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PubMed2026

Azotobacter vinelandii AmrZ is a global regulator linking alginate production and c-di-GMP homeostasis.

Azotobacter vinelandii, a member of the Pseudomonadaceae, produces the exopolysaccharide alginate during vegetative growth; however, the circuitry linking alginate biosynthesis to lifestyle transitions remains poorly defined. Here, we show that the Ribbon-Helix-Helix transcription factor AmrZ coordinates alginate production, intracellular c-di-GMP levels and motility. Deletion of amrZ abolished alginate synthesis, whereas chromosomal complementation restored it. A PalgD-gusA fusion and RT-qPCR demonstrated that algD, the first gene in the alginate biosynthetic cluster, depends on AmrZ for expression. Motif analysis identified multiple AmrZ sites upstream of algD, and electrophoretic mobility-shift assays (EMSAs) confirmed specific binding to these regions. AmrZ also positively autoregulates: PamrZ-gusA activity decreased in ΔamrZ, and purified AmrZ bound the amrZ promoter in EMSA. Moreover, PamrZ activity required the sigma factor AlgU, consistent with the presence of an AlgU promoter; this positive, AlgU-dependent feedback may stabilize AmrZ under alginate-inducing conditions. To probe AmrZ control of c-di-GMP, we implemented a riboswitch-based biosensor in A. vinelandii. The ΔamrZ strain showed a markedly reduced signal, similar to a diguanylate cyclase (DGC) mutant, whereas a phosphodiesterase mutant displayed elevated output, validating the assay. RNA-seq and RT-qPCR identified two DGC genes, AVAEIV_RS11610 and AVAEIV_RS18795, as AmrZ-activated targets; EMSA verified direct binding at the RS11610 regulatory region. By contrast, transcription of the principal vegetative DGC AvGReg was not AmrZ-regulated. Lower c-di-GMP in ΔamrZ correlated with larger swimming halos. Collectively, these genetic, biochemical and transcriptomic data support a model in which AmrZ directly activates algD and elevates c-di-GMP via selected DGCs, thereby promoting alginate synthesis while reducing motility. RNA-seq data also indicate that AmrZ influences broader cellular programmes, including metabolism and iron homeostasis, positioning AmrZ as a central regulator that links c-di-GMP homeostasis to coordinated exopolysaccharide production in A. vinelandii. This work contributes to our understanding of the regulatory networks controlled by AmrZ outside the Pseudomonas genus and reveals important differences in its targets and regulatory mechanisms.

Azotobacter vinelandii↗

PubMed2026

Binding analysis of the response regulator NarL protein to the promoter of the O6-methylguanine-DNA methyltransferase (ogt) gene in Salmonella Typhimurium.

BACKGROUND: Salmonella Typhimurium (STM) is a gram-negative bacterium that causes severe gastrointestinal disorders in both animals and humans. The regulation of DNA repair genes is critical for maintaining genomic stability of the bacteria. O6-methylguanine DNA methyltransferase (Ogt), plays a vital role in repairing alkylated DNA in STM; however, the transcriptional regulation of ogt gene remains poorly characterized. Furthermore, NarL is a transcriptional regulator, involved in the pathogenesis of STM under anaerobic condition. Therefore, this study investigated the interaction between NarL protein and the promoter region of the ogt gene. METHODS: In this study, narl gene was cloned in pET32a vector and NarL protein was expressed in Escherichia coli BL21 (DE3). Subsequently, the ogt gene promoter (pogt) was selected, amplified, cloned and its activity was evaluated. Electrophoretic mobility shift assay (EMSA), isothermal titration calorimetry (ITC), molecular docking were employed to elucidate the interaction between NarL protein and ogt promoter. Furthermore, the regulatory role of NarL in ogt gene expression was validated in vivo using RT-qPCR and β-galactosidase assay. RESULTS: This study resulted that NarL protein interacts specifically with the ogt promoter, as confirmed by EMSA and ITC, with ΔG of - 9.42 kcal mol⁻¹. Furthermore, RT-qPCR and β-galactosidase assays demonstrated that deletion of narl significantly (P ≤ 0.01) decreased ogt transcript levels and promoter activity than wild Salmonella Typhimurium, whereas exogenous supplementation of recombinant NarL protein restored the expression. These findings suggest that NarL plays a potential regulatory role in ogt gene expression in response to environmental signals. CONCLUSION: These findings highlight an interaction between NarL protein and the promoter region of ogt gene in Salmonella Typhimurium, linking nitrogen metabolism with the DNA repair pathway in STM, which may contribute to the bacterial survival under nitrosative stress.

