Biology subjects

Wan, J.

Publications and source records attributed to Wan, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

A programmable genetic platform for engineering noninvasive biosensors

Creating genetic sensors for noninvasive visualization of biological activities in deep, optically opaque tissues holds immense potential for basic research and the development of genetic and cell-based therapies. MRI stands out among deep-tissue imaging methods for its ability to generate high-resolution images without ionizing radiation. However, the adoption of MRI as a mainstream biomolecular technology has been hindered by the lack of adaptable methods to link molecular events with genetically encodable MRI contrast. To address this challenge, we introduce universal reporter circuit-based activatable sensors (URCAS), a highly programmable platform for the systematic creation of genetic sensors for MRI. In developing URCAS, we engineered protease-activatable MRI reporters using two distinct approaches: protein stabilization and subcellular trafficking. We established the applicability of URCAS in five diverse mammalian cell types and showcased its versatility by assembling a toolkit of genetic sensors for viral proteins, small-molecule drugs, logic gates, protein-protein interactions, and calcium, without requiring new customization for each target. Our findings suggest that URCAS provides a modular, programmable platform for streamlining the development of noninvasive, nonionizing, and genetically encoded sensors for biomedical research and in vivo diagnostics.

bioengineering↗

bioRxiv2025

DeepADR: Multi-modal Prediction of Adverse Drug Reaction Frequency by Integrating Early-Stage Drug Discovery Information via Kolmogorov-Arnold Networks

Adverse drug reactions (ADRs) are a major cause of clinical trial failure and post-market withdrawal, posing significant risks to public health and impeding drug development. While computational methods offer an alternative to costly preclinical testing, existing models often fail with novel compounds by requiring pre-existing information such as drug-ADR associations or by inadequately integrating diverse data sources. Here, we introduce DeepADR, a multi-modal deep learning framework for predicting both the occurrence and frequency of ADRs using early-stage, readily available data. DeepADR integrates chemical structures and biological target profiles with semantic representations of ADR terms derived from a large language model. These heterogeneous parameters are fused using a Kolmogorov-Arnold Network (KAN), which effectively models complex, non-linear cross-modal interactions to capture underlying toxicological mechanisms. Our model outperforms existing methods in predicting both ADR occurrence and frequency, demonstrating robust generalization to new chemical entities. By effectively integrating chemical, biological, and semantic datasets, DeepADR provides a powerful, scalable tool for the early-stage safety assessment and candidate prioritization. This framework not only facilitates the prioritization of safer drug candidates but also offers a methodology for predicting the toxicity of other hazardous materials, holding significant promise for advancing public health.

bioinformatics↗

bioRxiv2025

Global analysis of membrane protein S-acylation in the model plant Arabidopsis thaliana

Protein S-acylation is the addition of fatty acids to the cysteine residues in a protein, catalyzed by protein S-acyltransferases (PATs). Despite extensive research on protein S-acylation in animals, our understanding of this process in plants remains limited. In this study, we sought to characterize the S-acylproteome of membrane proteins in Arabidopsis and identify potential substrates for two important plant immunity-related PATs (PAT5 and PAT9). To achieve this, S-acylated membrane proteins were first enriched via our optimized acyl-biotinyl exchange strategies at both the protein-level and peptide-level. The enriched samples were then analyzed by label-free quantitative liquid chromatography-mass spectrometry. The results from the two enrichment methods demonstrated that they were complementary in identifying S-acylated proteins and S-acylation sites. Using these methods, over 2500 S-acylation sites in more than 2000 putative S-acylated proteins were identified. Proteins involved in vesicle trafficking, plant phosphorylation, immune responses, and signal transduction pathways were significantly enriched. Additionally, certain amino acid patterns surrounding the S-acylation sites were identified. Comparisons of the S-acylproteomes between the wild type and the PAT5 and PAT9 mutants revealed over 100 potential substrates for both S-acyltransferases. The high quality of our data was supported by the significant overlap with the previously reported data and successful experimental verification of selected candidate proteins. Overall, our study revealed a well-represented S-acylproteome for Arabidopsis (especially its membrane proteins) and identified potential substrates for PAT5 and PAT9. These findings will facilitate the functional characterization of S-acylated proteins in plants.

plant biology↗

bioRxiv2025

Identification of senescence-related hub genes and potential therapeutic agents for chronic heart failure

