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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

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↗

PubMed2026

Maize ZmMYB59 inhibits post-germinative shoot and root elongation through ZmGA2ox3/10-mediated gibberellin catabolism.

Gibberellin (GA) promotes seed germination, but sustained or excessive GA signaling after germination can lead to aberrant root and shoot elongation. How GA homeostasis is transcriptionally restrained during post-germinative seedling development remains unclear. Using overexpression and gene-edited maize materials, we demonstrate that ZmMYB59 inhibits root and shoot elongation during post-germinative growth. Integrated RNA-Seq and CUT&Tag analyses identified the GA catabolism genes ZmGA2ox3 and ZmGA2ox10 as candidate direct targets of ZmMYB59. Hormone profiling analysis showed elevated bioactive GA1 and GA4 levels in the scutellum and aleurone layer cells of zmmyb59 mutants. Dual-luciferase assays, electrophoretic mobility shift assays, and ChIP-qPCR further confirmed that ZmMYB59 directly binds AC8 cis-elements in the ZmGA2ox3/10 promoters and activates their transcription. The zmga2ox3/10 double mutant, but neither single mutant, exhibited enhanced root and shoot elongation, accompanied by GA4 accumulation. This phenotype was suppressed by exogenous application of the GA biosynthesis inhibitor uniconazole. Transcriptomic and biochemical analyses further revealed enhanced starch degradation, reduced starch content, and increased soluble sugar accumulation in the double mutant. Taken together, these findings reveal that the ZmMYB59-ZmGA2ox3/10 module restrains GA accumulation and starch mobilization after germination, thereby coordinating reserve utilization with post-germinative root and shoot growth in maize.

Gibberellins↗

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

TGA6 directly activates ABF2 and ABF3 to promote leaf senescence in Arabidopsis thaliana.

Leaf senescence is a tightly regulated developmental process governed by a complex transcriptional network. Although the TGACG motif-binding (TGA) family of basic leucine zipper (bZIP) transcription factors are well-characterized regulators of plant defense responses, their roles in leaf senescence remain poorly understood. Here, we report that overexpression of TGA6 in Arabidopsis thaliana promotes early leaf senescence. Independent TGA6-overexpressing lines displayed premature leaf yellowing and significantly lower chlorophyll levels than wild-type (WT) plants under both normal growth and dark-induced senescence conditions. At the molecular level, RT-qPCR analysis revealed significant upregulation of canonical senescence marker genes, including NYC1, PAO, SAG12, SAG13, SGR1, and SGR2, in the TGA6-OE lines relative to WT plants. Furthermore, we found that the transcript levels of ABA-responsive element binding factor 2 (ABF2) and ABF3, which act upstream of these senescence markers, were significantly elevated in the TGA6-OE lines. Dual-luciferase reporter assays and electrophoretic mobility shift assay demonstrated that TGA6 directly binds to the TGACG motifs within the promoters of ABF2 and ABF3 to activate their transcription. Collectively, these findings demonstrate that TGA6 functions as a positive regulator of leaf senescence.

Arabidopsis↗

PubMed2025

Non-coding single-nucleotide and structural variants affecting the EYS putative promoter cause autosomal recessive retinitis pigmentosa.

PURPOSE: Variants in untranslated genomic regions are difficult to identify as pathogenic but are capable of causing disease by interfering with gene expression. This study aimed to characterize the effect of variants identified in the 5'-untranslated region of EYS in patients with autosomal recessive retinitis pigmentosa (RP). METHODS: Variant screening included gene panels, Sanger, exome, and genome sequencing. Functional validation included an electrophoretic mobility shift assay and various luciferase assays. RESULTS: Patients with RP from 6 EYS biallelic Arab-Muslim families harbored a 5' noncoding EYS variant, c.-453G>T, and 4 harbored a structural variant affecting the 5' noncoding exons. Electrophoretic mobility shift assay analysis revealed an effect on binding of transcription factors for c.-453G>T and a neighboring variant c.-454G>T. Dual luciferase assays using overexpression of various transcription factors showed distinct effects on expression. c.-453G>T was associated with higher luciferase expression with CRX overexpression and c.-454G>C with OTX2 overexpression. In addition, the 2 variants were found to influence translation by affecting upstream initiation codons. Interestingly, visual function of EYS RP patients who harbor c.-453G>T are better than those with biallelic null EYS variants. CONCLUSION: Our analysis revealed both single-nucleotide and structural variants in the EYS promoter as the cause of autosomal recessive RP. These variants may affect EYS expression via a dual mechanism by altering transcription factor binding affinity at the EYS promoter and by affecting upstream open reading frames.

