Signal binding at both modules of its dCache domain enables the McpA chemoreceptor of Bacillus velezensis to sense different ligands.

Bacteria have evolved multiple signal transduction systems that permit an adaptation to changing environmental conditions. Chemoreceptor-based signaling cascades are very abundant in bacteria and are among the most complex signaling systems. Currently, our knowledge on the molecular features that determine signal recognition at chemoreceptors is limited. Chemoreceptor McpA of Bacillus velezensis SQR9 has been shown to mediate chemotaxis to a broad range of different ligands. Here we show that its ligand binding domain binds directly 13 chemoattractants. We provide support that organic acids and amino acids bind to the membrane-distal and membrane-proximal module of the dCache domain, respectively, whereas binding of sugars/sugar alcohols occurred at both modules. Structural biology studies combined with site-directed mutagenesis experiments have permitted to identify 10 amino acid residues that play key roles in the recognition of multiple ligands. Residues in membrane-distal and membrane-proximal regions were central for sensing organic acids and amimo acids, respectively, whereas all residues participated in sugars/sugar alcohol sensing. Most characterized chemoreceptors possess a narrow and well-defined ligand spectrum. We propose here a sensing mechanism involving both dCache modules that allows the integration of very diverse signals by a single chemoreceptor.

Listening to plant's Esperanto via root exudates: reprogramming the functional expression of plant growth-promoting rhizobacteria.

Rhizomicrobiome plays important roles in plant growth and health, contributing to the sustainable development of agriculture. Plants recruit and assemble the rhizomicrobiome to satisfy their functional requirements, which is widely recognized as the 'cry for help' theory, but the intrinsic mechanisms are still limited. In this study, we revealed a novel mechanism by which plants reprogram the functional expression of inhabited rhizobacteria, in addition to the de novo recruitment of soil microbes, to satisfy different functional requirements as plants grow. This might be an efficient and low-cost strategy and a substantial extension to the rhizomicrobiome recruitment theory. We found that the plant regulated the sequential expression of genes related to biocontrol and plant growth promotion in two well-studied rhizobacteria Bacillus velezensis SQR9 and Pseudomonas protegens CHA0 through root exudate succession across the plant developmental stages. Sixteen key chemicals in root exudates were identified to significantly regulate the rhizobacterial functional gene expression by high-throughput qPCR. This study not only deepens our understanding of the interaction between the plant-rhizosphere microbiome, but also provides a novel strategy to regulate and balance the different functional expression of the rhizomicrobiome to improve plant health and growth.

Nitrogen fertilization modulates beneficial rhizosphere interactions through signaling effect of nitric oxide.

Chemical nitrogen (N) fertilization is customary for increasing N inputs in agroecosystems. The nutritional effects of N fertilization on plants and soil microbes have been well studied. However, the signaling effects of N fertilization on rhizosphere plant-microbe interactions and the following feedback to plant performance remain unknown. Here, we investigated the effect of different N fertilizations on the behavior of the plant growth-promoting rhizobacteria (PGPR) Bacillus velezensis SQR9 in the cucumber (Cucumis sativus L.) rhizosphere. Moderate N fertilization promoted higher rhizosphere colonization of strain SQR9 than insufficient or excessive N input. Nitric oxide (NO) produced through the denitrification process under N fertilization was identified as the signaling molecule that dominates the root colonization of PGPR, and this effect could be neutralized by the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide. Gene expression analysis demonstrated that NO regulated the biofilm formation of strain SQR9 by affecting the synthesis of extracellular matrix γ-polyglutamic acid, consequently impacting its root colonization. Finally, we demonstrated that moderate N fertilization-modulated enhanced PGPR root colonization can significantly promote plant growth and nitrogen use efficiency. This study provides insights into our understanding of the beneficial rhizosphere plant-microbe interactions under N fertilization and suggests that rational fertilization is critical to promote beneficial rhizosphere interactions for sustainable agricultural production.

Identification of Adhesins in Plant Beneficial Rhizobacteria Bacillus velezensis SQR9 and Their Effect on Root Colonization.

Probiotic Bacillus colonization of plant root surfaces has been reported to improve its beneficial effect. Chemotaxis, adhesion, aggregation, and biofilm formation are the four steps of root colonization by plant growth-promoting rhizobacteria (PGPRs). Compared with the other three well-studied processes, adhesion of PGPRs is less known. In this study, using mutant strains deleted for potential adhesin genes in PGPR strain Bacillus velezensis SQR9, adherence to both cucumber root surface and abiotic surface by those strains was evaluated. Results showed that deletion mutations ΔlytB, ΔV529_10500, ΔfliD, ΔyhaN, and ΔsacB reduced the adhesion to root surfaces, while, among them, only ΔfliD had significant defects in adhesion to abiotic surfaces (glass and polystyrene). In addition, B. velevzensis SQR9 mutants defective in adhesion to root surfaces showed a deficiency in rhizosphere colonization. Among the encoded proteins, FliD and YhaN played vital roles in root adhesion. This research systematically explored the potential adhesins in a well-studied PGPR strain and also indicated that adhesion progress was required for root colonization, which will help to enhance rhizosphere colonization and beneficial function of PGPRs in agricultural production.[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 volatile cedrene from Trichoderma guizhouense modulates Arabidopsis root development through auxin transport and signalling.

Rhizosphere microorganisms interact with plant roots by producing chemical signals that regulate root development. However, the distinct bioactive compounds and signal transduction pathways remain to be identified. Here, we showed that sesquiterpenes are the main volatile compounds produced by plant-beneficial Trichoderma guizhouense NJAU4742. Inhibition of sesquiterpene biosynthesis eliminated the promoting effect of this strain on root growth, indicating its involvement in plant-fungus cross-kingdom signalling. Sesquiterpene component analysis identified cedrene, a highly abundant sesquiterpene in strain NJAU4742, to stimulate plant growth and root development. Genetic analysis and auxin transport inhibition showed that the TIR1 and AFB2 auxin receptors, IAA14 auxin-responsive protein, and ARF7 and ARF19 transcription factors affected the response of lateral roots to cedrene. Moreover, the AUX1 auxin influx carrier and PIN2 efflux carrier were also found to be indispensable for cedrene-induced lateral root formation. Confocal imaging showed that cedrene affected the expression of pPIN2:PIN2:GFP and pPIN3:PIN3:GFP, which might be related to the effect of cedrene on root morphology. These results suggested that a novel sesquiterpene molecule from plant-beneficial T. guizhouense regulates plant root development through the transport and signalling of auxin.

Overcoming diverse homologous recombinations and single chimeric guide RNA competitive inhibition enhances Cas9-based cyclical multiple genes coediting in filamentous fungi.

Deciphering the complex cellular behaviours and advancing the biotechnology applications of filamentous fungi increase the requirement for genetically manipulating a large number of target genes. The current strategies cannot cyclically coedit multiple genes simultaneously. In this study, we firstly revealed the existence of diverse homologous recombination (HR) types in marker-free editing of filamentous fungi, and then, demonstrated that sgRNA efficiency-mediated competitive inhibition resulted in the low integration of multiple genetic sites during coediting, which are the two major obstacles to limit the efficiency of cyclically coediting of multiple genes. To overcome these obstacles, we developed a biased cutting strategy by Cas9 to greatly enhance the desired HR type and applied a new selection marker labelling strategy for multiple donor DNAs, in which only the donor DNA with the lowest sgRNA efficiency was labelled. Combined with these strategies, we successfully developed a convenient method for cyclically coediting multiple genes in different filamentous fungi. In addition, diverse HRs resulted in a useful and convenient one-step approach for gene functional study combining both gene disruption and complementation. This research provided both a useful one-step approach for gene functional study and an efficient strategy for cyclically coediting multiple genes in filamentous fungi.

Bacillus velezensis tolerance to the induced oxidative stress in root colonization contributed by the two-component regulatory system sensor ResE.

Efficient root colonization of plant growth-promoting rhizobacteria is critical for their plant-beneficial functions. However, the strategy to overcome plant immunity during root colonization is not well understood. In particular, how Bacillus strains cope with plant-derived reactive oxygen species (ROS), which function as the first barrier of plant defence, is not clear. In the present study, we found that the homolog of flg22 in Bacillus velezensis SQR9 (flg22SQR9 ) has 78.95% identity to the typical flg22 (flg22P.s. ) and induces a significant oxidative burst in cucumber and Arabidopsis. In contrast to pathogenic or beneficial Pseudomonas, live B. velezensis SQR9 also induced an oxidative burst in cucumber. We further found that B. velezensis SQR9 tolerated higher H2 O2 levels than Pst DC3000, the pathogen that harbours the typical flg22, and that it possesses the ability to suppress the flg22-induced oxidative burst, indicating that B. velezensis SQR9 may exploit a more efficient ROS tolerance system than DC3000. Further experimentation with mutagenesis of bacteria and Arabidopsis showed that the two-component regulatory system, ResDE, in B. velezensis SQR9 is involved in tolerance to plant-derived oxidative stress, thus contributing to root colonization. This study supports a further investigation of the interaction between beneficial rhizobacteria and plant immunity.