BackgroundChronic heart failure (CHF), the terminal phase of cardiovascular disease progression, has emerged as an increasingly severe global public health concern. Despite current therapeutic approaches aimed at symptom relief, their long-term effectiveness remains limited, urgently necessitating the exploration of novel treatment strategies. This study endeavored to explore the role of cellular senescence in CHF, identify the characteristic genes linked to cellular senescence, and predict potential therapeutic agents. MethodsWe acquired CHF-related datasets from the Gene Expression Omnibus database and cell senescence-related genes from the CellAge database to identify differentially expressed cell senescence-related genes. We then conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses to elucidate the functions of these differentially expressed genes, constructed a protein-protein interaction network, and screened hub genes. Using receiver operating curve (ROC) analysis, we developed a diagnostic model based on these hub genes. Furthermore, we constructed networks for the hub genes involving miRNAs, lncRNAs, transcription factors, and drugs. Subsequently, we explored the potential mechanism of action of metformin in the treatment of CHF through molecular docking studies. Lastly, we verified the expression of the hub genes in a doxorubicin-induced CHF model in rats. Results We ultimately identified nine hub genes associated with cellular senescence: STAT1, MMP9, MAP2K1, SOCS1, SDC1, MET, EIF4EBP1, ATF3, and NAMPT. These genes exhibited significant differential expression between CHF and normal tissues. The constructed diagnostic model demonstrated robust diagnostic performance in ROC curve analysis, with an area under the curve exceeding 0.7, thereby providing biomarker support for early CHF diagnosis. Furthermore, we identified a regulatory network comprising 94 lncRNAs, 63 miRNAs, and 11 transcription factors and screened 42 potential therapeutic drugs. Subsequent molecular docking simulations revealed that metformin could effectively bind to the hub genes. Finally, we validated the expression of some hub genes in a rodent CHF model, in which gene expression regulation varied across diverse experimental settings. ConclusionsOur research identified nine genes cellular senescence-related genes with potential roles in CHF pathogenesis, offering fresh perspectives concerning the diagnosis and treatment of CHF.

bioinformatics↗

bioRxiv2025

The longevity effects of reduced IGF-1 signaling depend on the stability of the mitochondrial genome

Suppression of insulin-like growth factor-1 (IGF-1) signaling extends mammalian lifespan and protects against a range of age-related diseases. Surprisingly though, we found that reduced IGF-1 signaling fails to extend the lifespan of mitochondrial mutator mice. Accordingly, most of the longevity pathways that are normally initiated by IGF-1 suppression were either blocked or blunted in the mutator mice. These observations suggest that the pro-longevity effects of IGF-1 suppression critically depend on the integrity of the mitochondrial genome and that mitochondrial mutations may impose a hard limit on mammalian lifespan. Together, these findings deepen our understanding of the interactions between the hallmarks of aging and underscore the need for interventions that preserve the integrity of the mitochondrial genome.

molecular biology↗

bioRxiv2025

RABEP1 amplifies front signaling in neutrophil migration

Neutrophils are the first responders of our innate immune system, crucial for defense against various infections. The intricate regulation of neutrophil migration is essential for neutrophil function. However, a complete mechanistic understanding is missing. We performed a miRNA overexpression screen and identified miR-190 as a potent suppressor of neutrophil migration in zebrafish. Through a second round of small-scale screening using neutrophil-specific knockouts of putative miR-190 targets, we identified that rabep1 (Rabaptin-5, RAB GTPase binding effector protein 1) is essential for neutrophil motility and chemotaxis in zebrafish. Re-expressing full-length RABEP1 in the knockout, but not a truncated form lacking the Rab4/Rab5 binding domain, rescued cell motility. Knocking down RABEP1 in human dHL-60 cells consistently reduced cell motility. RABEP1-deficient dHL-60 cells accumulate excessive members inside the cells. The Rab5 GTP level is unaffected, but the RABEP1 knockdown cells displayed reduced PAK phosphorylation and overall reduced actin polymerization, but still appropriately polarized upon chemokine stimulation. Overexpression of dominant-negative Rab4 or Rab5 similarly inhibits neutrophil migration. Our data suggests that RABEP1 drives endosomal recycling, Rac activation, and leading-edge actin polymerization, providing significant insights into the role of the endocytic pathway in neutrophil motility.

cell biology↗

bioRxiv2025

Improved Injury Detection Through Harmonizing Multi-Site Neuroimaging Data after Experimental TBI: A Translational Outcomes Project in NeuroTrauma (TOP-NT) Consortium Study