Salmonella typhimurium↗

PubMed2025

The toxin-antitoxin system SavRS contributes to vancomycin resistance in vancomycin-intermediate Staphylococcus aureus by mediating cell wall thickening.

BACKGROUND: The emergence of vancomycin-intermediate Staphylococcus aureus (VISA) has significantly challenged the treatment of S. aureus infection. Toxin-antitoxin (TA) systems have been reported to mediate bacterial stress adaptation and virulence, but their role in vancomycin resistance remains elusive. This study investigated the vancomycin resistance mechanism regulated by the TA system SavRS in VISA. METHODS: savRS mutants in Mu50 and XN108 were generated via homologous recombination. To investigate the regulatory mechanism of vancomycin resistance mediated by savRS in VISA, phenotypic analyses including MICs, growth kinetics and cell wall thickness measurements were performed. Expression of cell wall synthesis-related genes was analysed using quantitative RT-PCR (RT-qPCR) and promoter-lacZ reporter assay. Electrophoretic mobility shift assay (EMSA) was performed to assess the binding of SavRS to the promoters of the cell wall synthesis-related genes. Pull-down assay identified an upstream regulatory element of savRS associated with vancomycin resistance. Quantitative assessment of bacterial burden in murine organ systems following vancomycin administration revealed the critical regulatory role of savRS in mediating vancomycin resistance in vivo. RESULTS: Compared with the WT, the savRS mutant exhibited enhanced vancomycin sensitivity, accelerated growth and reduced cell wall thickness. Correspondingly, RT-qPCR revealed marked down-regulation of the cell wall synthesis-related genes (glyS, dltA, scdA, pbp2, ddl). EMSA and promoter-lacZ reporter assay confirmed direct binding of SavRS to a conserved promoter motif, MGHYYTCCTCA. Pull-down assay identified UspA as an upstream regulator of SavRS, demonstrating that UspA directly controls savRS transcription and modulates VISA resistance. Mouse infection experiments showed that savRS promotes VISA to vancomycin resistance in vivo. CONCLUSIONS: SavRS critically regulates vancomycin resistance in VISA.

Cell Wall↗

PubMed2026

The transcriptional regulator CasR controls mycobacterial antioxidant defense and biofilm formation via multiple direct targets.

AIMS: The antioxidant defense system of Mycobacterium tuberculosis is critical for pathogenicity and persistence within macrophages, yet the regulatory networks remain poorly understood. This study aims to elucidate the molecular mechanism by which the transcription factor CasR regulates antioxidant defense in mycobacteria through delineation of the regulatory axis linking CasR activity, target gene expression, and the antioxidant phenotype. METHODS AND RESULTS: Using Mycobacterium smegmatis as a model organism, we demonstrate that overexpression of CasR renders the bacteria significantly susceptible to hydrogen peroxide. Electrophoretic mobility shift assay (EMSA) and β-galactosidase reporter analyses reveal that CasR directly binds and represses the promoter of cyp144, an uncharacterized cytochrome P450-encoding gene. Deletion of casRMsmreduces biofilm formation, consistent with the expected derepression of cyp144Msm, a gene that negatively regulates both biofilm and oxidative stress tolerance. EMSA and β-galactosidase activity assays also demonstrate that CasR negatively regulates antioxidant gene katGI, suggesting that CasR exerts a broader, global regulatory role within the mycobacterial antioxidant defense network. Furthermore, we identify isoleucine 18 as a critical residue for the DNA-binding and regulatory function of CasR. CONCLUSION: This study establishes CasR as a pleiotropic transcriptional regulator that directly controls multiple antioxidant genes, including cyp144 and katGI, in mycobacteria. We report a previously unrecognized role for a cytochrome P450 family member in suppressing bacterial antioxidant capacity, as cyp144 overexpression reduces biofilm formation. These findings provide a valuable reference for further investigation into mycobacterial antioxidant mechanisms and identify CasR and Cyp144 as potential targets for the development of anti-tuberculosis drugs.

