Biology subjects

Wu, C.-H.

Publications and source records attributed to Wu, C.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

A broad-host-range Rhizobium rhizogenes strain enables transient expression across diverse crops and establishes functional assays in faba bean

Agrobacterium-mediated transient expression has revolutionized plant research, enabling numerous landmark discoveries across diverse areas of plant biology. Yet this powerful approach remains largely confined to solanaceous species, leaving most economically important crop families without a comparable rapid assay platform. Here, we show that an engineered Rhizobium rhizogenes strain, AS109, mediates efficient transient expression across diverse dicot species spanning multiple taxonomic families, consistently outperforming commonly used laboratory agrobacterial strains. Leveraging the broad host range of AS109, we establish a suite of functional assays in faba bean (Vicia faba), including protein localisation, RNA interference-mediated gene silencing, cell-surface elicitor recognition screens, nucleotide-binding leucine-rich repeat receptor (NLR) activation, and infection cell biology at the host-pathogen interface. We further demonstrate that both singleton NLRs and sensor-helper NLR pairs from Solanaceae retain effector recognition and cell death activity when transferred into faba bean, establishing a rapid platform for evaluating cross-family transferability of disease-resistance genes. AS109 thus provides an accessible and versatile chassis for functional genomics in non-model crops, bridging the widening gap between hypothesis generation and experimental validation across diverse plant species.

plant biology↗

bioRxiv2026

Protein domain characterization reveals human MIC60 tolerates loss of helical bundle domain

The mitochondrial contact site and cristae organizing system (MICOS) is essential for cristae junction formation and inner mitochondrial membrane architecture. To define how MICOS integrity is established and maintained, we generated conditional deletion models of Immt (encoding MIC60), a core MICOS subunit, in tissue-specific settings and in cultured cells. Liver-specific deletion of Immt in mice induced profound defects in mitochondrial ultrastructure and function, establishing MIC60 as essential for mitochondrial integrity. Notably, despite the severity of the defects, we did not detect increased apoptosis in liver tissue or in cells. To directly link MIC60 structure to its function, we performed a systemic structure-function analysis of human MIC60 using domain-specific deletion mutants expressed in Immt-deleted cells. We identified that the transmembrane, coiled-coil, and mitofilin domains are required for MICOS assembly, mitochondrial morphology, and respiratory function. Unexpectedly, deletion of the predicted helical bundle (a region spanning 229 amino acids) substantially restored mitochondrial structure and function, nearly matching full-length MIC60. A mutation (K299E) associated with human disease within this domain similarly preserved most MIC60-dependent functions. Together, these results establish MIC60 as a non-redundant regulator of mitochondrial architecture while revealing that a large predicted structural domain is largely dispensable for MIC60s core functions, refining current models of MICOS organization and uncovering unexpected modularity within MIC60.

cell biology↗

bioRxiv2026

Crop-OCT: a Fully Integrated Imageomics Pipeline to Identify Regional and Focal Retinopathy in Murine Models

Imageomics uses machine learning to accelerate our understanding of biological traits and human disease processes. Some of the earliest imageomics applications used deep learning to assess human diseases. For example, retinal fundus images were analyzed to diagnose diabetic retinopathy. The imaging modality optical coherence tomography (OCT) is widely used to diagnose and monitor the progression of retinopathy in patients and preclinical models. The standardized instrumentation and image format of OCT lends itself to imageomics, but generalizable, automated pipelines for segmentation and quantitation of large numbers of OCT images are still in early development. Here, we present the automated, end-to-end pipeline Crop-OCT that extracts features from thousands of OCT images, while preserving their location within the eye. We used the Crop-OCT pipeline on a diverse dataset, including 13 genetic models of retinopathy, with more than 20,000 OCT images, which allowed us to analyze nearly 6 million measured features. The pipeline was generalized on an independent dataset that was analyzed in a blinded manner. The pipeline enabled us to monitor ocular changes associated with aging and progression of diseases, such as retinitis pigmentosa, Leber congenital amaurosis, achromatopsia, Stargardt disease, diabetic retinopathy, and age-related macular degeneration. We also characterized heterogeneity across animals and identified regional and focal lesions. Our pipeline will unify feature extraction for preclinical models of retinal disease and serve as a foundation for future multimodal data integration for artificial intelligence applications based on imageomics.

neuroscience↗

bioRxiv2025

Antimetastatic Sulfonate-Functionalized Mesoporous Silica Nanoparticles Enhance Irinotecan Stability and Delivery for Colorectal Cancer Treatment