Multi-site neuroimaging studies have become increasingly common in order to generate larger samples of reproducible data to answer questions associated with smaller effect sizes. The data harmonization model NeuroCombat has been shown to remove site effects introduced by differences in site-related technical variance while maintaining group differences, yet its effect on improving statistical power in pre-clinical models of CNS disease is unclear. The present study examined fractional anisotropy data computed from diffusion weighted imaging data at 3 and 30 days post-controlled cortical impact injury from 184 adult rats across four sites as part of the Translational-Outcome-Project-in-Neurotrauma (TOP-NT) Consortium. Findings confirmed prior clinical reports that NeuroCombat fails to remove site effects in data containing a high proportion-of-outliers (>5%) and skewness, which introduced significant variation in non-outlier sites. After removal of one outlier site and harmonization using a global sham population, harmonization displayed an increase in effect size in data that displayed group level effects (p<0.01) in both univariate and voxel-level volumes of pathology. This was characterized by movement toward similar distributions in voxel measurements (Kolmogorov-Smirnov p<<0.001 to >0.01) and statistical power increases within the ipsilateral cortex. Harmonization improved statistical power and frequency of significant differences in areas with existing group differences, thus improving the ability to detect regions affected by injury rather than by other confounds. These findings indicate the utility of NeuroCombat in reproducible data collection, where biological differences can be accurately revealed to allow for greater reliability in multi-site neuroimaging studies. Significance StatementThis project demonstrates the utility of NeuroCombat in reducing site effects in multi-site rodent imaging. We also demonstrate that harmonization improves the ability to distinguish between sham and injured rats at the voxel level and increase statistical power and effect size in areas of injury. Multi-center studies are becoming more common to allow for increased efficiency in data collection, and with conservative approaches and analysis into the datasets, NeuroCombat can be utilized to improve study reliability and reproducibility.

neuroscience↗

bioRxiv2025

Chemical inhibition of RPA gap protection sensitizes BRCA1-deficient cancers to PARP inhibition

Poly (ADP-ribose) polymerase inhibitors (PARPi) are standard of care for many BRCA1 deficient cancers, though few cures are achieved. We sought to determine if targeting the protection of the single-strand DNA gaps induced by PARPi in BRCA-deficient cancers could increase efficacy. Replication protein A (RPA) participates in critical protein-protein and protein-DNA interactions to protect single-stranded DNA (ssDNA) and support DNA metabolism. We have reported the optimization of small molecule RPA inhibitors (RPAi) that target protein-ssDNA interactions to chemically exhaust RPA and elicit single-agent anticancer activity. RPAi sensitizes cells to PARPi in BRCA1-deficient non-cancerous cells, where ssDNA gap formation drives therapeutic efficacy. We show that RPAi treatment abolishes PARPi-induced replication gap protection, resulting in genomic instability via replication fork degradation and chromosomal integrity, and that, in vivo, the RPA and PARP targeted combination abrogates cancer growth in a BRCA1-mutant breast cancer model. We find that genetic predispositions to ssDNA gap accumulation correlate with RPAi sensitivity, and BRCA1-proficient cells remain sensitive to combination treatment but require more PARP inhibition to increase ssDNA gaps. RPAi-PARPi combination activity in patient-derived ovarian cancer models demonstrates the utility of targeting gap protection to increase PARPi sensitivity and circumvent resistance. Collectively, this work provides a unifying mechanism of chemical RPA exhaustion as a cancer therapeutic strategy. One Sentence SummaryInhibition of single-strand DNA gap protection potentiates PARP targeted treatment of BRCA1 deficient cancers.

cancer biology↗

bioRxiv2025

DiCE: Differential Centrality-Ensemble Analysis Based on Gene Expression Profiles and Protein-Protein Interaction Network

Uncovering key genes that drive diseases and cancers is crucial for advancing understanding and developing targeted therapies. Traditional differential expression analysis often relies on arbitrary cutoffs, missing critical genes with subtle expression changes. Some methods incorporate protein-protein interactions (PPIs) but depend on prior disease knowledge. To address these challenges, we developed DiCE (Differential Centrality-Ensemble), a novel approach that combines differential expression with network centrality analysis, independent of prior disease annotations. DiCE identifies candidate genes, refines them with an information gain filter, and reconstructs a condition-specific weighted PPI network. Using centrality measures, DiCE ranks genes based on expression shifts and network influence. Validated on prostate cancer datasets, DiCE identified genes over-represented in key pathways and cancer fitness genes, significantly correlating with disease-free survival (DFS), despite DFS not being used in selection. DiCE offers a comprehensive, unbiased approach to identifying disease-associated genes, advancing biomarker discovery and therapeutic development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/638654v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1ce9b81org.highwire.dtl.DTLVardef@14352e1org.highwire.dtl.DTLVardef@f63caeorg.highwire.dtl.DTLVardef@a0086_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Two-Stage CD8+ CAR T-Cell Differentiation in Patients with Large B-Cell Lymphoma

