Search Results (1,117)

Cohen syndrome, caused by pathogenic variants in VPS13B, is characterized by microcephaly, developmental delay, and progressive retinal degeneration, yet the cellular mechanisms linking VPS13B dysfunction to disease pathology remain incompletely understood. Here, we used CRISPR-Cas9 to delete VPS13B exons 2–4 in Phoenix HEK293 cells, generating five independent knockout clones. In all mutant lines, VPS13B disruption caused a marked slowing of cell proliferation due to prolongation of the G1 phase. Immunocytochemistry and transmission electron microscopy revealed that VPS13B mutations causes Golgi apparatus fragmentation, loss of VPS13B Golgi localization, ER lumen dilation with rigid membrane morphology, mitochondrial damage, impaired autophagic maturation, and the appearance of cytoplasmic fibrillary inclusions located close to ER and absent from control cells. RNA-seq analysis identified 27 differentially expressed genes common to all four mutant clones, including downregulation of genes involved in transcriptional regulation, lipid metabolism, and neuronal signaling, alongside upregulation of the stress-response genes CLU and CDKN1A (p21). While our results do not support classical unfolded protein response activation, they are consistent with a model in which lipid bilayer stress and disrupted ER–Golgi trafficking may play a role in the pathophysiology of Cohen syndrome. Together, these findings demonstrate that VPS13B deficiency results in coordinated defects in organelle homeostasis, proteostasis, and cell-cycle progression, providing new dates for understanding Cohen syndrome pathogenesis. Full article

Sclerotinia sclerotiorum is a broad-spectrum soilborne fungal pathogen, causing substantial yield losses in crops worldwide. Auxiliary Activity Family 5 (AA5) copper radical oxidases (CROs) are members of the carbohydrate active enzymes (CAZymes), valued for their potential as biocatalysts. Although they were reported to contribute to fungal development and disease progression in some phytopathogenic fungi such as Colletotrichum graminicola, the roles of AA5 enzymes in S. sclerotiorum remain largely unexplored. Here, we systematically investigated S. sclerotiorum AA5 genes through reverse genetics and biochemical approaches. Phylogenetic analysis grouped the S. sclerotiorum AA5 proteins into two subfamilies, including three subfamily 1 (AA5_1) members and one subfamily 2 (AA5_2) member. Enzymatic characterization revealed that one AA5_1 enzyme, SsAA5c, exhibited the highest catalytic specificity towards D-glyceraldehyde compared with methylglyoxal and glycerol. In addition, a predicted peroxidase gene (SsPX1) was identified in tandem with the putative AA5_2 gene, SsAA5d. Phenotypic analysis of gene deletion mutants demonstrated that these genes differentially affect fungal development and virulence. While their exact physiological substrates await identification, our findings highlight important roles of AA5 oxidases and the associated peroxidase in the biology of S. sclerotiorum, providing insights that may contribute to future improved disease management strategies. Full article

