Identification and characterization of the TetR family transcriptional regulator NffT in Rhizobium johnstonii.

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF, nifA-fixA signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased fixA expression, was discovered through a fixA-promoter-ß-glucuronidase fusion assay performed with Rhizobium johnstonii. Real-time quantitative PCR analysis showed that nffT deletion increased the expression of symbiotic genes including nifA and fixA in nifA-fixA signaling, and fixL, fixK, fnrN, and fixN9 in fixL-fixN signaling. nffT overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091-93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located -32 to -99 bp upstream of RL0091-93 and NffT deletion significantly increased the expression of RL0091-93. nffT-promoter-ß-glucuronidase fusion assay indicated that nffT expression was regulated by the cobNTS genes and cobalamin. Mutations in cobNTS significantly decreased the expression of nffT, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of R. johnstonii-Pisum sativum.IMPORTANCESymbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of fixA in Rhizobium johnstonii. Furthermore, NffT was confirmed to play pleiotropic roles in R. johnstonii-Pisum sativum symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected R. johnstonii-P. sativum symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.

Enhancement of milbemycins production by phosphopantetheinyl transferase and regulatory pathway engineering in Streptomyces bingchenggensis.

Milbemycins (MILs), a group of 16-membered insecticidal macrocylic lactones, are widely used as the biological pesticide and the precursors of semi-synthetic veterinary drugs. Polyketide synthases (PKSs), which require phosphopantetheinyl transferases (PPTases) to activate their ACP domains from apo forms to holo forms, catalyze the backbone biosynthesis of MILs. Here we found there was a complex phosphopantetheinylation network mediated by five putative PPTases in Streptomyces bingchenggensis. Repression mutants of PpA27 and PpA62 via CRISPRi both produced significantly lower yields of MILs than that of the control strain. Repression mutant of PpA68 led to abolishment of the pigment production. MILs production was significantly enhanced by PpA27 overexpression, while not by the overexpression of other PPTases. PpA27 was thus proved a dedicated post-translational enzyme to activate PKSs involved in the MILs biosynthesis. MILs titer was further enhanced by co-overexpression of PpA27 and MilR, the pathway­specific transcriptional activator of MIL biosynthetic gene cluster. When PpA27 and MilR were co-overexpressed in the industrial S. bingchenggensis HMB, MILs production was increased by 40.5%. These results indicated that tuning the antibiotic biosynthetic pathway by co-engineering transcriptional regulation network and post-translational phosphopantetheinylation network is an effective strategy for antibiotic production improvement.

What determines symbiotic nitrogen fixation efficiency in rhizobium: recent insights into Rhizobium leguminosarum.

Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.

Characteristics, Roles and Applications of Proteinaceous Elicitors from Pathogens in Plant Immunity.

In interactions between pathogens and plants, pathogens secrete many molecules that facilitate plant infection, and some of these compounds are recognized by plant pattern recognition receptors (PRRs), which induce immune responses. Molecules in both pathogens and plants that trigger immune responses in plants are termed elicitors. On the basis of their chemical content, elicitors can be classified into carbohydrates, lipopeptides, proteinaceous compounds and other types. Although many studies have focused on the involvement of elicitors in plants, especially on pathophysiological changes induced by elicitors in plants and the mechanisms mediating these changes, there is a lack of up-to-date reviews on the characteristics and functions of proteinaceous elicitors. In this mini-review, we provide an overview of the up-to-date knowledge on several important families of pathogenic proteinaceous elicitors (i.e., harpins, necrosis- and ethylene-inducing peptide 1 (nep1)-like proteins (NLPs) and elicitins), focusing mainly on their structures, characteristics and effects on plants, specifically on their roles in plant immune responses. A solid understanding of elicitors may be helpful to decrease the use of agrochemicals in agriculture and gardening, generate more resistant germplasms and increase crop yields.

Roles of LuxR-family regulators in the biosynthesis of secondary metabolites in Actinobacteria.

Actinobacteria are well-known Gram-positive bacteria that produce approximately two-thirds of microbial bioactive natural products (NPs) through secondary metabolism. Usually, genes involved in the biosynthesis of NPs in actinobacteria are clustered, and their expression is regulated by an elaborate and stringent regulatory network formed by diverse regulators. These regulators can be classified into more than 50 superfamilies/families according to conserved amino acid sequences and biological functions. Among them, LuxR family regulators, which are widely distributed in microorganisms and feature an HTH_LUXR domain (PFAM00196, SMART00421), play key roles in quorum sensing (QS), bioluminescence, virulence and secondary metabolism. In this mini-review, we focus on their roles in regulating NP production in actinobacteria. First, the domain architecture and classification of LuxR proteins are summarized on the basis of their size and biological function diversity. Second, the landscape of the roles and action mechanism of LuxR regulators involved in NP production in actinobacteria is presented in detail. Finally, the application of LuxR is described from two perspectives: enhancement of NP production and discovery of novel NPs by engineering LuxR. This mini-review will help us comprehensively understand the role of LuxR in actinobacteria and promote the future application of LuxR family regulators in synthetic biology.