Metastatic colorectal cancer (mCRC) remains a leading cause of cancer-related mortality, with Irinotecan (IRI) serving as a backbone chemotherapeutic despite its dose-limiting toxicities, instability of the active lactone form, and lack of intrinsic antimetastatic activity. To overcome these barriers, we developed sulfonate-functionalized mesoporous silica nanoparticles (MSNs), referred to as (SO3-)-MSN-PEG/TA, as a multifunctional nanocarrier for IRI delivery. This nanoformulation markedly enhanced drug loading efficiency and preserved over 90% of the lactone form for up to six months, enabling sustained release and improved pharmacological stability. In vitro studies demonstrated superior cellular uptake, enhanced apoptosis, and a reduced IC50 compared to free IRI. Beyond drug delivery, (SO3-)-MSN-PEG/TA exhibited intrinsic antimetastatic activity by modulating focal adhesion kinase (FAK)/paxillin signaling, thereby impairing cell migration and suppressing angiogenesis, along with efficient tumor accumulation through the enhanced permeability and retention (EPR) effect. Pharmacokinetic analysis further revealed that IRI@(SO3-)-MSN-PEG/TA prolonged systemic retention, maintaining higher IRI plasma concentrations compared with free IRI. IRI@(SO3-)-MSN-PEG/TA significantly inhibited both primary tumor growth and metastatic dissemination in orthotopic and heterotopic colorectal cancer models, while markedly reducing systemic toxicities and preserving bone marrow cellularity relative to free IRI and liposomal IRI (Onivyde). Collectively, this dual-functional nanomedicine provides an innovative therapeutic strategy that not only augments IRI efficacy but also confers metastasis suppression and favorable pharmacokinetics, addressing critical unmet needs in mCRC treatment. These findings highlight the translational potential of IRI@(SO3-)-MSN-PEG/TA as a safer and more effective therapy for mCRC.

bioengineering↗

bioRxiv2025

Glutamine-Dependent Slc25a39-Nrf2 Axis Couples Mitochondrial Dynamics with Metabolic Reprogramming to Establish Myogenic Commitment

Myogenic commitment is a decisive and irreversible step in skeletal muscle regeneration, necessitating proliferating myoblasts to integrate metabolic cues with nuclear transcriptional programs. Among amino acids, glutamine is uniquely positioned to influence this transition by coupling energy production to macromolecule biosynthesis and epigenetic regulation. We reasoned that myoblasts must sense glutamine availability to ensure orderly progression toward commitment, and we tested this by examining the molecular consequences of acute glutamine withdrawal. We find that continued glutamine oxidation is required to sustain glycolysis, maintain mitochondrial fission, and preserve a redox balance that supports progression towards myogenic commitment. In its absence, myoblasts undergo a reductive shift, characterized by mitochondrial elongation, membrane depolarization, and suppression of glycolysis, ultimately leading to growth arrest. Transcriptomic profiling reveals reduced MyoD and MKi67, accompanied by increased Sprouty1 levels, defining a reversible non-proliferative state that resembles but is distinct from quiescent and reserve cells. We term this state Poised Metabolic Arrest (PMA), a cellular response to glutamine limitation during myogenic progression. Mechanistically, PMA is driven by Nrf2-dependent increased glutathione (GSH) biosynthesis and upregulation of mitochondrial GSH carrier Slc25a39 when glutamine is limited. Depleting mitochondrial glutathione or silencing Slc25a39 forces exit from PMA. However, this premature exit compromises subsequent differentiation potential, indicating PMA serves to preserve differentiation competence when glutamine is limited. Consistent with this, both loss and overexpression of Slc25a39 impair myoblast differentiation in vitro and disrupt regeneration in vivo. Together, these data suggest that a reciprocal Slc25a39-Nrf2 redox axis functions as a nutrient-dependent checkpoint, coupling glutamine availability to mitochondrial remodeling and metabolic reprogramming, necessary to establish irreversible myogenic commitment.

biochemistry↗

bioRxiv2025

Learning-Associated Flexibility of Cortical Taste Coding Is Impaired in Shank3 Knockout Mice

The ability to update the valence of sensory perception to influence behavior is crucial for survival. A common phenotype in autism spectrum disorders (ASDs) is defects in sensory processing, but whether these defects impair flexible sensory encoding is largely unexplored. In particular, how genetic risk factors such as Shank3 deletion affect the adaptability of cortical taste processing and downstream behavior is unknown. To address this gap, we performed two-photon calcium imaging during a conditioned taste aversion (CTA) learning paradigm, an ethologically relevant form of associative learning that depends on taste processing in the gustatory cortex (GC), to examine how Shank3 knockout alters taste-related neuronal activity in GC and influences CTA learning. We found that GC neurons in Shank3 knockout mice exhibited reduced stimulus-evoked suppression and increased trial-to-trial correlated variability during the acquisition of CTA memory. These activity changes, which likely reduced signal-to- noise ratio in GC, were associated with slower CTA acquisition in knockout mice. In both genotypes, CTA learning enhanced, while subsequent extinction reduced, taste discriminability in GC, and both extinction and the associated reduction in discriminability were faster in knockout than in wild-type mice. Together, these results show that Shank3 loss is associated with destabilized cortical activity dynamics in GC, which may contribute to inefficient encoding and maintenance of learned taste aversion. These findings show that loss of Shank3 compromises the ability of animals to update behavior to incorporate negative outcomes, and suggest this loss of flexibility may be an important feature of monogenic ASDs.

