The Type IV Secretion System (T4SS) Mediates Symbiosis between Bradyrhizobium sp. SUTN9-2 and Legumes.

There has been little study of the type IV secretion system (T4SS) of bradyrhizobia and its role in legume symbiosis. Therefore, broad host range Bradyrhizobium sp. SUTN9-2 was selected for study. The chromosome of Bradyrhizobium sp. SUTN9-2 contains two copies of the T4SS gene, homologous with the tra/trb operons. A phylogenetic tree of the T4SS gene traG was constructed, which exemplified its horizontal transfer among Bradyrhizobium and Mesorhizobium genera. They also showed similar gene arrangements for the tra/trb operons. However, the virD2 gene was not observed in Mesorhizobium, except M. oppotunistum WSM2075. Interestingly, the orientation of copG, traG, and virD2 cluster was unique to the Bradyrhizobium genus. The phylogenetic tree of copG, traG, and virD2 demonstrated that copies 1 and 2 of these genes were grouped in different clades. In addition, the derived mutant and complementation strains of T4SS were investigated in representative legumes Genistoids, Dalbergioids, and Millettiods. When T4SS copy 1 (T4SS1) was deleted, the nodule number and nitrogenase activity decreased. This supports a positive effect of T4SS1 on symbiosis. In addition, delayed nodulation was observed 7 dpi, which was restored by the complementation of T4SS1. Therefore, T4SS plays an important role in the symbiotic interaction between Bradyrhizobium sp. SUTN9-2 and its leguminous hosts. IMPORTANCE SUTN9-2 is a broad host range strain capable of symbiosis with several legumes. Two copies of T4SS clusters belonging to the tra/trb operon are observed on chromosomes with different gene arrangements. We use phylogenetic tree and gene annotation analysis to predict the evolution of the tra/trb operon of rhizobia. Our finding suggests that the gene encoding the T4SS gene among Bradyrhizobium and Mesorhizobium may have coevolution. In addition, Bradyrhizobium has a uniquely arranged copG, traG, and virD2 gene cluster. The results of T4SS1 gene deletion and complementation revealed its positive effect on nodulation. Therefore, T4SS seems to be another determinant for symbiosis. This is the first report on the role of T4SS in Bradyrhizobium symbiosis.

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid.

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.

nifDK Clusters Located on the Chromosome and Megaplasmid of Bradyrhizobium sp. Strain DOA9 Contribute Differently to Nitrogenase Activity During Symbiosis and Free-Living Growth.

Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobium sp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth.

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

Exploring the cellular surface polysaccharide and root nodule symbiosis characteristics of the rpoN mutants of Bradyrhizobium sp. DOA9 using synchrotron-based Fourier transform infrared microspectroscopy in conjunction with X-ray absorption spectroscopy.

The functional significance of rpoN genes that encode two sigma factors in the Bradyrhizobium sp. strain DOA9 has been reported to affect colony formation, root nodulation characteristics, and symbiotic interactions with Aeschynomene americana. rpoN mutant strains are defective in cellular surface polysaccharide (CSP) production compared with the wild-type (WT) strain, and they accordingly exhibit smaller colonies and diminished symbiotic effectiveness. To gain deeper insights into the changes in CSP composition and the nodules of rpoN mutants, we employed synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy. FTIR analysis of the CSP revealed the absence of specific components in the rpoN mutants, including lipids, carboxylic groups, polysaccharide-pyranose rings, and ß-galactopyranosyl residues. Nodules formed by DOA9WT exhibited a uniform distribution of lipids, proteins, and carbohydrates; mutant strains, particularly DOA9∆rpoNp:ΩrpoNc, exhibited decreased distribution uniformity and a lower concentration of C=O groups. Furthermore, Fe K-edge X-ray absorption near-edge structure and extended X-ray absorption fine structure analyses revealed deficiencies in the nitrogenase enzyme in the nodules of DOA9∆rpoNc and DOA9∆rpoNp:ΩrpoNc mutants; nodules from DOA9WT and DOA9∆rpoNp exhibited both leghemoglobin and the nitrogenase enzyme. IMPORTANCE This work provides valuable insights into how two rpoN genes affect the composition of cellular surface polysaccharides (CSPs) in Bradyrhizobium sp., which subsequently dictates root nodule chemical characteristics and nitrogenase production. We used advanced synchrotron methods, including synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy (XAS), for the first time in this field to analyze CSP components and reveal the biochemical changes occurring within nodules. These cutting-edge techniques confer significant advantages by providing detailed molecular information, enabling the identification of specific functional groups, chemical bonds, and biomolecule changes. This research not only contributes to our understanding of plant-microbe interactions but also establishes a foundation for future investigations and potential applications in this field. The combined use of the synchrotron-based FTIR and XAS techniques represents a significant advancement in facilitating a comprehensive exploration of bacterial CSPs and their implications in plant-microbe interactions.

Insights into Antifungal Mechanisms of Bacillus velezensis S141 against Cercospora Leaf Spot in Mungbean (V. radiata).