Biofilms↗

PubMed2026

An XRE-type regulator in Streptococcus mutans plays an important role in brpA expression and oxidative stress tolerance response.

This study used a functional genomics approach to explore the role of a xenobiotic response element (XRE)-type regulator (SMU.405c) in Streptococcus mutans physiology, including the expression of biofilm regulatory protein BrpA. Results showed that deletional mutation of xre significantly reduced the ability of the deficient mutant to grow in the presence of methyl viologen, a commonly used oxidative stressor (P < 0.001). When challenged in a hydrogen peroxide killing assay, the survival rate of the ∆xre mutant was >2-log less than the parent strain after 60 min (P < 0.001). Luciferase reporter fusion assays showed that xre deficiency had no significant effect on luciferase expression when it was under the control of the intact brpA promoter, but the reporter activity increased by >6-fold (P < 0.001) when the reporter gene was fused to a brpA promoter derivative with deletion of a putative XRE-binding box. Electrophoretic mobility shift assay (EMSA) showed that recombinant XRE interacted with the brpA promoter, resulting in an electrophoretic shift of the promoter probes. In vitro transcription assay also showed that inclusion of XRE caused transcription to fall off, significantly reducing full-length brpA transcripts. RNA-seq analysis revealed that deficiency of XRE led to altered expression of >102 genes by >2-fold (P < 0.05), including 28 with increased expression, and 74 with decreased expression. Among the down-regulated were genes for DNA repair and oxidative stress tolerance response. These results suggest that XRE (SMU.405c) in S. mutans plays an important role in brpA expression and oxidative stress tolerance response.IMPORTANCEStreptococcus mutans, a keystone pathogen in human dental caries, primarily lives in the highly diverse microbiota on tooth surfaces, where the conditions are often harsh and fluctuate frequently. Locus SMU.405c was annotated to encode a xenobiotic response element (XRE)-like transcriptional regulator, but no information is available concerning the role of this protein in S. mutans pathophysiology. This study used a functional genomics approach along with molecular and transcriptomic analysis to characterize a deletional xre mutant, and the results showed that xre deficiency in S. mutans resulted in weakened oxidative stress tolerance response and alterations in transcription of >102 genes, including those known to play an important role in cell envelope biogenesis and stress tolerance response. Reporter fusion assay, electrophoretic mobility shift assay (EMSA), and in vitro transcription further demonstrated that the XRE-like regulator encoded by SMU.405c is a repressor of brpA expression and plays an important role in oxidative stress tolerance response.

Streptococcus mutans↗

PubMed2026

Transcription factor LbUBC positively regulates salt gland development and salt tolerance by directly binding to the LbTTG1 promoter and repressing its transcription.

KEY MESSAGE：: LbUBC enhances salt tolerance by promoting salt gland development via repressing LbTTG1, revealing a synergisticregulatory mechanism in Limonium bicolor. In the context of increasingly severe soil salinization, salt-tolerant genetic resources from halophytes show great application potential. In particular, the recretohalophyte Limonium bicolor, which possesses specialized salt gland structures, has become a key model for deciphering the molecular mechanisms underlying salt tolerance and salt gland development. In this study, using LbTTG1-overexpressing and -silenced lines, we demonstrate that LbTTG1 negatively regulates salt-gland development and salt tolerance. Through yeast one-hybrid, EMSA, and dual-luciferase assays, Lb7G33228 (LbUBC) was screened and verified as an upstream transcriptional regulator of LbTTG1. LbUBC enhances salt tolerance in L. bicolor by positively regulating salt-gland development, verified using LbUBC silence and overexpression strains. Interestingly, LbUBC represses the expression of its downstream target LbTTG1, thereby releasing the inhibitory effect of LbTTG1 on salt-gland development. In this manner, LbUBC positively regulates salt-gland development, achieving a dynamic balance in the regulation of salt-gland development and salt tolerance in L. bicolor. This study reveals a synergistic regulatory mechanism involving multiple genes, offering new insights for comprehensively dissecting the molecular regulatory network of salt-gland development.