neuroscience↗

bioRxiv2025

Wntless interacts with Notch signaling to balance the generation of neurons and gliocytes in vertebrate dorsal diencephalon

Wnt chaperon Wntless (Wls) mediates the intracellular transport of Wnts and plays important roles in early vertebrate brain development. Spatially restricted induction of Wls denotes the earliest differentiation of non-telencephalic cells in human brain organoids. In zebrafish developing diencephalon, loss-of-Wls reduces the formation of habenula (HA) neurons but how Wls influences HA neurogenesis is unclear and whether Wls regulates gliocyte development is unknown. Here we report that the formations of cholinergic, substance P-ergic or glutamatergic neurons in HA are reduced differentially but the generation of gliocyte-derived choroid plexus (ChP) epithelia is increased in wls null mutants. At earlier stage, three-dimensional gene expression analyses revealed that while neurog1 expressions in HA progenitor zones are reduced, the expressions of Notch downstream effector her6 are increased and expanded into HA progenitor zones in wls mutants. Over-expressing Her6 in neurog1-positive cells reduced neurog1 expressions in HA progenitor zones. These results indicate that Wls restricts the expressions of Notch effector her6 to promote the specification of neurog1 proneurons and demotes the generation of ChP epithelia in zebrafish embryonic dorsal diencephalon.

developmental biology↗

bioRxiv2025

A plant pathogen effector blocks stepwise assembly of a helper NLR resistosome

Helper NLRs function as central nodes in plant immune networks. Upon activation, they oligomerize into inflammasome-like resistosomes to initiate immune signaling, yet the dynamics of resistosome assembly remain poorly understood. Here, we show that the virulence effector AVRcap1b from the Irish potato famine pathogen Phytophthora infestans suppresses immune activation by directly engaging oligomerization intermediates of the tomato helper NLR SlNRC3. Cryo-EM structures of SlNRC3 in AVRcap1b-bound and unbound states reveal that AVRcap1b bridges multiple protomers, stabilizing a stalled intermediate and preventing formation of a functional resistosome. Leveraging AVRcap1b as a molecular tool, we also capture an additional SlNRC3 resistosome intermediate showing that assembly proceeds in a stepwise manner from dissociated monomers. These findings uncover a previously unrecognized vulnerability in NLR activation and reveal a pathogen strategy that disrupts immune complex assembly. This work advances mechanistic understanding of resistosome formation and uncovers a previously unrecognized facet of pathogen-plant coevolution.

plant biology↗

bioRxiv2025

Virus-induced upregulation of mitochondrial metabolism modulates cytosolic redox balance and defense responses

Plants possess a remarkable capacity to reprogram their metabolism in response to pathogen attacks. However, the mechanisms by which metabolic reprogramming modulates defense signaling remain poorly understood. In this study, we leverage a multifaceted omics approach to investigate the metabolic shifts induced by the Bamboo mosaic virus (BaMV), a positive-sense single-stranded RNA virus that depends on host factors from multiple organelles for its replication. Metabolic profiling revealed an accumulation of hexose phosphates and Krebs cycle intermediates in Nicotiana benthamiana plants following BaMV infection. Fluxomic analysis uncovered an orchestrated redirection of metabolic flux toward glycolysis and the Krebs cycle during infection. Proteomic data further highlighted a concerted upregulation of mitochondrial enzymes, with three mitochondrial proteins showing markedly increased accumulation in BaMV-infected tissues. These integrated omics results suggest that BaMV infection triggers a metabolic shift toward energy-generating pathways. Notably, functional analysis revealed that silencing mitochondrial NAD+-dependent malic enzyme 1 significantly enhanced BaMV accumulation, accompanied by alterations in cytoplasmic NADH-to-NAD+ ratio and changes in the landscape of defense gene expression. Collectively, our findings underscore the pivotal role of mitochondrial metabolism in governing cytoplasmic redox balance, finely tuning defense responses to viral infection. One sentence summaryBaMV infection enhances mitochondrial metabolism to regulate cytoplasmic redox balance and promote antiviral defense.

plant biology↗

bioRxiv2025

Single-cell-resolved calcium and organelle dynamics in resistosome-mediated cell death

