Identification of immune-responsive circular RNAs in shrimp (Litopenaeus vannamei) upon yellow head virus infection.

Circular RNAs (circRNAs) are a subclass of non-coding RNAs (ncRNAs) formed through a process known as back-splicing. They play a crucial role in the genetic regulation of various biological processes. Currently, circRNAs have been identified as participants in the antiviral response within mammalian cells. However, circRNAs in shrimp infected with the yellow head virus (YHV) remain largely unexplored. Therefore, this study aims to identify circRNAs in the hemocytes of Litopenaeus vannamei during YHV infection. We discovered 358 differentially expressed circRNAs (DECs), with 177 of them being up-regulated and 181 down-regulated. Subsequently, eight DECs, including circ_alpha-1-inhibitor 3, circ_CDC42 small effector protein 2, circ_hemicentin 2, circ_integrin alpha V, circ_kazal-type proteinase inhibitor, circ_phenoloxidase 3, circ_related protein rab-8B, and circ_protein toll-like, were randomly selected for analysis of their expression patterns during YHV infection using qRT-PCR. Furthermore, the circRNAs' characteristics were confirmed through PCR, RNase R treatment, and Sanger sequencing, all of which were consistent with the features of circRNAs. These findings contribute to a better understanding of circRNAs' involvement in the antiviral response in shrimp.

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.

Transcriptome-based insights into the regulatory role of immune-responsive circular RNAs in Litopanaeus vannamei upon WSSV infection.

Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

The interferon-like proteins, Vagos, in Fenneropenaeus merguiensis elicit antimicrobial responses against WSSV and VPAHPND infection.

The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VPAHPND). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VPAHPND challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VPAHPND-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.

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.

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.

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.

The evolutionary dynamics of ancient and recent polyploidy in the African semiaquatic species of the legume genus Aeschynomene.

The legume genus Aeschynomene is notable in the ability of certain semiaquatic species to develop nitrogen-fixing stem nodules. These species are distributed in two clades. In the first clade, all the species are characterized by the use of a unique Nod-independent symbiotic process. In the second clade, the species use a Nod-dependent symbiotic process and some of them display a profuse stem nodulation as exemplified in the African Aeschynomene afraspera. To facilitate the molecular analysis of the symbiotic characteristics of such legumes, we took an integrated molecular and cytogenetic approach to track occurrences of polyploidy events and to analyze their impact on the evolution of the African species of Aeschynomene. Our results revealed two rounds of polyploidy: a paleopolyploid event predating the African group and two neopolyploid speciations, along with significant chromosomal variations. Hence, we found that A. afraspera (8x) has inherited the contrasted genomic properties and the stem-nodulation habit of its parental lineages (4x). This study reveals a comprehensive picture of African Aeschynomene diversification. It notably evidences a history that is distinct from the diploid Nod-independent clade, providing clues for the identification of the specific determinants of the Nod-dependent and Nod-independent symbiotic processes, and for comparative analysis of stem nodulation.

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.

Comparative study of endophytic and endophytic diazotrophic bacterial communities across rice landraces grown in the highlands of northern Thailand.

Communities of bacterial endophytes within the rice landraces cultivated in the highlands of northern Thailand were studied using fingerprinting data of 16S rRNA and nifH genes profiling by polymerase chain reaction-denaturing gradient gel electrophoresis. The bacterial communities' richness, diversity index, evenness, and stability were varied depending on the plant tissues, stages of growth, and rice cultivars. These indices for the endophytic diazotrophic bacteria within the landrace rice Bue Wah Bo were significantly the lowest. The endophytic bacteria revealed greater diversity by cluster analysis with seven clusters compared to the endophytic diazotrophic bacteria (three clusters). Principal component analysis suggested that the endophytic bacteria showed that the community structures across the rice landraces had a higher stability than those of the endophytic diazotrophic bacteria. Uncultured bacteria were found dominantly in both bacterial communities, while higher generic varieties were observed in the endophytic diazotrophic bacterial community. These differences in bacterial communities might be influenced either by genetic variation in the rice landraces or the rice cultivation system, where the nitrogen input affects the endophytic diazotrophic bacterial community.