Salt Tolerance↗

PubMed2026

CgMYC2 directly activates jasmonate-induced naringin biosynthesis in Citrus grandis 'Tomentosa'.

CgMYC2 links jasmonate signaling to naringin biosynthesis by binding G-box motifs and activating flavonoid-pathway promoters in Citrus grandis 'Tomentosa' Naringin, the predominant bitter-flavanone glycoside in Citrus grandis 'Tomentosa', has well-characterized biosynthetic enzymes, yet the transcriptional regulators coupling hormonal signals to pathway activation remain poorly understood. We demonstrate that CgMYC2, a jasmonate-responsive bHLH transcription factor, functions as a central activator of naringin biosynthesis. Exogenous methyl jasmonate (MeJA) treatment increased naringin content 3.45-fold in seedlings, coinciding with a rapid 6.6-fold induction of CgMYC2 that preceded the peak transcription of five core biosynthetic genes (CgPAL5, CgCHS, CgFNS, Cg7GlcT, and Cg1,2RhaT). Physical interaction between CgMYC2 and the JAZ protein CgJAZ3 was confirmed by pull-down and Co-IP assays, placing CgMYC2 within the canonical jasmonate signaling cascade. Y1H confirmed CgMYC2 binding to the Cg1,2RhaT promoter, EMSA demonstrated direct G-box-dependent binding to all five pathway promoters, and dual-luciferase assays showed transactivation of all five promoters, with the strongest activation for CgCHS. As complementary chromatin-level support, a single-sample CUT&Tag profile revealed G-box-enriched CgMYC2-associated chromatin regions across jasmonate-responsive and secondary-metabolic loci. Virus-induced gene silencing (VIGS) of CgMYC2 reduced naringin content by ~21% and suppressed biosynthetic gene expression, supporting its positive contribution. Furthermore, heterologous overexpression in tomato activated the flavonoid pathway and elevated 16 flavonoid compounds, consistent with evolutionary conservation of the MYC2-G-box regulatory logic. These findings establish CgMYC2 as a central, JA-responsive activator bridging jasmonate perception and naringin biosynthesis, providing a molecular framework for the targeted improvement of bitter-flavonoid traits in citrus.

Citrus↗

PubMed2026

Inhibitory mechanism of phloretin on the AgrA LytTR domain-agr operon complex formation and its application in beef.

Staphylococcus aureus (S. aureus) represents a major foodborne pathogen whose enterotoxin production poses significant challenges to food safety due to its high environmental resistance and limited efficacy of conventional sterilization. Since the expression of enterotoxins is predominantly governed by the agr quorum sensing system, targeting this regulatory pathway has become a strategic choice for virulence control. This study elucidated the mechanism by which phloretin, a potential quorum sensing inhibitor, interferes with the agr system to attenuate virulence. To achieve this, the recombinant AgrA LytTR domain was expressed and purified, and its interaction with phloretin was characterized using thermal shift assays (TSA), electrophoretic mobility shift assays (EMSA), and molecular dynamics (MD) simulations. The results showed that phloretin specifically binds to the AgrA LytTR domain, enhancing its thermal stability and disrupting AgrA LytTR-agr operon binding by reducing the free energy of interaction between them, without causing significant structural rearrangement. Mechanistic analysis indicated that phloretin sterically hinders key β-sheet turn residues (HIS169, ASN201, ARG233), thereby impairing DNA recognition, downregulating RNAIII transcription, and inhibiting agr signaling. In cooked beef, phloretin significantly inhibited the secretion of enterotoxins and α-hemolysin, while delaying lipid oxidation and protein degradation, and maintaining the meat texture. These findings suggested that phloretin is a multifunctional substance with anti-virulence, antioxidant, and preservative properties, demonstrating its potential as a natural food preservative.

Phloretin↗

PubMed2025

Multi-omics reveals an ecdysone-activated Eip75B-FABP signaling axis coordinating nutrient metabolism for development in Hermetia illucens.