Photobacterium damselae subsp. damselae (PDD), a marine fish pathogen, has an unclear contribution of tonB genes to iron acquisition and pathogenicity. ΔtonB1-PDD and ΔtonB2-PDD mutants as well as their complemented strains were generated via homologous recombination from the virulent strain PDD1605. Growth was evaluated by monitoring OD₆₀₀ with viable-count calibration, intracellular iron was measured using a colorimetric assay, biofilm was detected by crystal violet staining, transcription was analyzed through qRT-PCR, and pathogenicity was assessed via an 8-day intramuscular challenge in black rockfish (Sebastes schlegelii). Under iron limitation induced by 100 μM 2,2′-dipyridyl, the maximum density decreased from 4.51 × 10⁸ CFU/mL in wild-type (WT-PDD) to 4.00 × 10⁸ and 3.64 × 10⁸ CFU/mL in ΔtonB1-PDD and ΔtonB2-PDD respectively, and was largely restored after complementation. The intracellular iron content declined from 3.75 × 10⁻⁴ ± 4.00 × 10⁻⁶ nmol/10⁶ cells in WT-PDD to 3.13 × 10⁻⁴ ± 1.05 × 10⁻⁵ nmol/10⁶ cells in ΔtonB1-PDD and 2.55 × 10⁻⁴ ± 2.00 × 10⁻⁵ nmol/10⁶ cells in ΔtonB2-PDD. Compared with WT-PDD, biofilm formation was reduced by 22.1% in ΔtonB1-PDD and by 24.8% in ΔtonB2-PDD. Deletion of tonB2 induced mild yet statistically significant transcriptional alterations in multiple iron acquisition and virulence-related genes, while colony morphology, swarming motility, hemolysis, phospholipase activity, biochemical traits, and antimicrobial susceptibility remained unchanged. In the high-dose challenge assay, WT-PDD caused 100% mortality within 2 days, whereas ΔtonB1-PDD and ΔtonB2-PDD caused 17/20 and 18/20 deaths respectively; all infected groups reached 100% mortality by day 3. In the low-dose challenge assay, WT-PDD, ΔtonB1-PDD, and ΔtonB2-PDD caused 12/20, 12/20, and 13/20 deaths by day 2, with cumulative mortalities reaching 80%, 70%, and 80% by day 8, respectively. Kaplan–Meier analysis detected no significant differences among the survival curves at either challenge dose. These findings suggest that tonB1 and tonB2 play roles in iron acquisition, low-iron adaptation, and biofilm formation, with tonB2 deletion exerting greater effects on growth and intracellular iron accumulation under iron-limited conditions. Full article

Avian pathogenic Escherichia coli (APEC) induces avian colibacillosis and brings huge economic losses to global poultry production. The small alarmone (p)ppGpp mediates the bacterial stringent response, a vital pathway modulating microbial stress adaptation and pathogenic capacity. The functions of the (p)ppGpp synthase gene relA in APEC pathogenesis remain poorly characterized. In this study, we constructed a relA deletion mutant (ΔrelA) and its complemented strain (CΔrelA). The phenotypic and pathogenic characteristics of these strains were investigated. The results showed that deletion of relA did not significantly affect bacterial growth or motility. However, the ΔrelA strain showed increased susceptibility to aminoglycoside antibiotics. Furthermore, the enhanced interbacterial competition of the mutant was associated with the upregulation of core genes in the type VI secretion system (T6SS). Importantly, relA was essential for APEC adhesion to and invasion of avian DF-1 cells, as well as for colonization and virulence in ducklings, where ΔrelA exhibited significantly attenuated infectivity and reduced bacterial loads in the liver and spleen. Furthermore, transcriptomic analysis revealed that RelA deletion downregulated genes involved in integral components of the membrane, and further assays confirmed compromised membrane integrity in the mutant strain. These findings suggest that RelA maintains membrane integrity, which underpins its contributions to antibiotic resistance and virulence. These findings indicate that relA plays a key role in APEC virulence, antibiotic resistance, and membrane homeostasis, and could provide a theoretical basis for targeting the stringent response as a potential strategy to control avian colibacillosis. Full article

Fusarium graminearum, the major pathogen causing maize ear rot, severely threatens food security. Targeted gene deletion was used to characterize physiological functions of FgSRE1 in this pathogen. Transcriptomic profiling was conducted on axenic wild-type and ΔFgSRE1 cultures, plus maize ear tissues colonized by both strains. Nine core DEGs functionally annotated to transmembrane transport, membrane homeostasis and oxidative stress pathways were selected for qRT-PCR validation, including four upregulated genes encoding putative transferases and five downregulated transporter genes. Phenotypic assays revealed that the ΔFgSRE1 mutant displayed severe defects in conidial and ascospore production, along with drastically weakened pathogenicity on maize ears and leaves versus wild-type and complemented strains. qRT-PCR analysis confirmed consistent expression trends with the RNA-seq data: four putative transferase-encoding genes were up-regulated, whereas five genes related to membrane and redox transport were repressed. Together, the genetic and phenotypic results demonstrate that FgSRE1 contributes to reproductive development and virulence in F. graminearum, whereas the transcriptomic data suggest potential associations with metabolic, redox, and membrane-transport-related pathways. Our findings provide a basis for investigating the FgSRE1-associated virulence network and developing eco-friendly maize ear rot control strategies. Full article