Humans↗

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↗

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↗

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

Ecr positively regulates activity of the PhoQ/PhoP signalling system in Klebsiella pneumoniae.

BACKGROUND: The rising prevalence of polymyxin resistance in multidrug-resistant Klebsiella pneumoniae presents a critical situation with limited therapeutic options. METHODS: Methods Genomic sequencing of 15 clinical polymyxin-resistant K. pneumoniae strains with multidrug resistance revealed that MgrB inactivation, predominantly disrupted by insertion sequences (ISs) in the IS1, IS4, and IS5 families, was the leading cause of polymyxin resistance. Comparative transcriptomics of wild-type, ΔmgrB, and ΔmgrBΔphoP were performed to elucidate the MgrB-PhoPQ regulatory network. RESULTS: This study conducted a system-wide analysis of the regulatory network and identified a species-specific PhoPQ regulon in K. pneumoniae.Beyond the classical MgrB-PhoPQ-ArnBCADTEF pathway, we identified a previously unannotated PhoPQ-regulated gene, 144 bp LN739_RS09850, encoding an Ecr homologue from Enterobacter cloacae. This protein has been reported to confer colistin heteroresistance, with the underlying mechanism not yet functionally validated. This study revealed that overexpression of Ecr homologues decreased colistin susceptibility in both K. pneumoniae and E. cloacae, but this phenotype was abolished upon phoP deletion, confirming PhoP's essential role. Consistent with this dependency, comparative transcriptomics of Ecr-overexpressing K. pneumoniae vs. control revealed significant upregulation of mgrB, phoPQ, arnBCADTE, and pmrD. Two-hybrid bacterial assays further demonstrated direct Ecr-PhoQ interaction. Electrophoretic mobility shift assay confirmed that PhoP directly binds to the ecr promoter in vitro, and a β-galactosidase reporter assay demonstrated that PhoP enhanced ecr promoter activity, indicating that PhoP regulates ecr expression by directly controlling its transcription. CONCLUSION: Collectively, these findings suggest that PhoP may directly activate the transcription of Ecr, with Ecr feedback activating the PhoPQ system via interaction with PhoQ, leading to induction of the arn operon and consequent polymyxin resistance.

Klebsiella pneumoniae↗

PubMed2026

IFNL1 gene promoter single nucleotide polymorphism rs7247086 enhances transcription through a STAT-binding site.

A single nucleotide polymorphism (SNP) within the human interferon lambda 1 (IFN-L1, IFN-λ1) gene promoter, rs7247086 (C/T) has been reported to be associated with severe dengue and possibly with psoriasis and COVID-19. However, its functional nature is unknown. The present study was undertaken to examine the effect of rs7247086 on transcription, by utilizing promoter and enhancer-reporter assays. We see that the T allele completes a consensus signal transducer and activator of transcription (STAT)-binding site. While we did not find strong evidence to show that the STAT-binding site drove transcription from the IFNL1 gene promoter, we saw that it acts like an enhancer in reporter assays. The T allele of rs7247086 within the STAT-binding site significantly increased transcription of the reporter gene compared to the C allele when incorporated into enhancer-reporter constructs in both HEK293 and A549 cell lines. Mechanistically, we obtained evidence from electrophoretic mobility shift assays to show that the T allele binds to STAT proteins more strongly than the C allele. In a cohort of healthy individuals, we saw that the T allele carriers, specifically males but not females, had significantly increased secretion of IFN-λ1 from their peripheral blood mononuclear cells after stimulation. Lastly, rs7247086 significantly associated with psoriasis, only in males but not in females.