INTRODUCTION: Efficient nutrient storage is essential for insect development and energy homeostasis; however, the mechanisms coordinating nutrient allocation during ontogeny are not well understood. Elucidating these systems may yield valuable insights to insect metabolic adaptation. OBJECTIVES: This study aimed to identify regulatory modules governing nutrient metabolism in insects, focusing on hormonal and metabolic interplay. METHODS: Multi-omics profiling (proteomics, phosphoproteomics, and transcriptomics) was conducted throughout the life cycle, from egg to adult, to identify metabolic regulators. RNAi was utilized for gene knockdown, followed by qRT-PCR and mitochondrial DNA quantification to evaluate knockdown efficiency and its metabolic implications. Assessments of nutrient metabolism were performed using assays for triglycerides, crude protein, and fatty acid synthase. EMSA and BODIPY staining examined transcriptional regulation and lipid droplet dynamics. RESULTS: Utilizing an integrative multi-omics approach, this study elucidates the temporal metabolic regulators in insects. A conserved regulatory module was identified in which the PPAR homolog, ecdysone-induced protein 75B (Eip75B), functions as a transcriptional activator of fatty acid binding protein (FABP), sustaining lipid metabolic homeostasis during the larval stage. PPARγ modulators (rosiglitazone and GW9662) alter lipid accumulation, along with the expression of Eip75B and FABP, which was measured by qRT-PCR. Furthermore, the deficiency of FABP may reprogram metabolic pathways by inhibiting lipid storage and promoting mitochondrial β-oxidation, as supported by increased mitochondrial DNA copy number, as well as enhancing protein synthesis. This metabolic change could be modulated by ecdysone signaling, as hormonal supplementation effectively rescued the lipid loss phenotype. Our results establish the ecdysone-Eip75B-FABP signaling axis as a central regulatory module that integrates hormonal and nutrient-sensing signals to control insect nutritional metabolism. CONCLUSION: The ecdysone-Eip75B-FABP axis integrates hormonal and nutrient signals to regulate metabolic plasticity, underscoring a universal strategy for developmental energy allocation. The data also offer potential implications for research on metabolic disorders and bioenergy applications.

Animals↗

PubMed2026

The transcription factor SbWRKY6 confers cadmium tolerance via activating SbPLAC8-17 expression in sorghum.

Cadmium (Cd) is a widespread environmental pollutant that severely threatens crop productivity and food safety. However, the regulatory mechanisms underlying Cd detoxification and tolerance in sorghum remain largely elusive. Herein, we functionally characterized SbWRKY6, a Cd‑induced WRKY transcription factor that localizes to the nucleus and functions as a transcriptional activator. Stable overexpression of SbWRKY6 significantly enhanced Cd tolerance in sorghum, as evidenced by improved growth performance, mitigated oxidative damage, and decreased Cd concentration in plant tissues, whereas silencing of SbWRKY6 resulted in a Cd-hypersensitive phenotype with exacerbated toxicity symptoms. Mechanistically, we identified SbPLAC8-17, a member of the Plant Cadmium Resistance (PCR)/PLAC8 family, as a critical downstream target of SbWRKY6. Heterologous expression of SbPLAC8-17 functionally complemented the Cd‑sensitive phenotype of the yeast mutant ∆ycf1 and reduced intracellular Cd accumulation. Further yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter (Dual-LUC) assays confirmed that SbWRKY6 directly binds to the SbPLAC8-17 promoter and transcriptionally activates its expression. In vivo silencing of SbPLAC8-17 significantly impaired cellular Cd²⁺ efflux and aggravated Cd toxicity in sorghum. Additionally, the rapid Cd-induced transcriptional upregulation of SbMPK3 and its direct physical interaction with SbWRKY6 suggest a potential upstream regulatory module that remains to be functionally validated. Collectively, this study elucidates a novel SbWRKY6‑SbPLAC8-17 transcriptional cascade that positively regulates Cd tolerance by facilitating Cd²⁺ efflux, providing promising genetic targets for phytoremediation and molecular breeding of safe sorghum cultivars for Cd-contaminated fields.

Sorghum↗

PubMed2026

GmMYB29 activates Gm4CL3 to enhance soybean resistance to Heterodera glycines.

