhizobia are coordinated by a newly considered nitrate-related NLP pathway that is evolutionarily linked to regulatory circuits described in legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans.

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rhizobial Exopolysaccharides: Genetic Regulation of Their Synthesis and Relevance in Symbiosis with Legumes.

Rhizobia are soil proteobacteria able to engage in a nitrogen-fixing symbiotic interaction with legumes that involves the rhizobial infection of roots and the bacterial invasion of new organs formed by the plant in response to the presence of appropriate bacterial partners. This interaction relies on a complex molecular dialogue between both symbionts. Bacterial N-acetyl-glucosamine oligomers called Nod factors are indispensable in most cases for early steps of the symbiotic interaction. In addition, different rhizobial surface polysaccharides, such as exopolysaccharides (EPS), may also be symbiotically relevant. EPS are acidic polysaccharides located out of the cell with little or no cell association that carry out important roles both in free-life and in symbiosis. EPS production is very complexly modulated and, frequently, co-regulated with Nod factors, but the type of co-regulation varies depending on the rhizobial strain. Many studies point out a signalling role for EPS-derived oligosaccharides in root infection and nodule invasion but, in certain symbiotic couples, EPS can be dispensable for a successful interaction. In summary, the complex regulation of the production of rhizobial EPS varies in different rhizobia, and the relevance of this polysaccharide in symbiosis with legumes depends on the specific interacting couple.

How Rhizobia Adapt to the Nodule Environment.

Rhizobia are a phylogenetically diverse group of soil bacteria that engage in mutualistic interactions with legume plants. Although specifics of the symbioses differ between strains and plants, all symbioses ultimately result in the formation of specialized root nodule organs that host the nitrogen-fixing microsymbionts called bacteroids. Inside nodules, bacteroids encounter unique conditions that necessitate the global reprogramming of physiological processes and the rerouting of their metabolism. Decades of research have addressed these questions using genetics, omics approaches, and, more recently, computational modeling. Here, we discuss the common adaptations of rhizobia to the nodule environment that define the core principles of bacteroid functioning. All bacteroids are growth arrested and perform energy-intensive nitrogen fixation fueled by plant-provided C4-dicarboxylates at nanomolar oxygen levels. At the same time, bacteroids are subject to host control and sanctioning that ultimately determine their fitness and have fundamental importance for the evolution of a stable mutualistic relationship.

Investigating the Involvement of Cytoskeletal Proteins MreB and FtsZ in the Origin of Legume-Rhizobial Symbiosis.

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants; however, they don't carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homolog from a pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular, and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small, ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA sequencing analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, an FtsZ2 homolog was commonly found in Rhizobium, Sinorhizobium, and Mesorhizobium spp. but not in closely related nonsymbiotic species. Bacterial two-hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived nodule-specific cysteine-rich peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1 and FtsZ2 causes the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of members of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of the metabolites regulated in soybean-Rhizobia symbiosis through solid phase microextraction coupled with LC-MS.

Legumes provide one of the uniquely nutrient-rich food sources to the population and are one of the primary field crops that play significant roles in agricultural sustainability. Inoculation with Bradyrhizobium japonicum is necessary for the high yield of leguminous crops, i.e. soybean. Nodulation of soybean by Bradyrhizobium japonicum is a complex process that is essential for cultivation of these legumes and external stress factors, such as draught and soil acidity, that influence the nodulation and crop yield. Alterations in the nodule metabolites are known to identify the type of stress that mitigates nodulation and lowers crop yield. Current techniques aimed at understanding the metabolic activities in the symbiont, such as in the case of metabolic regulations in varying nodule growth phases, rely on exhaustive techniques based on the removal of nodules or other plant tissue. Aiming to capture a more in-depth, accurate profile of this system without quenching the metabolic activity in the nodules, or removing the nodules, a workflow was prepared for the metabolite sampling through in vivo solid phase microextraction in thin film format (TF-SPME). This technique was followed by LC-QTOF-MS instrumental analysis with subsequent metabolite annotation and reference standard validation. Our approach is unique in terms of eliminating the effects that arise due to analyte partition coefficients. We show that the symbiont undergoes metabolic regulations throughout the cultivation period, displaying the efficacy of TF-SPME as a non-exhaustive sampling method that can be used as a tool to investigate the metabolic alterations in nodules. These alterations would potentially fingerprint the environmental effects on soybean yield.

RibBX of Bradyrhizobium ORS285 Plays an Important Role in Intracellular Persistence in Various Aeschynomene Host Plants.

Bradyrhizobium ORS285 forms a nitrogen-fixating symbiosis with both Nod factor (NF)-dependent and NF-independent Aeschynomene spp. The Bradyrhizobium ORS285 ribBA gene encodes for a putative bifunctional enzyme with 3,4-dihydroxybutanone phosphate (3,4-DHBP) synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps in the riboflavin biosynthesis pathway. In this study, we show that inactivating the ribBA gene does not cause riboflavin auxotrophy under free-living conditions and that, as shown for RibBAs from other bacteria, the GTP cyclohydrolase II domain has no enzymatic activity. For this reason, we have renamed the annotated ribBA as ribBX. Because we were unable to identify other ribBA or ribA and ribB homologs in the genome of Bradyrhizobium ORS285, we hypothesize that the ORS285 strain can use unconventional enzymes or an alternative pathway for the initial steps of riboflavin biosynthesis. Inactivating ribBX has a drastic impact on the interaction of Bradyrhizobium ORS285 with many of the tested Aeschynomene spp. In these Aeschynomene spp., the ORS285 ribBX mutant is able to infect the plant host cells but the intracellular infection is not maintained and the nodules senesce early. This phenotype can be complemented by reintroduction of the 3,4-DHBP synthase domain alone. Our results indicate that, in Bradyrhizobium ORS285, the RibBX protein is not essential for riboflavin biosynthesis under free-living conditions and we hypothesize that its activity is needed to sustain riboflavin biosynthesis under certain symbiotic conditions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Sinorhizobium fredii HH103 type III secretion system effector NopC blocks nodulation with Lotus japonicus Gifu.

The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.