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.

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.

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.

Mechanisms of Rice Endophytic Bradyrhizobial Cell Differentiation and Its Role in Nitrogen Fixation.

Bradyrhizobium sp. strain SUTN9-2 is a symbiotic and endophytic diazotrophic bacterium found in legume and rice plants and has the potential to promote growth. The present results revealed that SUTN9-2 underwent cell enlargement, increased its DNA content, and efficiently performed nitrogen fixation in response to rice extract. Some factors in rice extract induced the expression of cell cycle and nitrogen fixation genes. According to differentially expressed genes (DEGs) from the transcriptomic analysis, SUTN9-2 was affected by rice extract and the deletion of the bclA gene. The up-regulated DEGs encoding a class of oxidoreductases, which act with oxygen atoms and may have a role in controlling oxygen at an appropriate level for nitrogenase activity, followed by GroESL chaperonins are required for the function of nitrogenase. These results indicate that following its exposure to rice extract, nitrogen fixation by SUTN9-2 is induced by the collective effects of GroESL and oxidoreductases. The expression of the sensitivity to antimicrobial peptides transporter (sapDF) was also up-regulated, resulting in cell differentiation, even when bclA (sapDF) was mutated. This result implies similarities in the production of defensin-like antimicrobial peptides (DEFs) by rice and nodule-specific cysteine-rich (NCR) peptides in legume plants, which affect bacterial cell differentiation.

Type III Secretion System of Bradyrhizobium sp. SUTN9-2 Obstructs Symbiosis with Lotus spp.

The rhizobial type III secretion system secretes effector proteins into host plant cells, which may either promote or inhibit symbiosis with legumes. We herein demonstrated that the type III secretion system of Bradyrhizobium sp. SUTN9-2 obstructed symbiosis with Lotus japonicus Miyakojima, L. japonicus Gifu, and Lotus burttii. A mutant of SUTN9-2 that is unable to secrete effector proteins showed better nodulation and plant growth promotion than wild-type SUTN9-2 when paired with these Lotus spp. We propose that SUTN9-2 is a useful strain for understanding the mechanisms by which effector proteins obstruct symbiosis between Bradyrhizobium and Lotus spp.

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.

Potential of Rice Stubble as a Reservoir of Bradyrhizobial Inoculum in Rice-Legume Crop Rotation.

Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with leguminous and nonleguminous plants, such as rice. Therefore, it can be expected that rice endophytic bradyrhizobia can be applied in the rice-legume crop rotation system. Some endophytic bradyrhizobial strains were isolated from rice (Oryza sativa L.) tissues. The rice biomass could be enhanced when supplementing bradyrhizobial strain inoculation with KNO3, NH4NO3, or urea, especially in Bradyrhizobium sp. strain SUTN9-2. In contrast, the strains which suppressed rice growth were photosynthetic bradyrhizobia and were found to produce nitric oxide (NO) in the rice root. The expression of genes involved in NO production was conducted using a quantitative reverse transcription-PCR (qRT-PCR) technique. The nirK gene expression level in Bradyrhizobium sp. strain SUT-PR48 with nitrate was higher than that of the norB gene. In contrast, the inoculation of SUTN9-2 resulted in a lower expression of the nirK gene than that of the norB gene. These results suggest that SUT-PR48 may accumulate NO more than SUTN9-2 does. Furthermore, the nifH expression of SUTN9-2 was induced in treatment without nitrogen supplementation in an endophytic association with rice. The indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase produced in planta by SUTN9-2 were also detected. Enumeration of rice endophytic bradyrhizobia from rice tissues revealed that SUTN9-2 persisted in rice tissues until rice-harvesting season. The mung bean (Vigna radiata) can be nodulated after rice stubbles were decomposed. Therefore, it is possible that rice stubbles can be used as an inoculum in the rice-legume crop rotation system under both low- and high-organic-matter soil conditions.IMPORTANCE This study shows that some rice endophytic bradyrhizobia could produce IAA and ACC deaminase and have a nitrogen fixation ability during symbiosis inside rice tissues. These characteristics may play an important role in rice growth promotion by endophytic bradyrhizobia. However, the NO-producing strains should be of concern due to a possible deleterious effect of NO on rice growth. In addition, this study reports the application of endophytic bradyrhizobia in rice stubbles, and the rice stubbles were used directly as an inoculum for a leguminous plant (mung bean). The degradation of rice stubbles leads to an increased number of SUTN9-2 in the soil and may result in increased mung bean nodulation. Therefore, the persistence of endophytic bradyrhizobia in rice tissues can be developed to use rice stubbles as an inoculum for mung bean in a rice-legume crop rotation system.

The Type III Secretion System (T3SS) is a Determinant for Rice-Endophyte Colonization by Non-Photosynthetic Bradyrhizobium.

Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6-24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection.

Preferential association of endophytic bradyrhizobia with different rice cultivars and its implications for rice endophyte evolution.

Plant colonization by bradyrhizobia is found not only in leguminous plants but also in nonleguminous species such as rice. To understand the evolution of the endophytic symbiosis of bradyrhizobia, the effect of the ecosystems of rice plantations on their associations was investigated. Samples were collected from various rice (Oryza sativa) tissues and crop rotational systems. The rice endophytic bradyrhizobia were isolated on the basis of oligotrophic properties, selective medium, and nodulation on siratro (Macroptilium atropurpureum). Six bradyrhizobial strains were obtained exclusively from rice grown in a crop rotational system. The isolates were separated into photosynthetic bradyrhizobia (PB) and nonphotosynthetic bradyrhizobia (non-PB). Thai bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese bradyrhizobial strains. This implies that the rice cultivars possess characteristics that govern rice-bacterium associations. To examine whether leguminous plants in a rice plantation system support the persistence of rice endophytic bradyrhizobia, isolates were tested for legume nodulation. All PB strains formed symbioses with Aeschynomene indica and Aeschynomene evenia. On the other hand, non-PB strains were able to nodulate Aeschynomene americana, Vigna radiata, and M. atropurpureum but unable to nodulate either A. indica or A. evenia. Interestingly, the nodABC genes of all of these bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium sp. strain ORS285. From these results, we discuss the evolution of the plant-bradyrhizobium association, including nonlegumes, in terms of photosynthetic lifestyle and nod-independent interactions.