Soybean cyst nematode is a devastating soil-borne pathogen that severely limits soybean yield worldwide. To uncover downstream target genes of the resistance-associated transcription factor GmMYB29, we combined ChIP-seq and RNA-seq data from T3-generation GmMYB29-overexpressing soybean plants, alongside Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, to screen candidate genes carrying transcription factor binding peaks within the 2000 bp region upstream of transcription start sites (TSS). Four orthogonal molecular assays-yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), dual-luciferase reporter (LUC) system, and GUS histochemical staining-collectively confirmed the specific physical interaction between GmMYB29 and the promoter of Gm4CL3. We generated transgenic soybean hairy roots overexpressing Gm4CL3 (OX-Gm4CL3) and CRISPR-Cas9-mediated Gm4CL3 knockout lines (KO-Gm4CL3), with wild-type (WT) plants serving as controls. Inoculation assays using SCN 3 demonstrated that OX-Gm4CL3 roots displayed substantially improved SCN resistance, while KO-Gm4CL3 roots were hypersusceptible to nematode infection. Mechanistic investigations revealed that Gm4CL3 promotes lignin deposition in root tissues to block SCN penetration. Furthermore, GmMYB29 and Gm4CL3 act synergistically to activate lignin biosynthetic pathways and strengthen soybean resistance against SCN 3 (SCN Race 3, the dominant physiological race in Northeast China). In summary, this study functionally characterizes Gm4CL3 and defines a previously unreported GmMYB29-Gm4CL3 regulatory cascade that mediates plant defense against SCN. This module functions independent of classic SCN resistance loci rhg1/Rhg4, providing new genetic resources for SCN-resistant soybean molecular breeding.

Glycine max↗

PubMed2025

Phosphoregulation of RAD51AP1 function in homology-directed repair.

Homology-directed DNA repair (HDR) is critical for genome stability and tumor suppression. HDR is initiated by the RAD51 single-stranded (ss)DNA nucleoprotein filament which conducts the homology search and invades a homologous DNA template, creating a displacement-loop (D-loop). The RAD51 filament is assisted in these processes by several proteins. One such protein is RAD51-Associated-Protein 1 (RAD51AP1) which binds DNA and RNA and directly interacts with RAD51. Of note, RAD51AP1 overexpression is associated with poor prognosis in several different cancer types. Here, we show that RAD51AP1 activity is regulated by phosphorylation. RAD51AP1 bearing S277/282A mutations is more proficient in the stimulation of D-loop formation than wild type RAD51AP1 or phosphomimetic RAD51AP1-S277/282D. In EMSAs, RAD51AP1 with S277/282A mutations more avidly binds ssDNA, double-stranded (ds)DNA, and the nucleosome core particle than wild type RAD51AP1 or RAD51AP1-S277/282D. In cells, RAD51AP1-S277/282A confers no rescue of RAD51AP1 deficiency in toxicity tests and DNA replication assays. In contrast, RAD51AP1-S277/282D fully rescues RAD51AP1 deficiency. We provide evidence that RAD51AP1-S277 is a CDK2 target and propose a model in which RAD51AP1-S277/282 phosphorylation ensures RAD51AP1 flexibility for dynamic engagement in consecutive steps of the HDR reaction. Our results provide new mechanistic insights into RAD51AP1 regulation by a CDK.

CDK1/2↗

PubMed2025

A six-repeat PPR protein WPR directly binds target RNAs and coordinates chloroplast RNA processing via dual recruitment of MORF1, MORF8b, and CAF2 proteins in rice.

Pentatricopeptide repeat (PPR) proteins are key regulators of organelle RNA metabolism in plants, yet their precise mechanisms in chloroplast RNA processing remain unclear. Here, we identify WPR, a unique P-type PPR protein in rice (Oryza sativa L.), as a critical factor in chloroplast RNA splicing and editing. A ~112-kb chromosomal inversion upstream of WPR causes an albino panicle rachis phenotype (wpr mutant), while complete loss of WPR function leads to seedling lethality. WPR deficiency disrupts the splicing of multiple group II introns (atpF, ndhA, ndhB, petB, rpl2, and rps12) and impairs RNA editing in transcripts such as ndhA, ndhB, ndhG, rps14, and ycf3. Electrophoretic mobility shift assay (EMSA) data confirm that WPR directly binds to precursor mRNAs of atpF, ndhA, petB, rpl2, and rps12. Strikingly, WPR interacts with both RNA editing factors (MORF1, MORF8b) and the splicing factor CAF2, but not with other PPR proteins targeting the same transcripts. Unlike most PPR proteins, WPR contains only six PPR repeats, which is the fewest among all functionally characterized rice PPR proteins. With few informative repeats, WPR likely possesses a broad, low-specificity RNA-binding activity. Moreover, WPR may act on chloroplast RNA maturation by recruiting MORFs and CAF2 rather than other PPR proteins, highlighting a novel regulatory mode in which P-type PPR protein may act as an RNA-binding scaffold to integrate diverse RNA-processing machineries. This study advances the understanding of PPR protein diversity and provides new insights into the molecular mechanisms of chloroplast RNA processing in rice.