Humans↗

PubMed2026

Circadian- and light-regulated oscillatory expression of CSA in rice leaves is required for pollen fertility.

The oscillatory expression of CSA in rice leaves is regulated by the circadian clock and red/far-red light signals, mediated through DOF5 and PIL11, and is required for normal pollen fertility. Photoperiod-sensitive male-sterile lines represent a pivotal innovation in the development of hybrid rice. However, the underlying mechanisms governing photoperiod-sensitive male reproductive development remain poorly understood. Our previous studies demonstrated that the carbon starved anther (csa) mutant exhibits male sterility under short-day (SD) conditions but partial fertility under long-day (LD) conditions. In this study, we report that CSA expression follows an oscillatory rhythm in rice leaves under both SD and LD conditions, a pattern regulated by both circadian clock and light signals. Tissue-specific RNA interference knockdown of CSA in leaves was associated with reduced pollen viability, suggesting that CSA expression in leaves contributes to normal male fertility. Promoter truncation assay results indicate that distinct regions of the CSA promoter contribute differentially to the regulation of CSA expression in leaves versus anthers, and that both the CSA expression level in anthers and the rhythmic expression pattern of CSA in leaves are associated with the restoration of male fertility. Using dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assays, we identified two proteins, PIL11 and DOF5, which directly bind to specific motifs (an E-box and T/AAAAG motif) within the CSA promoter truncation, thereby regulating its transcription. These findings elucidate novel mechanisms linking light sensing to the expression of circadian-controlled genes, thus connecting photoperiod with male reproductive development in rice.

Oryza↗

PubMed2014

Upregulation of TLR2 expression is induced by estrogen via an estrogen-response element (ERE).

TLR2 and estrogen are both thought to be involved in the pathogenesis of RA; however, it is unknown if there is an association between estrogen and TLR2. In this report, we treated PMA-differentiated THP-1 cells with 17β-estradiol (E2) and observed increases in TLR2 mRNA and protein levels by real-time quantitative PCR and western blot. Transfection of THP-1 cells with a series of 5'-deleted TLR2 promoter-luciferase constructs revealed that E2 enhanced TLR2 transcriptional activity in an estrogen receptor alpha (ERα)-dependent pattern. An estrogen receptor response element (ERE) was identified 251 bases upstream of the TLR2 promoter, and electrophoretic mobility shift assay and chromatin immunoprecipitations showed ERα binding was increased by E2. In summary, this work demonstrated that TLR2 is a new estrogen-regulated gene whose expression is upregulated through the interaction of ERα with an ERE in the promoter region.

Cell Line↗

PubMed2026

A novel DNA-protective function of Escherichia coli thioredoxin 2 mediated by its N-terminal zinc-binding domain.

Thioredoxins are ubiquitous thiol-disulfide oxidoreductases that maintain intracellular redox homeostasis. In addition to its conserved catalytic domain, Escherichia coli thioredoxin 2 (EcTrx2) possesses a unique N-terminal zinc-binding domain whose physiological function remains largely unknown. Here, we identify a previously unrecognized DNA-binding activity of EcTrx2 and demonstrate its role in protecting DNA during oxidative stress. Electrophoretic mobility shift assays showed that EcTrx2 bound plasmid DNA in a concentration-dependent and GST-tag-independent manner, whereas EcTrx1 exhibited no detectable DNA-binding activity. DNA binding was abolished by deletion of the N-terminal zinc-binding domain and was blocked by zinc occupancy, indicating that this unique domain is essential for DNA interaction. Consistent with these findings, EcTrx2 significantly protected plasmid DNA from DNase I digestion and hydroxyl radical-mediated oxidative damage in vitro. Furthermore, EcTrx2 enhanced bacterial tolerance to the DNA-damaging agents zeocin and diamide, supporting the physiological relevance of its DNA-binding activity. Our results reveal a DNA-binding role for EcTrx2 and identify its N-terminal zinc-binding domain as a key determinant of DNA binding and protection against oxidative DNA damage.