Cercospora leaf spot (CLS) is caused by Cercospora canescens and is one of the most important diseases of mungbean (Vigna radiata). Cercospora leaf spot may result in economic loss in production areas. The present study investigated the potential of Bacillus velezensis S141 as a biocontrol agent for C. canescens PAK1 growth on culture plates. Cell-free secretions from a dual culture of S141+PAK1 inhibited fungal growth more than those from a single culture of S141. The biocontrol efficiency of S141 against Cercospora leaf spot on mungbean was then evaluated by spraying. The disease severity of Cercospora leaf spot was significantly reduced in plants treated with S141, with a control efficiency of 83% after 2 days of infection. Comparative transcriptomics and qRT-PCR ana-lyses of S141 during C. canescens inhibition were performed to elucidate the antifungal mechanisms underlying its antifungal activity against Cercospora leaf spot. According to the differentially expressed genes, most up-regulated genes involved in the biosynthetic genes encoding enzymatic hydrolases, including protease, ß-glucanase, and N-acyl glucosaminase, were detected in strain S141 following its interaction. Moreover, genes related to secondary metabolites (surfactin, bacilysin, and bacillomycin D) were up-regulated. Collectively, these results suggest that S141 exhibited strong antifungal activity against C. canescens due to multiple enzymatic hydrolases and secondary metabolites. Therefore, the present study provides insights into the biological network responsible for the antifungal activity of B. velezensis S141 against C. canescens.

Enhancing the Efficiency of Soybean Inoculant for Nodulation under Multi-Environmental Stress Conditions.

The development of rhizobial inoculants with increased resistance to abiotic stress is critical to mitigating the challenges related to climate change. This study aims at developing a soybean stress-tolerant Bradyrhizobium inoculant to be used under the mixed stress conditions of acidity, high temperature, and drought. Six isolates of Bradyrhizobium with high symbiotic performance on soybean were tested to determine their growth or survival abilities under in vitro conditions. The representative stress-tolerant Bradyrhizobium isolates 184, 188, and 194 were selected to test their ability to promote soybean growth under stress conditions compared to the type strain Bradyrhizobium diazoefficiens USDA110. The plant experiment indicated that isolate 194 performed better in symbiosis with soybean than other Bradyrhizobium strains under stress conditions. Based on the stress tolerance index, soybeans inoculated with isolate 194 showed a high growth performance and significantly better nodulation competition ability than USDA110 under several stress conditions. Interestingly, supplementation of sucrose in the culture medium significantly enhances the survival of the isolate and leads to improved plant biomass under various stress conditions. Analysis of the intra-cellular sugars of isolate 194 supplemented with sucrose showed the accumulation of compatible solutes, such as trehalose and glycerol, that may act as osmoprotectants. This study indicates that inoculation of stress-tolerant Bradyrhizobium together with sucrose supplementation in a medium could enhance bacterial survival and symbiosis efficiency under stress conditions. Although it can be applied for inoculant production, this strategy requires validation of its performance in field conditions before adopting this technology.

Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species.

The Bradyrhizobium vignae strain ORS3257 is an elite strain recommended for cowpea inoculation in Senegal. This strain was recently shown to establish symbioses on some Aeschynomene species using a cocktail of Type III effectors (T3Es) secreted by the T3SS machinery. In this study, using a collection of mutants in different T3Es genes, we sought to identify the effectors that modulate the symbiotic properties of ORS3257 in three Vigna species (V. unguiculata, V. radiata and V. mungo). While the T3SS had a positive impact on the symbiotic efficiency of the strain in V. unguiculata and V. mungo, it blocked symbiosis with V. radiata. The combination of effectors promoting nodulation in V. unguiculata and V. mungo differed, in both cases, NopT and NopAB were involved, suggesting they are key determinants for nodulation, and to a lesser extent, NopM1 and NopP1, which are additionally required for optimal symbiosis with V. mungo. In contrast, only one effector, NopP2, was identified as the cause of the incompatibility between ORS3257 and V. radiata. The identification of key effectors which promote symbiotic efficiency or render the interaction incompatible is important for the development of inoculation strategies to improve the growth of Vigna species cultivated in Africa and Asia.

Homocitrate Synthase Genes of Two Wide-Host-Range Bradyrhizobium Strains are Differently Required for Symbiosis Depending on Host Plants.

The nifV gene encodes homocitrate synthase, the enzyme that catalyzes the formation of homocitrate, which is essential for arranging the FeMo-cofactor in the catalytic center of nitrogenase. Some host plants, such as Lotus japonicus, supply homocitrate to their symbionts, in this case, Mesorhizobium loti, which lacks nifV. In contrast, Bradyrhizobium ORS285, a symbiont of Aeschynomene cross-inoculation (CI) groups 2 and 3, requires nifV for symbiosis with Aeschynomene species that belong to CI group 3, and some species belonging to CI group 2. However, it currently remains unclear whether rhizobial nifV is required for symbiosis with Aeschynomene species belonging to CI group 1 or with other legumes. We generated nifV-disruption (ΔnifV) mutants of two wide-host-range rhizobia, Bradyrhizobium SUTN9-2 and DOA9, to investigate whether they require nifV for symbiosis. Both ΔnifV mutant strains showed significantly less nitrogenase activity in a free-living state than the respective wild-type strains. The symbiotic phenotypes of SUTN9-2, DOA9, and their ΔnifV mutants were examined with four legumes, Aeschynomene americana, Stylosanthes hamata, Indigofera tinctoria, and Desmodium tortuosum. nifV was required for the efficient symbiosis of SUTN9-2 with A. americana (CI group 1), but not for that of DOA9. SUTN9-2 established symbiosis with all three other legumes; nifV was required for symbiosis with I. tinctoria and D. tortuosum. These results suggest that, in addition to Aeschynomene CI groups 2 and 3, CI group 1 and several other legumes require the rhizobial nifV for symbiosis.

Regulation of Nitrogen Fixation in Bradyrhizobium sp. Strain DOA9 Involves Two Distinct NifA Regulatory Proteins That Are Functionally Redundant During Symbiosis but Not During Free-Living Growth.

The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (ß-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc.