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

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

Fusaricidins produced by Paenibacillus polymyxa are important lipopeptide antibiotics against fungi. The fusGFEDCBA (fusaricidin biosynthesis) operon is responsible for synthesis of fusaricidins. However, the regulation mechanisms of fusaricidin biosynthesis remain to be fully clarified. In this study, we revealed that fusaricidin production is controlled by a complex regulatory network including KinB-Spo0A-AbrB. Evidence suggested that the regulator AbrB represses the transcription of the fus gene cluster by direct binding to the fus promoter, in which the sequences (5′-AATTTTAAAATAAATTTTGTGATTT-3′) located from −136 to −112 bp relative to the transcription start site is required for this repression. Spo0A binds to the abrB promoter that contains the Spo0A-binding sequences (5′-TGTCGAA-3′, 0A box) and in turn prevents the further transcription of abrB. The decreasing concentration of AbrB allows for the derepression of the fus promoter repressed by AbrB. The genome of P. polymyxa WLY78 contains two orthologs (named Kin1508 and Kin4833) of Bacillus subtilis KinB, but only Kin4833 activates sporulation and fusaricidin production, indicating that this kinase may be involved in phosphorylating Spo0A to initiate sporulation and regulate the abrB transcription. Our results reveal that Kin4833 (KinB), Spo0A, and AbrB are involved in regulation of fusaricidin production and a signaling mechanism that links fusaricidin production and sporulation.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ralstonia solanacearum RSc0454 is predicted as a FAD-linked oxidase based on protein homologies, while it contains distinct domains of lactate dehydrogenase and succinate dehydrogenase. A previous study demonstrated that RSc0454 exhibits lactate dehydrogenase activity using pyruvate and NADH as substrates, and is essential for pathogenicity of R. solanacearum. Here, we genetically characterized involvement of RSc0454 on bacterial growth and expression of genes for the type III secretion system (T3SS, a pathogenicity determinant) in R. solanacearum. The RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate but not lactate could substantially restore some phenotypes of RSc0454 mutants, including faint growth in host plants, diminished growth in the minimal medium, and lost pathogenicity toward host plants. Expression of T3SS genes is directly controlled by a master regulator, HrpB, and hrpB expression is positively regulated by HrpG and PrhG in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, although through some novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells, and supplementary NADH partially restored diminished growth of the RSc0454 mutant in the minimal medium only in the presence of succinate at some moderate concentrations, indicating that the unbalanced redox in the RSc0454 mutant might be responsible for its diminished growth in the minimal medium. Taken together, these results provide novel insights into the understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Agrobacterium salinitolerans DG3-1 is an endophytic bacterium isolated from cotton root tissue. Our previous work has shown that it can inhibit the growth of Fusarium and Verticillium wilt pathogens as well as increase the chlorophyll content of cotton leaves. Here, we reported the complete genome sequence of strain DG3-1, which was analyzed by sequence reads generated from Nanopore PromethION and Illumina NovaSeq PE150 platforms. This genome sequence could be used to clarify the possible mechanism of DG3-1 at the gene level.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencing-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.[Graphic: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bacillus subtilis is a Gram-positive plant-growth-promoting rhizobacterium exerting many beneficial effects on plant health. Because they secrete antimicrobial compounds and elicit induced systemic resistance, B. subtilis and phylogenetically related species are of particular interest as antifungals in organic agriculture. These bacteria are also known for their capacity to differentiate phenotypically into endospores able to withstand many environmental stresses. However, although B. subtilis is often inoculated on plants as spores, dynamics of germination and sporulation on roots remain unexplored. Using a hydroponic culture system and a soil system for Arabidopsis thaliana, we observed that B. subtilis spores germinate rapidly on contact with plants. However, the vegetative cells are abundant on roots for only a few days before reversing back to spores. We observed that the germinant receptor GerK and sporulation kinases KinA and KinB identified in vitro control sporulation dynamics on plants. Surprisingly, when plants are inoculated with B. subtilis, free-living cells sporulate more rapidly than plant-associated cells. However, direct contact between plant and bacteria is required for the induction of sporulation in the surrounding B. subtilis. This study has fundamental implications for our understanding of interactions between Bacillus spp. and plants, and particularly for a more efficient usage of B. subtilis as a biofertilizer or biofungicide.

Plant-growth-promoting bacteria (PGPB) stimulate plant growth through diverse mechanisms. In addition to biological nitrogen fixation, diazotrophic PGPB can improve nutrient uptake efficiency from the soil, produce and release phytohormones to the host, and confer resistance against pathogens. The genetic determinants that drive the success of biological nitrogen fixation in nonlegume plants are understudied. These determinants include recognition and signaling pathways, bacterial colonization, and genotype specificity between host and bacteria. This review presents recent discoveries of how nitrogen-fixing PGPB interact with cereals and promote plant growth. We suggest adopting an experimental model system, such as the Setaria -diazotrophic bacteria association, as a reliable way to better understand the associated mechanisms and, ultimately, increase the use of PGPB inoculants for sustainable agriculture.[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 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.

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata . The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.

The potato common scab pathogen Streptomyces scabies produces N- coronafacoyl-l-isoleucine (CFA-Ile), which is a member of the coronafacoyl family of phytotoxins that are synthesized by multiple plant pathogenic bacteria. The CFA-Ile biosynthetic gene cluster contains a regulatory gene, cfaR , which directly controls the expression of the phytotoxin structural genes. In addition, a gene designated orf1 encodes a predicted ThiF family protein and is cotranscribed with cfaR , suggesting that it also plays a role in the regulation of CFA-Ile production. In this study, we demonstrated that CfaR is an essential activator of coronafacoyl phytotoxin production, while ORF1 is dispensable for phytotoxin production and may function as a helper protein for CfaR. We also showed that CFA-Ile inhibits the ability of CfaR to bind to the promoter region driving expression of the phytotoxin biosynthetic genes and that elevated CFA-Ile production by overexpression of both cfaR and orf1 in S. scabies increases the severity of disease symptoms induced by the pathogen during colonization of potato tuber tissue. Overall, our study reveals novel insights into the regulatory mechanisms controlling CFA-Ile production in S. scabies and it provides further evidence that CFA-Ile is an important virulence factor for this organism.

Azorhizobium caulinodans ORS571 can induce nodule formation on the roots and the stems of its host legume, Sesbania rostrata . Plant exudates are essential in the dialogue between microbes and their host plant and, in particular, amino acids can play an important role in the chemotactic response of bacteria. Histidine, arginine, and aspartate, which are the three most abundant amino acids present in S. rostrata seed exudates, behave as chemoattractants toward A. caulinodans . A position-specific-iterated BLAST analysis of the methyl-accepting chemotaxis proteins (MCPs) (chemoreceptors) in the genome of A. caulinodans was performed. Among the 43 MCP homologs, two MCPs harboring a dCache domain were selected as possible cognate amino acid MCPs. After analysis of relative gene expression levels and construction of a gene-deleted mutant strain, one of them, AZC_0821 designed as TlpH, was confirmed to be responsible for the chemotactic response to the three amino acids. In addition, it was found that these three amino acids can also influence chemotaxis of A. caulinodans independently of the chemosensory receptors, by being involved in the increase of the expression level of several che and fla genes involved in the chemotaxis pathway and flagella synthesis. Thus, the contribution of amino acids present in seed exudates is directly related to the role as chemoattractants and indirectly related to the role in the regulation of expression of key genes involved in chemotaxis and motility. This "dual role" is likely to influence the formation of biofilms by A. caulinodans and the host root colonization properties of this bacterium.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter . Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize ( Zea mays ), is investigated in Arabidopsis thaliana . RHG1 colonized both roots and shoots of Arabidopsis , promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.