Chimeric antigen receptor (CAR) T-cell therapy has expanded therapeutic options for patients with diffuse large B-cell lymphoma (DLBCL). However, progress in improving clinical outcomes has been limited by an incomplete understanding of CAR T-cell differentiation in patients. To comprehensively investigate CAR T-cell differentiation in vivo, we performed single-cell, multi-modal, and longitudinal analyses of CD28-costimulated CAR T cells from infusion product and peripheral blood (day 8-28) of patients with DLBCL who were successfully treated with axicabtagene ciloleucel. Here, we show that CD8+ CAR T cells undergo two distinct waves of clonal expansion. The first wave is dominated by CAR T cells with an exhausted-like effector memory phenotype during the peak expansion period (day 8-14). The second wave is dominated by CAR T cells with a terminal effector phenotype during the post-peak persistence period (day 21-28). Importantly, the two waves have distinct ontogeny and are biologically uncoupled. Furthermore, lineage tracing analysis via each CAR T cells endogenous TCR clonotype demonstrates that the two waves originate from different effector precursors in the infusion product. Precursors of the first wave exhibit more effector-like signatures, whereas precursors of the second wave exhibit more stem-like signatures. These findings suggest that pre-infusion heterogeneity mediates the two waves of in vivo clonal expansion. Our findings provide evidence against the intuitive idea that the post-peak contraction in CAR abundance is solely apoptosis or extravasation of short-lived CAR T cells from peak expansion. Rather, our findings demonstrate that CAR T-cell expansion and persistence are mediated by clonally, phenotypically, and ontogenically distinct CAR T-cell populations that serve complementary clinical purposes.

immunology↗

bioRxiv2025

Building an atlas of mechanobiology: high-throughput contractility screen of 2418 kinase inhibitors in five primary human cell types reveals selective divergent responses among related cell types

Cellular mechanical forces play crucial roles in both normal physiology and disease, yet drug discovery efforts targeting mechanobiology have been limited in part by assumptions about the conservation of contractile pathways across cell types. Here, we present the first high-throughput contractility screen of an annotated kinase inhibitor library, evaluating 2,418 compounds across five primary human cell types using the FLECS (Fluorescent Elastomer Contractility Sensors) platform. Quantification of contractile responses revealed selective divergent responses among related cell types. Clustering analysis identified distinct mechanobiological profiles and novel pathway associations that challenge the assumption that contractile pathways are too highly conserved for selective targeting. This systematic approach supports wider adoption of mechanical phenotypic screening as a viable strategy for discovering cell-type specific contractile pathway modulators for a broad range of mechanically-driven disease indications.

bioengineering↗

bioRxiv2025

WormID-Benchmark: Extracting Whole-Brain Neural Dynamics of C. elegans At the Neuron Resolution

The nematode C. elegans is a premier model organism for studying neural circuit function due to its fully mapped connectome and genetically identifiable neurons. Recent advances in 3D light microscopy and fluorescent protein tagging have enabled whole-brain imaging at single-neuron resolution. However, extracting meaningful neural dynamics from these high-resolution recordings requires addressing three fundamental challenges: (i) accurate detection of individual neurons in fluorescence images, (ii) precise identification of neuron classes based on anatomical and colorimetric cues, and (iii) robust tracking of neurons over time in calcium imaging videos. To systematically evaluate these challenges, we introduce WormID-Bench, a large-scale, multi-laboratory dataset comprising 118 worms from five distinct research groups, along with standardized evaluation metrics for detection, identification, and tracking. Our benchmark reveals that existing computational approaches show substantial room for improvement in sensitivity, specificity, and generalization across diverse experimental conditions. By providing an open and reproducible benchmarking framework1, WormID-Bench aims to accelerate the development of high-throughput and scalable computational tools for whole-brain neural dynamics extraction in C. elegans, setting the stage for broader advancements in functional connectomics.

