Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Schistosoma japonicum Histone Acetyltransferase 1 (SjHAT1): A Novel Anti-schistosomal Drug Target

Schistosomiasis remains a critical global health issue, necessitating novel therapies due to emerging praziquantel resistance. We previously developed a patented praziquantel derivative, DW-3-15, which demonstrated potent broad-spectrum schistosomicidal activity through SjHAT1 inhibition. Here, the full-length SjHAT1 cDNA is cloned by rapid amplification of cDNA ends methods. The amino acids encoded by the cDNA retains conserved catalytic residues of acetyltransferases although sharing only 34% identity with mammalian orthologs. Phylogenetic analyses place it in a distinct clade, indicating its divergence specific to Schistosoma species. The expression profiling of SjHAT1 reveals stage- and sex-specific patterns. Fluorescence in situ hybridization localizes SjHAT1 predominantly in female vitellaria and male parenchyma near the gynecophoral canal. Knockdown of SjHAT1 impairs worm survival, reduces female oviposition and disrupts ovarian and vitelline morphology both in vitro and in vivo. RNA-sequencing analysis reveals that SjHAT1 knockdown disrupts {beta}-alanyl-tryptamine pheromone signaling via downregulation of aromatic L-amino acid decarboxylase in males and multidrug resistance-associated protein 4 in females. Through exploring this dual-sex regulatory mechanisms, SjHAT1 may emerge as a promising therapeutic target for interrupting schistosomiasis transmission.

microbiology↗

bioRxiv2025

STAG2 cohesin cooperates with DREAM to maintain quiescence and suppress tumourigenesis in the urothelium

The maintenance of quiescence is essential for tissue homeostasis. STAG2 is one of the few genes mutated in the normal urothelium of organ donors, with mutant cells undergoing positive selection 1. STAG2 is also a major tumour suppressor gene 2-4 and its inactivation is an early event in bladder carcinogenesis 1,3. However, how STAG2, a cohesin component, regulates urothelial homeostasis remains largely unknown. Here, we demonstrate that Stag2 inactivation in normal murine urothelial cells interferes with differentiation programs, triggers transient cell cycle entry, and primes cells for clonal expansion under stress. Moreover, STAG2 loss enhances tumor formation in urothelial cells expressing mutant FGFR3 - the key oncogene in bladder cancer 5. We reveal that STAG2 cooperates with the DREAM transcriptional complex, a master regulator of quiescence 6,7, by binding to shared genomic sites, including cell cycle control genes. STAG2 loss alters DREAM target expression, complex composition, and chromatin distribution, and leads to rewiring of chromatin interactions involving DREAM binding motifs in genes critical for cell cycle entry. Our findings provide compelling evidence that STAG2 loss disrupts in 3D genome organization through a novel mechanism involving the DREAM complex, thereby impairing homeostatic quiescence and increasing oncogenic sensitivity.

cancer biology↗

bioRxiv2025

Stepwise firing mechanism of an extracellular contractile injection system

Contractile injection systems (CISs) mediate cell-cell interactions and are widespread among bacteria and archaea. These phage tail-like macromolecular machines puncture their target by a tube that is propelled by a contractile sheath. The mechanism underlying CIS firing, which starts with target binding and ends with sheath contraction, remains unclear. Here, we investigate the extracellular CIS from Algoriphagus machipongonensis (AlgoCIS) by a multimodal cryo-electron microscopy approach and structure-guided engineering, which allowed us to arrest AlgoCIS in multiple intermediate states of firing. Together with the post-firing structure, our data suggest a stepwise firing mechanism involving all modules: signal propagation starts with the binding of the tail-fibers, followed by opening of the cage, an expansion of the baseplate iris, and resulting in sheath contraction and the release of cap adaptor. Our study will serve as a framework for understanding the firing mechanism of diverse CISs and will facilitate the engineering of CISs for biomedical applications.

microbiology↗

bioRxiv2025

TWIST1 Modulates Cilia Length, Endocytic Vesicle Dynamics, and Cell-Cell Junctions during Neural Tube Morphogenesis