The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining.

Streptomyces is taken as an important resource for producing the most abundant antibiotics and other bio-active natural products, which have been widely used in pharmaceutical and agricultural areas. Usually they are biosynthesized through secondary metabolic pathways encoded by cluster situated genes. And these gene clusters are stringently regulated by interweaved transcriptional regulatory cascades. In the past decades, great advances have been made to elucidate the regulatory mechanisms involved in antibiotic production in Streptomyces. In this review, we summarized the recent advances on the regulatory cascades of antibiotic production in Streptomyces from the following four levels: the signals triggering the biosynthesis, the global regulators, the pathway-specific regulators and the feedback regulation. The production of antibiotic can be largely enhanced by rewiring the regulatory networks, such as overexpression of positive regulators, inactivation of repressors, fine-tuning of the feedback and ribosomal engineering in Streptomyces. The enormous amount of genomic sequencing data implies that the Streptomyces has potential to produce much more antibiotics for the great diversities and wide distributions of biosynthetic gene clusters in Streptomyces genomes. Most of these gene clusters are defined cryptic for unknown or undetectable natural products. In the synthetic biology era, activation of the cryptic gene clusters has been successfully achieved by manipulation of the regulatory genes. Chemical elicitors, rewiring regulatory gene and ribosomal engineering have been employed to crack the potential of cryptic gene clusters. These have been proposed as the most promising strategy to discover new antibiotics. For the complex of regulatory network in Streptomyces, we proposed that the discovery of new antibiotics and the optimization of industrial strains would be greatly promoted by further understanding the regulatory mechanism of antibiotic production.

Corrigendum: The Application of Regulatory Cascades in Streptomyces: Yield Enhancement and Metabolite Mining.

[This corrects the article DOI: 10.3389/fmicb.2020.00406.].

OsRLCK 57, OsRLCK107 and OsRLCK118 Positively Regulate Chitin- and PGN-Induced Immunity in Rice.

BACKGROUND: The receptor-like cytoplasmic kinase, OsRLCK176 has been reported to participate in both chitin- and PGN-induced immunity in rice. Here, we further researched the function of the homologous proteins, OsRLCK57, OsRLCK107 and OsRLCK118, in chitin- and PGN immunity in rice. FINDINGS: Silencing of OsRLCK57,OsRLCK107 and OsRLCK118 suppressed chitin- and PGN-induced immunity responses, including reactive oxygen species generation, defense gene expression. Furthermore, OsRLCK107 could interact with OsCERK1 in a MAMP induced way, which suggested a possible physiological relevance of OsRLCKs107 to OsCERK1 pathway. CONCLUSIONS: OsRLCK57, OsRLCK107 and OsRLCK118, positively regulate chitin- and PGN- induced responses in rice, similar to that observed in OsRLCK176.

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity.

Microbe-associated molecular pattern (MAMP)-triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif-containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor-like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN-triggered immunity in rice. Silencing of OsCERK1 suppressed PGN-induced (and chitin-induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor-binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand-induced heterooligomeric receptor complexes. Furthermore, the receptor-like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.

OsLYP4 and OsLYP6 play critical roles in rice defense signal transduction.

Plant innate immunity relies on successful detection of trespassing pathogens through recognizing their microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) at the cell surface. We recently reported two rice lysin motif (LysM)-containing proteins, OsLYP4 and OsLYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Here we further demonstrated the important roles of OsLYP4 and OsLYP6 in rice defense signaling, as silencing of either LYP impaired the defense marker gene activation induced by either bacterial pathogen Xanthomonas oryzaecola or fungal pathogen Magnaporthe oryzae. Moreover, we found that OsLYP4 and OsLYP6 could form homo- and hetero-dimers, and could interact with CEBiP, suggesting an unexpected complexity of chitin perception in rice.

Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity.

Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif-containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN- or chitin- but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity.

Rice MAPK phosphatase IBR5 negatively regulates drought stress tolerance in transgenic Nicotiana tabacum.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) are important negative regulators in the MAPK signaling pathways, which play crucial roles in plant growth, regulation of development and response to environment stresses. Several MAPKs have been reported to be involved in the drought stress response, however, there is no evidence for the specific function of MKPs in drought stress. Here, a putative MKP in rice (Oryza sativa), OsIBR5, was characterized. Expression of OsIBR5 was induced by PEG6000, abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)). Overexpression of OsIBR5 in tobacco plants resulted in hypersensitivity to drought and H(2)O(2) treatments. Drought and ABA-induced stomatal closure was significantly reduced in OsIBR5-overexpressing tobacco plants compared with controls. Moreover, OsIBR5 was found to interact with tobacco MAPKs SIPK and WIPK, and drought-induced WIPK activity was impaired in OsIBR5-overexpressing tobacco plants. These results indicated that OsIBR5 is a MKP which was induced by abiotic stresses and decreased tolerance to drought stress in transgenic tobacco plants.