The aquatic pathogen Aeromonas salmonicida SRW-OG1 is responsible for substantial mortality in Epinephelus coioides and many other cultured fish species. This study investigates the contribution of trpE to the pathogenicity of A. salmonicida SRW-OG1 in E. coioides. Specifically, trpE encodes a key enzyme in tryptophan biosynthesis; however, its function in regulating virulence in A. salmonicida remains uncertain. Phenotypic assays indicate that deletion of trpE markedly impaired bacterial growth and significantly reduced multiple virulence-associated traits, including swimming motility, biofilm formation, chemotaxis, hemolytic activity, and extracellular enzyme activity. Transmission electron microscopy revealed that the ΔtrpE mutant lacked flagella, indicating that trpE is involved in flagellar formation or maintenance. In an E. coioides infection model, fish infected with ΔtrpE showed a cumulative survival rate of 48.3%, whereas those infected with the wild-type SRW-OG1 suffered 100% mortality. The ΔtrpE mutant also exhibited reduced bacterial loads in target organs (liver, head kidney, and spleen), while pretreatment with ΔtrpE provided 53.3% protection against a subsequent wild-type challenge. Transcriptomic analysis identified 616 differentially expressed genes, mainly enriched in pathways including flagellar assembly, bacterial chemotaxis, two-component systems, quorum sensing, and biofilm formation. Metabolomic analysis further showed a significant decrease in N-butyryl-L-homoserine lactone, an acyl-homoserine lactone quorum-sensing signal in the ΔtrpE mutant. These findings suggest that trpE contributes to mesophilic A. salmonicida SRW-OG1’s virulence by partially affecting quorum-sensing signal production and motility-related pathways. The attenuated phenotype and protective effect of ΔtrpE indicate its potential as a live attenuated vaccine candidate for controlling A. salmonicida infection in aquaculture. Full article

Oil palm is a major commodity crop in Southeast Asia, particularly in Malaysia and Indonesia, but its productivity is severely threatened by basal stem rot (BSR) and upper stem rot (USR) caused by the white-rot fungus Ganoderma boninense. Infected palms can lose up to 80% yield and die within 6–24 months (young) or 2–3 years (mature). Despite extensive field management efforts, disease incidence continues to rise, especially after replanting. Understanding infection mechanisms and validating fungal virulence factors are crucial for effective control, yet functional genomics in G. boninense has been limited. Previous RNAi-based gene silencing provided initial insights but was constrained by off-target effects and transient activity. Here, we report the first successful application of CRISPR/Cas9 genome editing in G. boninense for functional gene studies. Two genes were targeted: pyrG, essential in uridine monophosphate (UMP) biosynthesis; and hyd-2, encoding a hydrophobin, a potential virulence determinant implicated in host invasion. The disruption of pyrG produced a uracil auxotroph, and the knockout was validated by screening on 5-FOA and validated our system as a functional molecular tool. Disruption of hyd-2 reduced infection capability of the fungus by ~72% to ~91% in vitro. Mutations in both gene disruptions, including insertions, deletions, and substitutions, were confirmed by sequencing. Sequencing analysis also revealed incomplete editing events, as wild-type gene sequences were detected alongside edited alleles in the mutants. Future enhancements should focus on improving editing efficiency of the system. This work establishes a robust platform for functional genetic analysis and dissecting pathogenicity in G. boninense, ultimately advancing strategies to mitigate basal stem rot disease in oil palm. Full article