BackgroundEndocytosis constitutes a fundamental cellular process governing development through coordinated regulation of plasma membrane remodeling and ciliogenesis, processes essential for cell shape changes and embryonic development. Although Twist1 null embryos display complete cranial neural tube closure defects and conditional knockout in neuroectoderm disrupts cranial neural crest cell fate determination and delamination, the function of TWIST1 in neural tube morphogenesis remains unknown. We investigated the basis underlying neuroectodermal morphological abnormalities in TWIST1 mutant embryos, specifically the formation of ectopic lateral bending points and cellular disorganization, by examining TWIST1 function in cilia formation, adherens junction integrity, and endocytic vesicle dynamics. ResultsImmunofluorescence analysis revealed that cytosolic TWIST1 colocalizes with {beta}-catenin and endocytic regulators LRP2 and RAB11B along the apical surface of cranial neuroectoderm. Twist1 knockout resulted in reduced ciliary length and number. Quantitative PCR and Western blot analyses demonstrated upregulation of RAB11B and {beta}-catenin at mRNA and protein levels in Twist1 mutants. This molecular dysregulation coincided with increased accumulation of apical endocytic vesicles and altered expression profiles of endocytic component genes, ultimately modifying the apical neuroectodermal cell-cell junctions. ConclusionOur findings establish TWIST1 as a regulator of neuroectodermal morphology, demonstrating its ability to modulate ciliogenesis, endocytic vesicle dynamics, and cell-cell integrity.

developmental biology↗

bioRxiv2025

Integrated analysis reveals neuro-immune pathway in the central nervous system that supports SGLT2i`s protective effects in treatment of cardiac remodeling

BackgroundDespite growing evidence of sodium-dependent glucose transporters 2 inhibitors (SGLT2i) improving heart failure, their mechanisms remain unclear. This study explored the effects of empagliflozin (EM) on sympathetic nerve activity and potential neuroimmune pathways. MethodsSingle-nucleus RNA sequencing (snRNA-seq) was performed in hypothalamic tissue of a DOCA-salt mouse model to screen for cellular changes. Validation of relevant mechanisms in DOCA-salt and high-salt diet (HSD) mouse models. Additionally, clinical data from 1,189 hospitalized heart failure patients were analyzed to evaluate the translational relevance of the experimental findings. ResultsEM treatment was associated with protective effects against cardiac remodeling in both DOCA-salt and HSD mice. Both models exhibited sympatho-excitation and neuronal hyperactivity in pre-autonomic brain regions, which appeared to be blunted by EM co-treatment. snRNA-seq analysis suggested that cellular interplay among vascular cells, microglia, and inhibitory neurons might be involved in these processes. Specifically, high-salt intake was linked to peripheral immune activation, including lymphocytosis and elevated plasma interferon-{gamma}, which potentially mediated microglial state transitions. EM might counteract the disease-associated upregulation of protein ubiquitination. Clinically, SGLT2i use was significantly associated with reduced lymphocyte counts in specific patient subgroups. ConclusionsEmpagliflozin may attenuate cardiac remodeling caused by high sodium intake through neuroimmune effects by restoring microglial homeostasis and inhibiting sympathetic nerve activity.

molecular biology↗

bioRxiv2025

METTL3 Uncouples Chromatin Accessibility from Transcription during Retinal Development

METTL3 is a key regulator of RNA metabolism, yet its genomic and epitranscriptomic roles in tissue development are largely unexplored. Using embryonic stem cell-derived 3D retinal organoids to model retinal progenitor cell (RPC) differentiation, we integrated transcriptome-wide m6A profiling (GLORI), protein-DNA (ChIP-seq and CUT&RUN) and chromatin accessibility (ATAC-seq) mapping, and targeted m6A engineering (dCas13b-FTO) to dissect METTL3 function. Loss of METTL3 nuclear m6A activity disrupted Rx+ retinal anlage formation in vitro, with dCas13b-FTO epitranscriptome engineering revealing that m6A at the Six3 3UTR governs its stability. Surprisingly, while METTL3 loss altered histone modifications and chromatin accessibility, its direct chromatin targets showed little transcriptional correlation. A degron-based METTL3 degradation strategy, paired with protein-RNA interaction profiling, exposed rapid regulatory shifts in RPCs, revealing a METTL3-Ythdf1 protein-RNA axis. Our multi-omics approach establishes METTL3-dependent m6A as a critical epitranscriptomic layer in retinal development, unveiling a genomic paradigm in which chromatin accessibility diverges from transcriptional output.

developmental biology↗

bioRxiv2025

NQO1 phase condensation promotes stress granule assembly to facilitate pancreatic carcinogenesis