Volatile compounds from beneficial rhizobacteria Bacillus spp. promote periodic lateral root development in Arabidopsis.

Lateral root formation is coordinated by both endogenous and external factors. As biotic factors, plant growth-promoting rhizobacteria can affect lateral root formation, while the regulation mechanism is unclear. In this study, by applying various marker lines, we found that volatile compounds (VCs) from Bacillus amyloliquefaciens SQR9 induced higher frequency of DR5 oscillation and prebranch site formation, accelerated the development and emergence of the lateral root primordia and thus promoted lateral root development in Arabidopsis. We demonstrated a critical role of auxin on B. amyloliquefaciens VCs-induced lateral root formation via respective mutants and pharmacological experiments. Our results showed that auxin biosynthesis, polar transport and signalling pathway are involved in B. amyloliquefaciens VCs-induced lateral roots formation. We further showed that acetoin, a major component of B. amyloliquefaciens VCs, is less active in promoting root development compared to VC blends from B. amyloliquefaciens, indicating the presence of yet uncharacterized/unknown VCs might contribute to B. amyloliquefaciens effect on lateral root formation. In summary, our study revealed an auxin-dependent mechanism of B. amyloliquefaciens VCs in regulating lateral root branching in a non-contact manner, and further efforts will explore useful VCs to promote plant root development.

Participating mechanism of a major contributing gene ysnE for auxin biosynthesis in Bacillus amyloliquefaciens SQR9.

The phytohormone indole-3-acetic acid (IAA) has been demonstrated to contribute to the plant growth-promoting effect of rhizobacteria, but the IAA biosynthesis pathway in rhizobacteria remains unclear. The ysnE gene, encoding a putative tryptophan acetyltransferase, has been demonstrated to be involved in and strongly contribute to IAA production in Bacillus, but the mechanism is unknown. In this study, to investigate how ysnE participates in IAA biosynthesis in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens SQR9, differences in the produced IAA biosynthesis intermediates between wild-type SQR9 and ΔysnE were analyzed and compared, and the effects of different intermediate compounds on the production of IAA and the accumulation of other intermediates were also investigated. The results showed that the mutant ΔysnE produced more indole-3-lactic acid (ILA) and tryptamine (TAM) than the SQR9 wild-type strain (nearly 1.6- and 2.1-fold), while the production of tryptophol (TOL) was significantly decreased by 46%. When indole-3-pyruvic acid (IPA) served as the substrate, the concentration of ILA in the ΔysnE fermentation broth was much higher than that of the wild type, while IAA and TOL were significantly lower, and ΔysnE was lower than SQR9 in IAA and TOL with the addition of TAM. The TOL content in the ΔysnE fermentation broth was much lower than that in the wild-type SQR9 with the addition of ILA. We suggest that ysnE may be involved in the IPA and TAM pathways and play roles in indole acetaldehyde (IAAld) synthesis from IPA and TAM and in the conversion of ILA to TOL.

Chemotaxis of Beneficial Rhizobacteria to Root Exudates: The First Step towards Root-Microbe Rhizosphere Interactions.

Chemotaxis, the ability of motile bacteria to direct their movement in gradients of attractants and repellents, plays an important role during the rhizosphere colonization by rhizobacteria. The rhizosphere is a unique niche for plant-microbe interactions. Root exudates are highly complex mixtures of chemoeffectors composed of hundreds of different compounds. Chemotaxis towards root exudates initiates rhizobacteria recruitment and the establishment of bacteria-root interactions. Over the last years, important progress has been made in the identification of root exudate components that play key roles in the colonization process, as well as in the identification of the cognate chemoreceptors. In the first part of this review, we summarized the roles of representative chemoeffectors that induce chemotaxis in typical rhizobacteria and discussed the structure and function of rhizobacterial chemoreceptors. In the second part we reviewed findings on how rhizobacterial chemotaxis and other root-microbe interactions promote the establishment of beneficial rhizobacteria-plant interactions leading to plant growth promotion and protection of plant health. In the last part we identified the existing gaps in the knowledge and discussed future research efforts that are necessary to close them.

Root-Secreted Spermine Binds to Bacillus amyloliquefaciens SQR9 Histidine Kinase KinD and Modulates Biofilm Formation.

The signal molecules in root exudates that are sensed by plant growth-promoting rhizobacteria (PGPR) are critical to regulate their root colonization. Phosphorylated Spo0A is an important global transcriptional regulator that controls colonization and sporulation in Bacillus species. In this study, we found that deletion of kinD from PGPR strain Bacillus amyloliquefaciens SQR9, encoding an original phosphate donor of Spo0A, resulted in reduced biofilm formation in root exudates compared with the wild-type strain, indicating that KinD is responsible for sensing root exudates. Ligands of B. amyloliquefaciens SQR9 KinD in cucumber root exudates were determined by both the nontargeted ligand fishing method and the targeted surface plasmon resonance detection method. In total, we screened 80 compounds in root exudates for binding to KinD and found that spermine and guanosine could bind to KinD with dissociation constant values of 213 and 51 µΜ, respectively. In addition, calcium l-threonate, N-acetyl-l-aspartic acid, sodium decanoic acid, and parabanic acid could also bind weakly to KinD. The three-dimensional binding models were then constructed to demonstrate the interactions between the root-secreted signals and KinD. It was observed that exogenous spermine reduced the wrinkles of biofilm when kinD was deleted, indicating that KinD might be involved in sensing root-secreted spermine and stabilizing biofilm in response to this negative effector. This study provided a new insight of interaction between a rhizobacterial sensor and root-secreted signals.

Induced root-secreted D-galactose functions as a chemoattractant and enhances the biofilm formation of Bacillus velezensis SQR9 in an McpA-dependent manner.

Chemotaxis towards root exudates and subsequent biofilm formation are very important for root colonization and for providing the beneficial functions of plant growth-promoting rhizobacteria (PGPRs). In this study, in comparison with other root-secreted compounds, D-galactose in the root exudates of cucumber was found to be a strong chemoattractant at the concentration of 1 µM for Bacillus velezensis SQR9. Chemotaxis assays with methyl-accepting chemotaxis proteins (MCPs) deletion strains demonstrated that McpA was solely responsible for chemotaxis towards D-galactose. Interestingly, D-galactose significantly enhanced the biofilm formation of SQR9 in an McpA-dependent manner. Further experiment showed that D-galactose also enhanced root colonization by SQR9. In addition, the secretion of D-galactose by cucumber roots could be induced by inoculation with SQR9, indicating that D-galactose may be an important signal in the interaction between plant and SQR9. These findings suggested that the root-secreted D-galactose was a signal, the secretion of which was induced by the beneficial bacteria, and which in turn induced colonization of the bacteria.

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth-promoting rhizobacteria.

Chemotaxis to plant root exudates is supposed to be a prerequisite for efficient root colonization by rhizobacteria. This is a highly multifactorial process since root exudates are complex compound mixtures of which components are recognized by different chemoreceptors. Little information is available as to the key components in root exudates and their receptors that drive colonization related chemotaxis. We present here the first global assessment of this issue using the plant growth-promoting rhizobacterium (PGPR) Bacillus velezensis SQR9 (formerly B. amyloliquefaciens). This strain efficiently colonizes cucumber roots, and here, we show that chemotaxis to cucumber root exudates was essential in this process. We conducted chemotaxis assays using cucumber root exudates at different concentrations, individual exudate components as well as recomposed exudates, taking into account their concentrations detected in root exudates. Results indicated that two key chemoreceptors, McpA and McpC, were essential for root exudate chemotaxis and root colonization. Both receptors possess a broad ligand range and recognize most of the exudate key components identified (malic, fumaric, gluconic and glyceric acids, Lys, Ser, Ala and mannose). The remaining six chemoreceptors did not contribute to exudate chemotaxis. This study provides novel insight into the evolution of the chemotaxis system in rhizobacteria.

Identification of Chemotaxis Compounds in Root Exudates and Their Sensing Chemoreceptors in Plant-Growth-Promoting Rhizobacteria Bacillus amyloliquefaciens SQR9.