Klebsiella pneumoniae (K. pneumoniae) is a major opportunistic pathogen associated with a broad spectrum of hospital-and community-acquired infections. Biofilm formation contributes to bacterial persistence, stress tolerance, and host immune clearance. In this study, we constructed a transposon mutant library of the clinical K. pneumoniae strain KP20 using a mariner-based mutagenesis system and screened for mutants exhibiting defective biofilm formation. Targeted knockout of thrA significantly reduced biofilm formation and attenuated the adhesion and invasion of K. pneumoniae with respect to human airway epithelial Calu-3 cells and lung adenocarcinoma A549 cells. These phenotypic changes are associated with altered transcription of the gene clusters responsible for type I and type III fimbria biosynthesis. Moreover, deletion of thrA compromised bacterial tolerance to acidic and alkaline stresses and increased susceptibility to phagocytosis by dendritic cells. In a murine infection model, thrA deletion significantly attenuated the virulence of K. pneumoniae and decreased bacterial colonization in target organs. Collectively, these findings indicate that thrA substantially contributes to biofilm formation, pH stress tolerance, host–cell interaction, and virulence in K. pneumoniae. Full article

Sporulation is essential for asexual reproduction and dispersal in filamentous fungi, but the roles of upstream sporulation regulators in nematode-trapping (NT) fungi remain poorly understood. In this study, we characterized two Flb homologs, AoFlbB and AoFlbD, in Arthrobotrys oligospora. AoFlbB contains a bZIP-type domain, whereas AoFlbD contains a Myb-like DNA-binding domain, and both proteins are conserved among NT fungi. Deleting AoflbB or AoflbD had little effect on hyphal growth but markedly reduced spore production and altered spore germination, and real-time quantitative PCR analysis showed that the central sporulation regulators were significantly differently expressed, collectively indicating that AoFlbB and AoFlbD play positive roles in sporulation. Furthermore, both mutants produced more traps and showed enhanced nematode predation efficiency. The mutants also showed altered cell wall stress responses, hyphal cell length, nuclear number, endocytic trafficking, and metabolite profiles. The ΔAoflbB mutant showed irregular cell wall wrinkling, whereas the ΔAoflbD mutant displayed increased vesicle-like structures and accelerated FM4-64 uptake. Extracellular protease activity was comparable to the WT in ΔAoflbB but reduced in ΔAoflbD. Transcriptomic analysis of ΔAoflbD showed that the differentially expressed genes were significantly enriched in metabolism-related pathways. These findings indicate that AoFlbB and AoFlbD promote sporulation and contribute to the coordination of asexual reproduction and trap formation in A. oligospora. Full article

Pseudomonas sp. MUP55, isolated from rainfall water in Western Australia, was characterized by polyphasic taxonomy and functional assays. Whole-genome and 16S rRNA phylogeny placed Pseudomonas sp. MUP55 in the Pseudomonas fluorescens species group. Massetolide A/D was identified as the leading candidate bioactive compound(s), consistent with its biosynthetic gene cluster, GNPS library matching, and loss of activity in regulatory mutants. The strain showed broad-spectrum antimicrobial activity against bacterial (Escherichia coli and Xanthomonas campestris) and fungal (Fusarium oxysporum and Rhizoctonia solani) plant pathogens. GacA regulates Massetolide production: a P58L mutation abolished synthesis and reduced biocontrol efficacy. Metabolomic and transcriptomic analysis of a ΔpvfC mutant revealed that the pvf cluster regulates specialized metabolism while also contributing to secreted growth-inhibitory activity. The pvf cluster differentially regulates dual siderophore systems and uncouples the co-regulated small RNAs rsmY and rsmZ in the Gac/Rsm cascade. Deletion of pvfC partially reduced the growth-inhibitory activity of Pseudomonas sp. MUP55 supernatants against bacterial pathogens, indicating that pvfC also influences secreted antimicrobial activity beyond its global regulatory role. These findings establish Pseudomonas sp. MUP55 as a taxonomically novel, mechanistically characterized biocontrol agent with potential for sustainable agriculture. Full article