G3BP1 promotes pancreatic ductal adenocarcinoma (PDAC) tumorigenesis driven by oncogenic KRAS mutations through liquid-liquid phase separation (LLPS)-mediated assembly of stress granules (SGs). However, the regulatory mechanisms remain elusive. We identify the antioxidant enzyme NAD(P)H quinone dehydrogenase 1 (NQO1) as a novel SG regulator that enhances SG assembly in pancreatic cancer cells independently of reactive oxygen species (ROS). Mechanistic studies reveal that NQO1 does not regulate the expression of G3BP1 or other SG-associated genes. Instead, NQO1 undergoes LLPS dependent on its RNA-binding K homology (KH) like domain. Further analysis demonstrates that residues 121-131 of NQO1 directly interact with G3BP1s RNA-binding domain (RBD), enhancing the multivalency of G3BP1 complexes to potentiate LLPS-driven SG assembly. This interaction accelerates cell proliferation, KRAS mutation-induced acinar-to-ductal metaplasia (ADM), and pancreatic carcinogenesis. Notably, the interaction between NQO1 residues 121-131 and G3BP1s RBD is essential for NQO1 phase condensation under stress condition. Integrative analysis of human PDAC transcriptomic datasets reveals a weak association between NQO1 and G3BP1 levels. Importantly, both NQO1 and G3BP1 formed biological condensates and co-localized in the lesions of human PDAC tissue sections. Our study uncovers a novel KRAS mutation-driven mechanism of pancreatic carcinogenesis through the lens of phase separation, transcending conventional gene expression regulation and offering new insights into non-canonical KRAS-NQO1-G3BP1-SG regulatory networks in PDAC initiation.

cancer biology↗

bioRxiv2025

Deletion of Cd248 in Postn positive myofibroblast Fails to Attenuate Cardiac Remodeling and Fibrosis in a Murine Model of Pressure-Overload Cardiomyopathy

BackgroundCardiac fibrosis, driven by activated Periostin (Postn)-expressing myofibroblasts, is a key pathophysiological feature of heart failure induced by pressure overload. The clinic trial tested tumor vascular regulator CD248 has been validated as a potential anti-fibrotic target in ischemic heart injury. We therefore tested the hypothesis that targeting Cd248 specifically in Postn+ myofibroblasts ameliorates pressure-overload cardiomyopathy. MethodsSingle-cell RNA-sequencing data from murine hearts subjected to transverse aortic constriction (TAC) were analyzed to quantify the Cd248+/Postn+ fibroblast population. Subsequently, Postn+ myofibroblast-specific inducible Cd248 knockout mice were subjected to TAC. Gene deletion was induced with tamoxifen from the day of surgery. Cardiac remodeling and function were analyzed 8 weeks after TAC. ResultsAnalysis of scRNAseq data confirmed that about 1/3 of the fibroblasts in the 14-day TAC heart are Cd248+Postn+ fibroblasts. However, comprehensive echocardiographic analysis revealed no significant differences in key parameters of cardiac function or dimensions, including left ventricular ejection fraction, ventricular volumes, wall thickness and global muscle strain between PostnMCM+/-;Cd248fl/fl and PostnMCM+/- controls. Histological analysis at 8 weeks also showed no significant alteration in the extent of interstitial or perivascular fibrosis, myocardial vessel density, or cardiomyocyte hypertrophy. ConclusionThe specific deletion of Cd248 in Postn-expressing myofibroblasts does not mitigate pathological remodeling or fibrosis in response to chronic pressure overload. These findings suggest the role of Cd248 in this cell lineage is not a primary driver of pressure-overload pathology. Therefore, a better understanding of the context-dependent functions of CD248 is critical to successfully guide the clinical translation of CD248-based anti-fibrotic therapies.

pathology↗

bioRxiv2025

Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T cell therapies

Patients with T-cell lymphomas and leukemias have overall poor outcomes due to the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. Development of chimeric antigen receptor (CAR) T-cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective anti-tumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, using anti-CD2 CAR T-cells engineered without CD2 expression (CART2), would support CAR T-cell manufacturability and preclinical efficacy. Optimized CD2-knockout CART2, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T-cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, co-stimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel PD-1:CD2 switch receptor that rescues intracellular CD2 signaling, particularly when PD-L1 is engaged, resulting in improved in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T-cells and provides an effective strategy to overcome CD2-signaling deficiencies.