DNA binding↗

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↗

PubMed2025

The Tartary Buckwheat FtMYB46-FtNRAMP3 Module Enhances Plant Lead and Cadmium Tolerance.

The presence of toxic heavy metals lead (Pb) and cadmium (Cd) in polluted soil damage crop production and consequently harms human and livestock health. Tartary buckwheat (Fagopyrum tataricum) is a potential model plant for heavy metal phytoremediation because of its valuable characteristics of high heavy metal tolerance and abundant biomass production. Here, we report that the Tartary buckwheat FtMYB46-FtNRAMP3 module enhances plant Pb and Cd tolerance. RNA sequencing analysis showed that Pb treatment specifically induced expression of FtNRAMP3, a member of the NRAMP (Natural Resistance-Associated Macrophage Protein) transporter gene family. Further cytological and biochemical analysis revealed that FtNRAMP3 was localised to the plasma membrane and significantly contributed to increased tolerance to Pb and Cd in yeast cells. Consistently, transgenic overexpression of FtNRAMP3 in Arabidopsis significantly increased plant tolerance to Pb and Cd applications, reducing Pb concentration but increasing Cd concentration in the overexpression transgenic plants. Subsequent yeast one-hybrid and electrophoretic mobility shift assays showed that the transcription factor FtMYB46 directly binds to the FtNRAMP3 promoter. Further, FtMYB46 promoted FtNRAMP3 expression and increased plant Pb and Cd tolerance. Overall, this study demonstrates the important role of the FtMYB46-FtNRAMP3 module and its potential value in the phytoremediation of Pb and Cd stress.

Cadmium↗

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↗

PubMed2026

TagR, a newly identified member of the MarR family of transcriptional regulators, represses the NRPS operon in Klebsiella oxytoca.

Toxigenic Klebsiella oxytoca strains produce the pyrrolobenzodiazepine enterotoxins tilimycin (TM) and tilivalline (TV), which contribute to the development of antibiotic-associated hemorrhagic colitis. The biosynthesis of these toxins depends on the nonribosomal peptide synthetase (NRPS) operon located within the til pathogenicity island. Although several global and signal-responsive regulators of NRPS operon expression have been identified, the regulatory network governing enterotoxin biosynthesis remains incompletely characterized. In this study, we identified a previously unrecognized transcriptional regulator encoded within the til pathogenicity island of K. oxytoca. This protein, designated TagR (Tilivalline-associated genes repressor), is a member of the MarR family and acts as a negative regulator of NRPS operon expression. Structural prediction, molecular dynamics simulations, and biochemical analyses demonstrated that TagR exhibits the characteristic architecture of MarR family regulators and forms a stable homodimer. Deletion of tagR led to significant upregulation of the NRPS-associated genes npsA, thdA, and npsB, while complementation restored transcriptional repression. Electrophoretic mobility shift assays confirmed that TagR binds directly and specifically to the regulatory region upstream of the NRPS operon, supporting a mechanism of direct transcriptional repression. Consistent with these findings, loss of TagR significantly increased the cytotoxicity of K. oxytoca culture supernatants toward HeLa cells. Collectively, these results identify TagR as a direct repressor of the NRPS operon and expand the regulatory framework governing enterotoxin biosynthesis in toxigenic K. oxytoca. This study provides new insight into the transcriptional control of virulence-associated genes and establishes TagR as a previously unrecognized component of the regulatory network controlling TM and TV production.IMPORTANCEElucidating the mechanisms by which toxigenic Klebsiella oxytoca regulates enterotoxin production is critical for understanding the pathogenesis of antibiotic-associated hemorrhagic colitis. TagR is identified as a previously unrecognized MarR family regulator that directly represses the nonribosomal peptide synthetase (NRPS) operon responsible for tilimycin (TM) and tilivalline (TV) biosynthesis. This discovery uncovers a novel regulatory mechanism governing toxin production and offers new perspectives on virulence regulation in this emerging intestinal pathogen.

Klebsiella oxytoca↗