Cotton is an economically important cash crop severely affected by Verticillium wilt caused by Verticillium dahliae. Secreted cell-wall-degrading enzymes act as key virulence factors of this pathogen, yet the biological function of glucan 1,3-β-glucosidase remains largely uncharacterized. The gene VDAG_02814 (designated Vdbgl1) was previously found to be strongly induced during host infection. Here, combined approaches including gene knockout, host-induced gene silencing (HIGS), and transcriptomic analysis were utilized to characterize the function of Vdbgl1. The results showed that Vdbgl1 deletion retarded colony growth on PDA medium by 20.1–21.6%, decreased conidial yield by 20.2–46.7%, lowered spore germination rate by 30.7–32.9%, and weakened utilization of diverse carbon sources by 5.0–14.9%. The ΔVdbgl1 mutants also exhibited significantly increased sensitivity to cell wall-perturbing, osmotic, and membrane-damaging agents. Additionally, the ΔVdbgl1 mutants exhibited reduced disease index by approximately 19.1–27.7% and decreased fungal biomass by 42.7–61.8% in cotton; consistently, HIGS-mediated silencing of Vdbgl1 reduced the disease index by 25.7% and 26.7% and decreased fungal biomass by 48.1–71.5%. Transcriptome profiling of cotton roots infected by the ΔVdbgl1 mutant and the wild-type strain revealed 1007 down-regulated genes enriched in carbohydrate metabolism, cell wall degradation, and energy pathways, including 27 carbohydrate-active enzyme genes and 58 genes encoding cysteine-rich secreted proteins. Collectively, these findings indicated that Vdbgl1 coordinates carbon utilization, cell wall remodeling, and stress responses to regulate fungal development and pathogenicity, representing a promising target for cotton Verticillium wilt control. Full article

Glucocorticoids are stress hormones released into circulation that play central roles in the immediate and slower adaptive responses of the brain and body to environmental and internal stimuli, whether perceived or imagined. Receptors for glucocorticoids translocate from the cytosol to the nucleus and mitochondria to regulate the expression of numerous genes involved in neuroplasticity, metabolism, inflammation, immunity, and the cytoskeleton. Rapid effects of glucocorticoids are mediated by distinct receptor-associated mechanisms to facilitate or suppress neurotransmitter release. Feedforward and feedback mechanisms in neurotransmission thus depend on glucocorticoid receptor localization and functional states across synapses. Post-translational modifications of glucocorticoid receptors generate specific docking sites for signalling effectors which vary between active and inactive neurons. Diversity of glucocorticoid reactivity produces heterogeneous cellular responses that integrate prior experience. The activity-dependent trans-synaptic neurotrophic messenger BDNF emerges as a key modulator of glucocorticoid receptor signalling diversity. These mechanisms have been investigated with deletion mutants in a variety of preclinical models, pointing not only at possible causes of human diseases but also at potential strategies for mitigating them. This review specifically focuses on neurotrophic glucocorticoid signalling as a coincidence detection mechanism between BDNF and glucocorticoids, extending beyond classical genomic and non-genomic frameworks. Full article

Background: Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing the hemagglutinin (HA) gene of H3N8 EIV (sub-lineage: Florida clade 2) was generated and characterized in vitro. Methods: The HA gene of EIV (Florida clade 2) was used for antigen gene cloning. The expression cassette for the HA gene was commercially synthesized and inserted into the backbone of EHV1∆IR6 BAC using an En passant mutagenesis strategy. Recombinant clones were selected using antibiotic selection, PCR, and RFLP. Further, the recombinant virus was regenerated in RK-13 cells via transfection and characterized in vitro for plaque size, growth kinetics and immunofluorescence antibody test (IFAT). Results: PCR and RFLP confirmed the successful insertion of the HA gene into pEHV1∆IR6/gE BAC. The recombinant virus, vEHV1∆IR6/gE-HA(FC2), was successfully rescued in RK13 cells and demonstrated expression of the EIV haemagglutinin proteins by immunofluorescence assay. Although plaque size was reduced in the generated mutant virus in comparison to parental virus, the growth kinetics of the recombinant viruses were comparable to those of vEHV1∆IR6/gE. Conclusions: These findings demonstrate the successful expression of immunodominant hemagglutinin protein of EIV by recombinant EHV-1 and indicate the potential suitability of EHV-1 BAC as a vector platform for foreign gene expression. Full article