cancer biology↗

bioRxiv2025

Rat somatic genome editing enables ER+ breast cancer modeling

Genetically engineered mouse models have advanced cancer research but often fail to capture key features of certain human tumors. Rats, with distinct physiology and tumor biology, offer a powerful alternative, yet their use has been constrained by technical barriers to genome editing. Here, we report efficient somatic genome editing in rats, enabling both Indel and substitution mutations. We then apply this approach to model estrogen receptor (ER)-positive breast cancer, which accounts for [~]70% of human cases but remains poorly represented in mice. The resulting rat tumors reproduce hallmarks of human ER+ breast cancer, including ductal histology, hormone responsiveness, and immune-microenvironmental features. By contrast, identical genetic alterations in mice failed to yield ER+ tumors, underscoring critical species differences in tumorigenesis. Together, this work establishes a versatile platform for rapid generation of clinically relevant rat tumor models, opening new avenues to dissect tumor biology, therapeutic response, and immune interactions in previously inaccessible cancer subtypes.

cancer biology↗

bioRxiv2025

Integrative Computational and Experimental Analysis of Curly Su Mutations in Drosophila melanogaster

The Curly Su (dMPO) protein, a homolog of the human myeloperoxidase (hMPO), is critical for wing development in Drosophila melanogaster. Like human peroxidases, dMPO is involved in various cellular and physiological processes, producing significant quantities of reactive oxygen species that contribute to both development and immunity in the fruit fly. Given the significant sequence and structural similarities between dMPO and hMPO, dMPO serves an ideal model for studying peroxidase functions and related pathologies. We performed saturated computational mutagenesis on dMPO, analyzing the effects of 11,191 missense mutations on its stability. Notably, the G378W mutation exhibited the greatest destabilizing effect, while the W621R, potentially pathogenic, also reduced dMPO stability. To investigate these effects in vivo, we used genome editing to generate the transgenic Drosophila with G378W, W621R, and deletion of residues 305-687. Remarkably, G378W mutants displayed significant alterations in wing morphology and reduced lifespan. RNA-seq analysis of transgenic and wild-type flies revealed differentially expressed genes (DEGs), as interpreted through gene ontology analysis. Our integrated computational and genetic approach identified dMPO mutations that disrupt protein stability and alter gene expression. These findings offer new insight into how single-point mutations can lead to systemic biological changes. Author SummaryProteins are the molecular machines that drive almost every process in living organisms, and even small genetic changes can disrupt their structure and function. In this study, we focused on Curly Su, a protein in fruit flies that is similar to human myeloperoxidase. We used a computational approach called saturation mutagenesis to model over 11,000 possible single mutations in the Curly Su protein and predict how they would affect its stability. We then used genome editing to introduce several destabilizing and potential disease-causing mutations into flies. These mutant flies developed abnormal wings, had shorter lifespans, and showed widespread changes in gene activity, particularly in metabolism and immune pathways. By combining computational predictions with experimental validation, our work demonstrates a powerful and generalizable strategy for linking specific genetic mutations to whole-organism outcomes. This approach not only sheds light on the biological role of Curly Su but also provides a framework that can be applied to other genes and organisms, offering insights relevant to human health and disease.

bioinformatics↗

bioRxiv2025

Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity required to robustly detect short or low-abundance targets, particularly in thick tissue with high autofluorescence. Here, we describe three HCR variants that combine the specificity of HCR v3.0 with additional signal amplification through catalytic reporter deposition (HCR-Cat), immunostaining (HCR-Immuno), or iterative HCR and immunodetection (HCR-Multi). These methods substantially enhance detection sensitivity, enabling robust spatial visualization of low-abundance transcripts, improved performance in challenging tissue environments, and compatibility with fluorescence- and alkaline phosphatase-based chromogenic detection. These methods enable transcript-resolution imaging, even when using a limited number of probes, thereby expanding the range of biological applications accessible to HCR.

neuroscience↗

bioRxiv2025

The role of intrinsically disordered domains in regulating G protein coupled receptor signaling