Enhancing Resistance to Cercospora Leaf Spot in Mung Bean (Vigna radiata L.) through Bradyrhizobium sp. DOA9 Priming: Molecular Insights and Bio-Priming Potential.

Mung bean (Vigna radiata L.), a vital legume in Asia with significant nutritional benefits, is highly susceptible to Cercospora leaf spot (CLS) caused by Cercospora canescens, leading to significant yield losses. As an alternative to chemical fungicides, bio-priming with rhizobacteria can enhance plant resistance. This study explores the potential of Bradyrhizobium sp. strain DOA9 to augment resistance in mung bean against CLS via root priming. The results reveal that short (3 days) and double (17 and 3 days) priming with DOA9 before fungal infection considerably reduces lesion size on infected leaves by activating defense-related genes, including Pti1, Pti6, EDS1, NDR1, PR-1, PR-2, Prx, and CHS, or by suppressing the inhibition of PR-5 and enhancing peroxidase (POD) activity in leaves. Interestingly, the Type 3 secretion system (T3SS) of DOA9 may play a role in establishing resistance in V. radiata CN72. These findings suggest that DOA9 primes V. radiata CN72's defense mechanisms, offering an effective bio-priming strategy to alleviate CLS. Hence, our insights propose the potential use of DOA9 as a bio-priming agent to manage CLS in V. radiata CN72, providing a sustainable alternative to chemical fungicide applications.

Exploring the therapeutic potential of Thai medicinal plants: in vitro screening and in silico docking of phytoconstituents for novel anti-SARS-CoV-2 agents.

BACKGROUND: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking. METHODS: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation. RESULTS: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-ß-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ - 9 kcal/mol. The stability of each ligand-protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

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.

Genome analysis suggests that the soil oligotrophic bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) is a nitrogen-fixing symbiont of Aeschynomene indica.

Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58(T) is a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments. Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165 bp with an average GC content of 65.1% lacking nodABC genes and the typical symbiosis island. The genome showed a high level of similarity to the genomes of Bradyrhizobium sp. ORS278 and Bradyrhizobium sp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant, Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nif and fix) with A. indica were well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with five A. oligotrophica strains (S58, S42, S55, S72, and S80). Surprisingly, all five strains of A. oligotrophica formed effective nitrogen-fixing nodules on the roots and/or stems of A. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwelling A. oligotrophica generally possesses the ability to establish symbiosis with A. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis with A. indica is a common trait of nodABC- and symbiosis island-lacking strains within the members of the photosynthetic Bradyrhizobium clade, including A. oligotrophica.

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.

Bacillus velezensis S141, a soybean growth-promoting bacterium, hydrolyzes isoflavone glycosides into aglycones.

Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodule bacteria, including Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydrolyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strains shared four genes encoding ß-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major ß-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely committed to the hydrolysis of isoflavone glycosides. Thus, an unknown ß-glucosidase exists, and our genetic knowledge of ß-glucosidases was insufficient to evaluate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation.

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.

Genetic diversity, symbiotic evolution, and proposed infection process of Bradyrhizobium strains isolated from root nodules of Aeschynomene americana L. in Thailand.

The diversity of bacteria nodulating Aeschynomene americana L. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK, recA, and glnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genus Bradyrhizobium, in which B. yuanmingense was the dominant species. Some of the other species, including B. japonicum, B. liaoningense, and B. canariense, were minor species. These isolated strains were divided into 2 groups-nod-containing and divergent nod-containing strains-based on Southern blot hybridization and PCR amplification of nodABC genes. The divergent nod genes could not be PCR amplified and failed to hybridize nod gene probes designed from B. japonicum USDA110, but hybridized to probes from other bradyrhizobial strains under low-stringency conditions. The grouping based on sequence similarity of nod genes was well correlated with the grouping based on that of nifH gene, in which the nod-containing and divergent nod-containing strains were obviously distinguished. The divergent nod-containing strains and photosynthetic bradyrhizobia shared close nifH sequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated from A. americana could nodulate Aeschynomene plants that belong to different cross-inoculation (CI) groups, including A. afraspera and A. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic and nod-containing strain) originating from CI group 1 nodulating roots of A. indica (CI group 3). An infection process used to establish symbiosis on Aeschynomene different from the classical one is proposed.