Chemotaxis-mediated response to root exudates, initiated by sensing-specific ligands through methyl-accepting chemotaxis proteins (MCP), is very important for root colonization and beneficial functions of plant-growth-promoting rhizobacteria (PGPR). Systematic identification of chemoattractants in complex root exudates and their sensing chemoreceptors in PGPR is helpful for enhancing their recruitment and colonization. In this study, 39 chemoattractants and 5 chemorepellents, including amino acids, organic acids, and sugars, were identified from 98 tested components of root exudates for the well-studied PGPR strain Bacillus amyloliquefaciens SQR9. Interestingly, mutant stain SQR9Δ8mcp, with all eight putative chemoreceptors completely deleted, lost the chemotactic responses to those 44 compounds. Gene complementation, chemotaxis assay, and isothermal titration calorimetry analysis revealed that McpA was mainly responsible for sensing organic acids and amino acids, while McpC was mostly for amino acids. These two chemoreceptors may play important roles in the rhizosphere chemotaxis of SQR9. In contrast, the B. amyloliquefaciens-unique chemoreceptor McpR was specifically responsible for arginine, and residues Tyr-78, Thr-131, and Asp-162 were critical for arginine binding. This study not only deepened our insights into PGPR-root interaction but also provided useful information to enhance the rhizosphere chemotaxis mobility and colonization of PGPR, which will promote their application in agricultural production.

Identification of Root-Secreted Compounds Involved in the Communication Between Cucumber, the Beneficial Bacillus amyloliquefaciens, and the Soil-Borne Pathogen Fusarium oxysporum.

Colonization of plant growth-promoting rhizobacteria (PGPR) is critical for exerting their beneficial effects on the plant. Root exudation is a major factor influencing the colonization of both PGPR and soil-borne pathogens within the root system. However, the tripartite interaction of PGPR, plant roots, and soil-borne pathogens is poorly understood. We screened root exudates for signals that mediate tripartite interactions in the rhizosphere. In a split-root system, we found that root colonization of PGPR strain Bacillus amyloliquefaciens SQR9 on cucumber root was significantly enhanced by preinoculation with SQR9 or the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum, whereas root colonization of F. oxysporum f. sp. cucumerinum was reduced upon preinoculation with SQR9 or F. oxysporum f. sp. cucumerinum. Root exudates from cucumbers preinoculated with SQR9 or F. oxysporum f. sp. cucumerinum were analyzed and 109 compounds were identified. Correlation analysis highlighted eight compounds that significantly correlated with root colonization of SQR9 or F. oxysporum f. sp. cucumerinum. After performing colonization experiments with these chemicals, raffinose and tryptophan were shown to positively affect the root colonization of F. oxysporum f. sp. cucumerinum and SQR9, respectively. These results indicate that cucumber roots colonized by F. oxysporum f. sp. cucumerinum or SQR9 increase root secretion of tryptophan to strengthen further colonization of SQR9. In contrast, these colonized cucumber roots reduce raffinose secretion to inhibit root colonization of F. oxysporum f. sp. cucumerinum.

pheS * , an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens.

Aside from applications in the production of commercial enzymes and metabolites, Bacillus amyloliquefaciens is also an important group of plant growth-promoting rhizobacteria that supports plant growth and suppresses phytopathogens. A host-genotype-independent counter-selectable marker would enable rapid genetic manipulation and metabolic engineering, accelerating the study of B. amyloliquefaciens and its development as both a microbial cell factory and plant growth-promoting rhizobacteria. Here, a host-genotype-independent counter-selectable marker pheS * was constructed through a point mutation of the gene pheS, which encodes the α-subunit of phenylalanyl-tRNA synthetase in Bacillus subtilis strain 168. In the presence of 5 mM p-chloro-phenylalanine, 100 % of B. amyloliquefaciens strain SQR9 cells carrying pheS * were killed, whereas the wild-type strain SQR9 showed resistance to p-chloro-phenylalanine. A simple pheS * and overlap-PCR-based strategy was developed to create the marker-free deletion of the amyE gene as well as a 37-kb bmy cluster in B. amyloliquefaciens SQR9. The effectiveness of pheS * as a counter-selectable marker in B. amyloliquefaciens was further confirmed through the deletion of amyE genes in strains B. amyloliquefaciens FZB42 and NJN-6. In addition, the potential use of pheS * in other Bacillus species was preliminarily assessed. The expression of PheS* in B. subtilis strain 168 and B. cereus strain ATCC 14579 caused pronounced sensitivity of both hosts to p-chloro-phenylalanine, indicating that pheS * could be used as a counter-selectable marker (CSM) in these strains.

Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates.

BACKGROUND: Bacillus amyloliquefaciens SQR9 is a plant growth-promoting rhizobacteria (PGPR) with outstanding abilities to enhance plant growth and to control soil-borne diseases. Root exudates is known to play important roles in plant-microbe interactions. To explore the rhizosphere interactions and plant-beneficial characteristics of SQR9, the complete genome sequence as well as the transcriptome in response to maize root exudates under biofilm-forming conditions were elucidated. RESULTS: Maize root exudates stimulated SQR9 biofilm formation in liquid culture, which is known to be positively correlated with enhanced root colonization. Transcriptional profiling via RNA-sequencing of SQR9 under static conditions indicated that, at 24 h post-inoculation, root exudates stimulated the expression of metabolism-relevant genes, while at 48 h post-inoculation, genes related to extracellular matrix production (tapA-sipW-tasA operon) were activated by root exudates. The individual components in maize root exudates that stimulated biofilm formation included glucose, citric acid, and fumaric acid, which either promoted the growth of SQR9 cells or activated extracellular matrix production. In addition, numerous groups of genes involved in rhizosphere adaptation and in plant-beneficial traits, including plant polysaccharide utilization, cell motility and chemotaxis, secondary antibiotics synthesis clusters, and plant growth promotion-relevant, were identified in the SQR9 genome. These genes also appeared to be induced by the maize root exudates. CONCLUSIONS: Enhanced biofilm formation of B. amyloliquefaciens SQR9 by maize root exudates could mainly be attributed to promoting cell growth and to inducing extracellular matrix production. The genomic analysis also highlighted the elements involved in the strain's potential as a PGPR. This study provides useful information for understanding plant-rhizobacteria interactions and hence for promoting the agricultural applications of this strain.

Enhanced control of cucumber wilt disease by Bacillus amyloliquefaciens SQR9 by altering the regulation of Its DegU phosphorylation.

Bacillus amyloliquefaciens strain SQR9, isolated from the cucumber rhizosphere, suppresses the growth of Fusarium oxysporum in the cucumber rhizosphere and protects the host plant from pathogen invasion through efficient root colonization. In the Gram-positive bacterium Bacillus, the response regulator DegU regulates genetic competence, swarming motility, biofilm formation, complex colony architecture, and protease production. In this study, we report that stepwise phosphorylation of DegU in B. amyloliquefaciens SQR9 can influence biocontrol activity by coordinating multicellular behavior and regulating the synthesis of antibiotics. Results from in vitro and in situ experiments and quantitative PCR (qPCR) studies demonstrate the following: (i) that the lowest level of phosphorylated DegU (DegU∼P) (the degQ mutation) impairs complex colony architecture, biofilm formation, colonization activities, and biocontrol efficiency of Fusarium wilt disease but increases the production of macrolactin and bacillaene, and (ii) that increasing the level of DegU∼P by degQ and degSU overexpression significantly improves complex colony architecture, biofilm formation, colonization activities, production of the antibiotics bacillomycin D and difficidin, and efficiency of biocontrol of Fusarium wilt disease. The results offer a new strategy to enhance the biocontrol efficacy of Bacillus amyloliquefaciens SQR9.

Design and analysis of tubular permanent magnet linear wave generator.

Due to the lack of mature design program for the tubular permanent magnet linear wave generator (TPMLWG) and poor sinusoidal characteristics of the air gap flux density for the traditional surface-mounted TPMLWG, a design method and a new secondary structure of TPMLWG are proposed. An equivalent mathematical model of TPMLWG is established to adopt the transformation relationship between the linear velocity of permanent magnet rotary generator and the operating speed of TPMLWG, to determine the structure parameters of the TPMLWG. The new secondary structure of the TPMLWG contains surface-mounted permanent magnets and the interior permanent magnets, which form a series-parallel hybrid magnetic circuit, and their reasonable structure parameters are designed to get the optimum pole-arc coefficient. The electromagnetic field and temperature field of TPMLWG are analyzed using finite element method. It can be included that the sinusoidal characteristics of air gap flux density of the new secondary structure TPMLWG are improved, the cogging force as well as mechanical vibration is reduced in the process of operation, and the stable temperature rise of generator meets the design requirements when adopting the new secondary structure of the TPMLWG.

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE: Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.