The 2A adrenergic receptor (2AAR) is a clinically important target for various diseases including hypertension, diabetes and chronic pain. Here, using single-molecule fluorescence resonance energy transfer imaging, we show how agonist-specific activation dynamics in both structured transmembrane domain (TMD) and intrinsically disorders regions (IDRs) of 2AAR lead to diverse signaling profiles. Through seven pairs of strategically designed fluorophore labels, we systematically investigate the real-time conformational changes of 2AAR. Our study reveals unique TM6 dynamics in 2AAR, featured by a high energy barrier for agonist-induced outward movements essential for activation. In contrast, we identify agonist-specific conformational dynamics of a partially disordered extracellular loop (ECL2), highlighting its role as a dynamic regulatory module that controls receptor function. Moreover, we characterize the conformational landscapes of the long third intracellular loop (ICL3), revealing its compact structural features and membrane-proximal localization in the basal state, where it acts as a negative allosteric regulator in transducer coupling. Furthermore, we identify multiful functional sub-states of ICL3 that are dynamically modulated by both kinase phosphorylation and drug efficacy. These findings offer previously underappreciated structural and dynamic insights into 2AAR function governed by both TMD and IDRs, and may open up new avenues for the development of better therapeutics.

biophysics↗

bioRxiv2025

Dysregulation of innate immunity and cellular metabolism through virus-induced deISGylation

Interferon-stimulated gene 15 (ISG15) regulates diverse cellular responses including antiviral immunity through its conjugation to proteins, a process known as ISGylation. Several pathogens, including SARS-CoV-2, subvert ISGylation by encoding deISGylating enzymes. However, the direct targets and physiological consequences of coronaviral deISGylation remain poorly defined. Here, we ablated the deISGylating activity of the SARS-CoV-2 papain-like protease (PLpro) and found that loss of deISGylation boosted innate immune activation, attenuated virus replication, and promoted viral clearance in human cells and in mice. Through untargeted metabolomics and ISGylome proteomics analyses, combined with functional studies, we discovered in molecular detail how the activities of key metabolic enzymes in glycolysis, the pentose phosphate pathway, and oxidative stress are controlled by PLpro deISGylation. These findings provide fundamental new insight into how reversible ISGylation regulates immunity and metabolic processes at the molecular level and highlight viral deISGylation as a major viral tactic for rewiring immunometabolism.

microbiology↗

bioRxiv2025

QuantaMind MD enables protein modeling with ab initio accuracy

Accurate biomolecular simulations are essential for understanding chemical processes and advancing applications such as protein engineering and drug design. While quantum mechanical (QM) methods can provide chemical accuracy, their computational cost limits their scalability. Machine learning force fields (MLFFs) offer a promising alternative by achieving similar accuracy with vastly improved efficiency, but their effectiveness is often constrained by limited conformational and chemical diversity in training. We introduce QuantaMind, a robust MLFF workflow designed to extend chemical and conformational coverage, particularly in transition-state regions. QuantaMind enables quantum-accurate molecular dynamics simulations, successfully capturing complex phenomena such as proton diffusion, water dissociation, and acid-base neutralization. We show that QuantaMind can be applied to large biomolecular systems, enabling accurate protein structure optimization and improving the prediction of residue contacts and hydrogen bonds. A pocket-centric simulation strategy further allows QuantaMind to efficiently model protein-ligand interactions with high structural accuracy. These results establish QuantaMind as a versatile and scalable tool for atomistic simulations at ab initio accuracy.

biochemistry↗

bioRxiv2025

Stress-Responsive Transcriptomic Signatures in Human iPSC-Derived Microglia Reveal Links to Alzheimer's Disease Risk Genes

Commercially available human iPSC lines with inducible CRISPR interference (CRISPRi) systems offer scalable platforms for gene function studies. One widely available line, the AICS-0090 dCas9-KRAB iPSC line developed by the Allen Institute, has been extensively validated for genomic integrity and stem cell potency. However, its utility in modeling specialized immune cell types such as microglia -- and in assessing their functional responses to disease-relevant stimuli -- has not been fully established. Here, we evaluated the AICS-0090 line for its ability to differentiate into microglia-like cells, support efficient gene knockdown, and respond to environmental stressors. We assessed its differentiation capacity by qPCR, flow cytometry, and immunocytochemistry, confirming reproducible expression of microglial surface markers at early and late timepoints. Gene knockdown efficiency was validated both at the single-gene level and in pooled CRISPRi screens. Focusing on functional responsiveness, we exposed the microglia-like cells to two distinct stimuli: amyloid-{beta} (A{beta}), a disease-associated trigger in Alzheimers disease, and lipopolysaccharide (LPS), a classical inflammatory signal. Transcriptomic and functional analyses revealed stimulus-specific responses: A{beta} induced limited activation of stress and inflammation pathways, whereas LPS elicited broader transcriptional reprogramming and cytokine release. Signatures from both conditions partially overlapped with ex vivo human and mouse microglial states. Together, these findings support the use of the AICS-0090 dCas9-KRAB iPSC-derived microglia-like cells as a flexible and tractable model for gene function interrogation under defined inflammatory contexts, with potential for future applications in neuroimmune modeling and perturbation-based screening.