neuroscience↗

bioRxiv2024

Insights into Intestinal Barrier Disruption During Long-Term Gut Chlamydia Colonization in Mice: A Single-Cell Transcriptomic Approach

Chlamydia trachomatis, an intracellular pathogen, stands as the most prevalent sexually transmitted bacterial infection among women globally. Traditionally recognized as a genital pathogen, recent research indicates that the gastrointestinal tract may also act as a reservoir for its long-term colonization. However, the mechanisms underlying Chlamydias ability to persist in the gut remain poorly understood. This gap in knowledge limits our ability to develop effective treatments for persistent Chlamydia infections. In this study we utilized single-cell RNA sequencing to analyze the gene expression profiles and cellular heterogeneity of mouse colonic tissues during Chlamydia long-term infection. This approach provided detailed insights into the transcriptional changes and cellular interactions involved in the persistence of Chlamydia in the gut. Our results revealed significant alterations in the gene expression profiles of various intestinal cell populations, with distinct molecular pathways contributing to Chlamydia persistence. Notably, we observed a reduction in the expression of markers associated with epithelial tight junctions, indicating a potential breakdown of the intestinal epithelial barrier. This impairment may facilitate the penetration of Chlamydia into deeper tissues and contribute to the initiation of infection. We also found dysregulation of the transcriptional networks in goblet cells and an imbalance in communication between immune and epithelial cells. These disruptions were linked to the pathogens ability to establish persistent colonization and infection. IMPORTANCEFew studies have explored Chlamydia persistence in the gastrointestinal tract. In this study, we use single-cell RNA sequencing to identify the molecular and cellular mechanisms driving the pathogens long-term colonization. Our findings provide crucial insights into how Chlamydia overcomes the hosts immune defenses and epithelial barriers to establish chronic infection in the gut. Notably, we identify disruption of epithelial tight junctions and an imbalance in immune-cell interactions, offering new avenues for therapeutic interventions aimed at restoring mucosal integrity and preventing persistent infection.

microbiology↗

bioRxiv2024

Epromoters bind key stress-related transcription factors to regulate clusters of stress response genes

Stress insults trigger the rapid and global reprogramming of gene transcription by coordinated recruitment of a limited number of key inducible transcription factors to cis-regulatory elements. Here, we performed a comprehensive analysis of different stress models and observed that co-induced genes are generally located in close genomic proximity. By integrating gene expression and transcription factor binding resources in different stress models, we found an enrichment for clusters whereby only one of the promoters of the cluster recruits the key transcription factors, reminiscent of Epromoters, a type of cis-regulatory elements displaying both promoter and enhancer function. Epromoter-regulated clusters were frequently found irrespectively of the stress or inflammatory response. Predicted Epromoters displayed enhancer activity and regulated clusters of stress-response genes independently of their genomic location. These findings have significant implications for understanding complex gene regulation following the response to acute perturbations. TeaserWhen cells face stress, they undergo rapid changes in gene expression, orchestrated by a handful of key transcription factors. But how do these factors coordinate such a complex response? Our study reveals that "Epromoters"--cis-regulatory elements that combine the functions of both promoters and enhancers help organize stress-response genes into tightly regulated clusters. This discovery not only deepens our understanding of gene regulation in the face of stress but also offers exciting implications for studying inflammation and other acute cellular responses.

genomics↗

bioRxiv2024

Impact of oral Chlamydia Vaccination on Host Gut Microbiome and Metabolites Composition

Chlamydia trachomatis, an intracellular pathogen, is recognized as the most common sexually transmitted bacterial infection among women worldwide. Chlamydia infections can lead to undesirable clinical outcomes, including pelvic inflammatory disease and infertility. Recently, the gut has been identified as a niche for Chlamydia colonization; however, despite the biological impact on the host remaining under investigation, oral inoculation of Chlamydia as a whole-organism vaccine has been reported as a promising strategy for preventing genital Chlamydia infections. Few studies have evaluated the impact of oral Chlamydia vaccination on the gut microbiome and metabolite changes. In this study, we assessed time-series alterations in the gut microbiome and metabolites following oral Chlamydia inoculation, and we analyzed the composition and correlation between serum immune parameters and the sequencing profiles in the host. We identified 129 microbial changes and 186 significantly different metabolites in the gut across various vaccination approaches during the 30-day immunization process. Additionally, we discussed potential biomarkers of effective immunization based on correlation analysis. IMPORTANCEChlamydia infections primarily lead to morbidity rather than mortality. Consequently, in developing and implementing a Chlamydia vaccine, the utmost priority is ensuring safety. As a promising yet controversial approach, live oral vaccination for Chlamydia raises concerns regarding its impact on the hosts gut environment. Our study not only investigates changes in the gut microbiome and metabolites during vaccination but also identifies potential biomarkers during immunization. These findings are crucial for the development of whole-organism oral Chlamydia vaccines and provide valuable insights into the long-term colonization of Chlamydia in the gut.