Oryza↗

PubMed2025

The SlWRKY39-SlZF61 module synergistically regulates SlGSTU42 to enhance low-temperature tolerance in tomato.

Low-temperature stress affects plant growth, and WRKY transcription factors alleviate such damage by regulating downstream genes. This study found that tomato SlWRKY39 significantly responds to low temperatures: its overexpression enhances seedling low-temperature tolerance by promoting ROS scavenging, while knockout exacerbates ROS accumulation and increases sensitivity to low temperatures. Transcriptome analysis indicated induction of glutathione metabolic pathway genes in slwrky39 plants under low-temperature stress. Y1H, EMSA, and Dual-LUC experiments confirmed that SlWRKY39 specifically binds to and activates the SlGSTU42 promoter; silencing SlGSTU42 attenuated the low-temperature tolerance conferred by SlWRKY39 overexpression, verifying that SlWRKY39 improves low-temperature tolerance via direct regulation of SlGSTU42. Additionally, SlZF61 interacts with SlWRKY39, enhancing its regulatory effect on SlGSTU42. SlZF61 overexpression strengthens low-temperature tolerance, while knockout increases sensitivity to low temperatures. In summary, under low-temperature stress, SlWRKY39 and SlZF61 are upregulated expression in tomato; SlWRKY39 binds to the SlGSTU42 promoter, and SlZF61 interacts with SlWRKY39 to form a protein complex, enhancing this binding. They synergistically activate SlGSTU42 transcription, thereby improving seedling low-temperature tolerance by scavenging ROS. This coordinated regulatory mechanism provides a new theoretical basis and practical insights for enhancing tomato low-temperature tolerance and ensuring stable production under low-temperature stress conditions.

Solanum lycopersicum↗

PubMed2025

The NmpRSTU multi-component signaling system of Myxococcus xanthus regulates expression of an oxygen utilization regulon.

UNLABELLED: Myxococcus xanthus has numerous two-component signaling systems (TCSs), many of which regulate the complex social behaviors of this soil bacterium. A subset of TCSs consists of NtrC-like response regulators (RRs) and their cognate histidine sensor kinases (SKs). We have previously demonstrated that a multi-component, phosphorelay TCS named NmpRSTU plays a role in M. xanthus social motility. NmpRSTU was discovered through a screen that identified mutations in nmp genes that restored Type-IV pili-dependent motility to a nonmotile strain. The Nmp pathway begins with the SK NmpU, which is predicted to be active in the presence of oxygen. NmpU phosphorylates another SK, NmpS, a hybrid kinase containing an RR domain and a HisKA-CA domain. These two kinases work in a reciprocal fashion: when NmpU is active, NmpS is inactive, and vice versa. Finally, the phosphorelay culminates in NmpS phosphorylating the NtrC-like RR NmpR. To better understand the role of NmpRSTU in M. xanthus physiology, we determined the NmpR regulon by combining in silico predictions of the NmpR consensus binding sequence with in vitro electromobility shift assays (EMSAs) and in vivo transcriptional reporters. We identified several NmpR-dependent, upregulated genes likely to be important in oxygen utilization. Additionally, we demonstrate NmpRSTU plays a role in fruiting body development, suggesting a role for oxygen sensing in this behavior. We propose that NmpRSTU senses oxygen-limiting conditions, and NmpR upregulates genes associated with optimal utilization of that oxygen. This may be necessary for M. xanthus physiology and behaviors in the highly dynamic soil where oxygen concentrations vary dramatically. IMPORTANCE: Bacteria use two-component signaling systems (TCSs) to respond to a multitude of environmental signals and subsequently regulate complex cellular physiology and behaviors. Myxococcus xanthus is a ubiquitous soil bacterium that encodes numerous two-component systems to respond to the conditions of its soil environment and coordinate multicellular behaviors such as coordinated motility, microbial predation, fruiting body development, and sporulation. To better understand how this bacterium uses a two-component system that has been linked to the sensing of oxygen concentrations, NmpRSTU, we determined the gene regulatory network of this system. We identified several genes regulated by NmpR that are likely important in oxygen utilization and for the M. xanthus response to varied oxygen concentrations in the dynamic soil environment.