neuroscience↗

bioRxiv2025

GSDME unlocks astrocyte-driven neurotoxicity in Alzheimer's Disease

Astrocytic calcium dysregulation and reactivity precede A{beta} deposition in amyloid-{beta} deposition in Alzheimers disease (AD) but the neurotoxic mechanisms remain unclear. We show that GSDME acts as a switch, linking MAM-mediated calcium release to astrocyte-driven neurotoxicity. Specifically, A{beta}-activated microglial signals activate astrocytic GSDME, releasing its N-terminal fragment, which targets MAMs and triggers ER calcium efflux. This induces biphasic CaMKII phosphorylation, initially boosting NRF2 defenses, then activating NF-{kappa}B-driven inflammation, shifting astrocytes from protective to toxic states. GSDME activation also drives astrocyte-derived exosomes (ADEs) to carry neurotoxic tau, proinflammatory miRNAs, and toxic lipids, propagating toxicity. GSDME deletion in AD mice reduces A{beta} burden, restores NF-{kappa}B/NRF2 balance, reprograms astrocytes and ADEs to protective states, and rescues cognition. Multi-omics profiling of serum ADEs from AD patients reveals a disease-specific signature with central neurotoxicity and peripheral immune regulation. These findings position GSDME as a promising dual diagnostic and therapeutic target for early AD invention.

neuroscience↗

bioRxiv2025

Quantitative Analysis of Meloidogyne incognita Population Density Using Real-Time PCR and Its Correlation with Root-Knot Disease Index in Tabacco (Nicotiana tabacum)

Tobacco root-knot disease represents a significant threat to tobacco production, particularly in the western Henan region, where Meloidogyne incognita is the predominant species. This study collected samples of M. incognita and soil from Luoyang, Henan, and designed specific primers MiF and MiR based on the amplified 735 bp sequence of the ITS1-5.8S-ITS2 region. These primers exhibit mismatches with related species, including M. javanica, M. minor, M. hapla, and M. arenaria, showing 1, 6, 10, and 10 base differences in the forward direction, respectively, 3, 10, and 9 base mismatches in the reverse direction. Although the primers were used to detect corn-wheat soil samples, no amplification was observed. Additionally a real-time quantitative PCR curve for M. incognita in soil was constructed, revealing a negative correlation between the Ct value (Y) and the log-transformed number of nematodes (x) per 20 g of dry soil, represented by the equation y = -0.9757x + 35.565; (R2 = 0.9999, P < 0.01). According to the decomposition efficiency of nematode DNA in soil, the results showed that nematode DNA degrades rapidly in soil, with a degradation rate of approximately 87.3% at 3 days and 99.97% at 14 days. Furthermore, significant differences were observed in the real-time PCR detection efficiency among various nematode forms: the Ct value of J1 was significantly higher than that of J2, while abnormal eggs (empty eggs or internal bubble eggs) exhibited the highest Ct value. The proportion of abnormal eggs in the soil before planting was significantly higher at 64.08% compared to only 15.3% at harvest, indicating that the activity of nematodes in the soil is significantly reduced after harvesting in October and planting in March of the following year. A survey of 126 tobacco plants indicated a significant positive correlation between the root-knot index (RKI) and root-knot nematode density (r = 0.80, p < 0.01). The study identified a minimum disease threshold of 234 individuals / 20 g soil at harvest and revealed a nonlinear relationship between disease severity and nematode density. Specifically, a weak correlation was observed at low density (Low RKI: 0-1; nematode density<2000 individuals/20 g soil, r = 0.49, P<0.05), while a significant correlation was noted at moderate density (High RKI: nematode density>2000 individuals/20 g soil, r = 0.69, P<0.05). This study showed that as the RKI increases, the rate of increase in nematode density in the soil diminishes. These findings provide valuable insights for the development of effective scientific strategies for nematode control.

plant biology↗