microbiology↗

bioRxiv2024

High-throughput development and characterization of new functional nanobodies for gene regulation and epigenetic control in human cells

Controlling gene expression and chromatin state via the recruitment of transcriptional effector proteins to specific genetic loci has advanced the potential of mammalian synthetic biology, but is still hindered by the challenge of delivering large chromatin regulators. Here, we develop a new method for generating small nanobodies against human chromatin regulators that can repress or activate gene expression. We start with a large and diverse nanobody library and perform enrichment against chromatin regulatory complexes using yeast display, followed by high-throughput pooled selection for transcriptional control when recruited to a reporter in human cells. This workflow allows us to efficiently select tens of functional nanobodies that can act as transcriptional repressors or activators in human cells.

synthetic biology↗

bioRxiv2024

Effect of thermoneutral housing on MASLD severity, hepatic gene expression, and BAT activation during beta3-adrenergic stimulation in mice

Metabolic dysfunction-associated steatotic liver disease (MASLD), and its more advanced stage metabolic dysfunction-associated steatohepatitis, is the most common chronic liver disease, constituting a major public health issue. No medication is approved for MASLD treatment, and relevant preclinical models are needed to define molecular mechanisms underlying MASLD pathogenesis, and evaluate therapeutic approaches. Here we demonstrated that compared to standard temperature housing, thermoneutral housing aggravated western diet (WD)-induced obesity, diabetes, and steatosis in male mice, which was associated with increased hepatic expression of inflammation- and fibrosis-related genes. Accordingly, compared to standard-housed mice, thermoneutral-housed WD-fed mice developed more severe hepatic inflammation and fibrosis. The liver is the central metabolic organ in whole-body metabolic homeostasis. We used thermoneutrally housed mice with WD-induced MASLD to examine the effect of MASLD during {beta}3- adrenergic stimulation, and found that diet-induced MASLD was associated with defective inter- organ metabolic cross-talk, leading to impaired brown adipose tissue activation. HighlightsO_LIThermoneutral housing promotes WD-induced obesity and MASLD in mice C_LIO_LIThermoneutral housing fosters WD-induced change in gene expression C_LIO_LIThermoneutral housing fosters hepatic inflammation and fibrosis C_LIO_LIMASLD is associated with defective BAT response to {beta}3-adrenergic stimulation C_LI

physiology↗

bioRxiv2024

Universal function of WRI1 involved in integrated regulation of multiple latex metabolism pathways in Natural Rubber

Hevea brasiliensis and Taraxacum kok-saghyz are sources of nature rubber (NR) and recent research has focused on finding a method to increase NR quality and production. NR consists mainly of cis-1,4-polyisoprene, which is synthesized from sucrose by a series of reactions during carbon metabolism. WRINKLED1 (WRI1) is a transcription factor (TF) that coordinates many genes involved in carbon metabolism and lipid biosynthesis. Here, we isolated and characterized two orthologues of WRI in rubber-producing plants H. brasiliensisa and T. kok-saghyz, which are highly expressed in their latex. Subcellular localization and ectopic expression in Arabidopsis thaliana indicated that the TFs, HbWRI1 and TkWRI1, are involved in lipid accumulation. Overexpression of HbWRI1 and TkWRI1 in T. kok-saghyz substantially enhanced NR quality and production, including dry rubber content, molecular weight, and the diameter of rubber particles in the latex. Conversely, they were decreased in TkWRI1 repression. Furthermore, based on the activated expression in transgenic T. kok-saghyz latex and the existence of AW-box element in the promoter regions, 16 direct downstream genes of HbWRI1 and TkWRI1 were identified by a dual-luciferase reporter assay and yeast one-hybrid assay, and their products were responsible for both NR biosynthesis and lipid metabolism. These results reveal a regulatory module that HbWRI1 and TkWRI1 TFs can positively regulate NR quality and production in T. kok-saghyz latex by synergistically regulating the entire NR biosynthesis. This module is expected to be utilized to develop superior varieties to enhance NR quality and production.

biochemistry↗