Myxococcus xanthus↗

PubMed2025

Mutations in the transcriptional regulator MAB_2885 confer tedizolid and linezolid resistance through the MmpS-MmpL efflux pump MAB_2302-MAB_2303 in Mycobacterium abscessus.

Mycobacterium abscessus (MAB) is a clinically significant multidrug-resistant (MDR) pathogen, particularly implicated in pulmonary infections among cystic fibrosis (CF) patients. Tedizolid (TZD), an oxazolidinone-class antibacterial drug, has been recommended as an alternative treatment for MAB-infected patients who are intolerant to or whose isolate is resistant to first-line drugs including linezolid (LZD). To investigate the TZD resistance mechanisms in MAB, we isolated 23 TZD-resistant MAB mutants and performed whole-genome sequencing (WGS) to identify resistance-associated genes. Frequent mutations were identified in MAB_2885, encoding a putative TetR transcriptional regulator, and MAB_2303, encoding a putative mycobacterial membrane protein large (MmpL). Drug susceptibility testing confirmed that MAB_2885 mutations contribute to both TZD and LZD resistance in MAB. RNA-seq analysis revealed that restoring wild-type MAB_2885 in mutants downregulated the MAB_2302-MAB_2303. Electrophoretic mobility shift assay (EMSA) showed the MAB_2885 protein binds to its target sequence upstream of MAB_2302-MAB_2303, further confirming their regulatory relationship. The W91R mutation in the MAB_2885 protein was found to impair its DNA-binding activity compared to the wild-type. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed that MAB_2302-MAB_2303 functions as a TZD efflux pump. Additionally, overexpression of MAB_2885 in M. abscessus subsp. bolletii and M. abscessus subsp. massiliense also increased their TZD susceptibility and downregulated their respective MmpS-MmpL orthologs. Overall, our study demonstrates that mutations in MAB_ 2885 contribute to TZD and LZD resistance by disrupting the negative regulation of the downstream MAB_2302-MAB_2303, which functions as a direct efflux pump for TZD. These findings provide new insights into oxazolidinone resistance mechanisms in MAB and identify potential biomarkers for detecting drug resistance.

Mycobacterium abscessus↗

PubMed2026

Integrated analysis of ATAC-seq and RNA-seq reveals the TCP-ARF molecular module related to pathogenic process of phytoplasma infection in Paulownia fortunei.

BACKGROUND: Witches’ broom is an important disease of the Paulownia fortunei. Understanding the pathogenesis of witches’ broom is a prerequisite for its prevention and control. Phytoplasma is the pathogen of Paulownia witches’ broom. RESULTS: We investigated the changes in chromatin accessibility before and after phytoplasma infection in Paulownia fortunei by analyzing the DNA accessibility (ATAC-seq). In phytoplasma-infected P. fortunei (PFI) compared to healthy samples (PF), the closed regions of chromatin(1187 regions) were three times more than the open regions (352 regions). Fifty one percent of the accessible chromatin regions were overlapped with either H3K27ac or H3K9ac peaks. The closed regions were enriched in the conserved motif TGGGC[CT] that is recognized by the TCP transcription factor family. The closed regions in PFI are intersected with ARF family gene locus. The gene PfARF3 was verified to interact with the PfTCP23 transcription factor. The PfTCP23 was predicted to be interacted with the effector pawb44 in the pathogen of phytoplasma. CONCLUSIONS: The phytoplasma infection in P. fortunei is involved in the chromatin changes of the DNA accessibility and histone modification. The binding regions of TCP23 were found to be changed mostly in the accessibility between PFI and PF. The TCP-ARF module was found to be the possible regulatory module inducing the crinkled leaf trait.

RNA-Seq↗