The bZIP type transcription factors (bZIPs) are global regulators governing vegetative growth, development, stress defense and secondary metabolism, including mycotoxin production in filamentous fungi. In this work, we constructed and phenotypically characterized gene deletion and complementation mutants of some bZIPs, including Afap1, AflatfA, LziP, AflatfB and bZIP6 in Aspergillus flavus. Environmental and fungicide stress responses, as well as aflatoxin production of the mutants in both surface cultures and infected maize kernels, were studied. Phenotypic characterization of the mutants revealed that Afap1 and AflatfA were involved in the oxidative (H₂O₂, menadione, tert-butyl hydroperoxide), cell wall integrity (Congo Red) and heavy metal (CdCl₂) stress responses of A. flavus. In addition, the Afap1 and AflatfA gene deletions decreased the diamide and prothioconazole tolerances of the fungus, respectively. The ΔLziP strain showed increased growth in the presence of diamide, while reduced colony diameters were observed after exposure to CdCl₂ and fludioxonil. The ΔAflatfB gene deletion mutant was also sensitive to tBOOH, while azoxystrobin and prothioconazole fungicides significantly inhibited the growth of ΔbZIP6. When aflatoxin (AFB1) production was measured in surface cultures, decreased AFB1 levels were detected only in the ΔAflatfA gene deletion mutant strain. However, in corn kernel infection assays, the ΔAfap1, ΔAflatfA, and ΔAflatfB mutants were characterized by significantly reduced aflatoxin production, while the deletion of bZIP6 almost completely abolished AFB1 biosynthesis. Our results suggest that Afap1 and AflatfA appear to be promising targets for the development of new antifungal agents, as their inhibition may increase the sensitivity of A. flavus to environmental stress, simultaneously reducing the aflatoxin production of the fungus and the use of azoles (AflatfA) in the antifungal protection of maize. In addition, bZIP6 may also be considered as an attractive target for further studies when aiming to eliminate aflatoxin production and minimize the use of azoxystrobin and prothioconazole in maize crop protection. Full article

Black soybean is an important functional crop valued for its high anthocyanin content and associated health benefits, attracting increasing interest in plant breeding and mutation-based approaches to enhance its nutritional and agronomic traits. Here, we investigated a DT26 black seed-coat mutant (DT26BS) derived from the Vietnamese cultivar DT26 following gamma irradiation. Genetic analysis of F₂ populations indicated that the phenotype is controlled by a single recessive mutation, with additional epistatic interactions observed in other genetic backgrounds. MutMap analysis based on whole-genome sequencing of pooled F₂ individuals identified a candidate region on chromosome 8 corresponding to the I locus. A large deletion (~176 kb) was identified in this region, affecting multiple Chalcone Synthase (CHS) gene repeats, and which may disrupt RNAi-mediated silencing of CHS, thereby triggering anthocyanin restoration in the seed coat. PCR-based markers confirmed tight linkage between this deletion and the black seed-coat phenotype. Crosses with elite lines and further selection until F7 generations showed that the mutation has no adverse effects on major agronomic traits and is useful as a donor for developing improved lines with black seed coat, shorter maturity, high yield, and enhanced anthocyanin content for nutritional improvement. These results demonstrate the utility of MutMap for detecting irradiation-induced structural variants and extend its application to a non-reference elite tropical soybean background, providing a useful genetic resource for black soybean breeding. Full article