Plant nucleotide-binding domain leucine-rich repeat-containing (NLR) proteins act as intracellular immune receptors that assemble into resistosomes to execute immune responses. However, the subcellular processes during cell death following resistosome activation remain unclear. Here, we visualized the changes in calcium signaling and organelle behavior after activation of the NRC4 (NLR-required for cell death 4) resistosome. We found that NRC4 membrane enrichment coincided with calcium influx. This is followed by sequential mitochondria and plastid disruption, endoplasmic reticulum fragmentation and cytoskeleton depolymerization. Subsequent loss of plasma membrane integrity, nuclear shrinkage, and vacuolar collapse mark the terminal stage of cell death. Our findings reveal a spatiotemporally-resolved cascade of subcellular events downstream of resistosome activation, providing new mechanistic insight into the execution phase of plant immune cell death.

plant biology↗

bioRxiv2025

Development and In Vivo Evaluation of a Novel Bioabsorbable Polylactic Acid Middle Ear Ventilation Tube

BackgroundOtitis media with effusion (OME) is a widespread condition causing hearing impairment, particularly in pediatric populations. Existing non-absorbable middle ear ventilation tubes frequently necessitate secondary surgical removal. Bioabsorbable polylactic acid (PLA) offers a promising alternative due to its inherent biocompatibility and tunable degradation characteristics. This study aimed to design, fabricate, and comprehensively evaluate a novel PLA middle ear ventilation tube. MethodsBioabsorbable PLA tubes were designed and fabricated based on commercial models. In vitro biocompatibility was assessed according to ISO 10993 guidelines. In vivo evaluations were performed in a guinea pig model, including otoscopic examinations, auditory brainstem response (ABR) measurements, micro-computed tomography (micro-CT) imaging, and histological analyses. ResultsThe PLA tubes were successfully designed and fabricated, exhibiting dimensions comparable to commercially available products. In vitro testing confirmed their biocompatibility. In vivo observations demonstrated that the PLA segments remained stable, with no significant inflammation detected. ABR measurements revealed no adverse impact on hearing function. Micro-CT imaging confirmed tube integrity and displayed initial signs of degradation over a 9-month period. Histological analyses indicated a favorable tissue response with minimal foreign body reaction. ConclusionThe developed PLA middle ear ventilation tube represents a highly promising alternative to conventional non-absorbable tubes. It demonstrates excellent biocompatibility, preserves auditory function, and exhibits a controlled degradation profile. This preclinical study provides strong support for further investigation and subsequent clinical trials to validate its safety and efficacy in human patients.

bioengineering↗

bioRxiv2025

Comparative Transcriptomics Reveals Context- and Strain-Specific Regulatory Programs of Agrobacterium During Plant Colonization

Agrobacterium is a genus of plant-associated bacteria capable of transferring DNA into host genomes to induce tumorigenesis. The process has been primarily studied in a few model strains, particularly C58, and developed into Agrobacterium-mediated transformation (AMT) for genetic manipulation. However, the diversity of wild-type strains and their context-specific regulatory responses remain poorly characterized. Here, we evaluated five wild-type strains and identified 1D1108 as superior in tumorigenesis on legumes and transient transformation in Nicotiana benthamiana. Under in vitro virulence induction with acetosyringone (AS), we identified 126 differentially expressed genes (DEGs) in 1D1108. Although the number of DEGs was comparable to those in C58 and the legume isolate 1D1609 under the same condition, only 22 DEGs, primarily within the vir regulon, were conserved, indicating extensive divergence among these Agrobacterium strains. Leaf infiltration of N. benthamiana revealed 1,134 DEGs specifically regulated in planta for 1D1108. These included genes involved in attachment, virulence regulation, type IV pilus, succinoglycan biosynthesis, and diverse nutrient transporters, providing new evidence on expression regulation during colonization. Comparative analyses of in planta transcriptomes with C58 and Pseudomonas syringae DC3000 revealed distinct secretion systems required for pathogenesis, namely type IV for Agrobacterium and type III for Pseudomonas, and only approximately 5-19% of DEGs were conserved. These limited transcriptomic overlaps underscore the importance of studying gene expression in strains and conditions directly relevant to the biological context, rather than relying on model systems. Together, this work reveals how environmental and host-associated cues shape transcriptional responses in plant-associated bacteria. IMPORTANCEAgrobacterium is a key tool for plant genetic engineering, yet much of our knowledge about its biology comes from a few strains studied under artificial conditions. This work combines tumor formation assays on legumes and transient transformation in Nicotiana benthamiana with transcriptomic analyses to explore how diverse Agrobacterium strains, particularly the high-performing wild-type strain 1D1108, respond to host and environmental cues. By comparing gene expression under in vitro and in planta conditions, we found that the bacterial response within plant tissue is more complex than previously appreciated. Cross-strain comparisons further revealed limited conservation in gene expression regulation, even under similar conditions. Comparative analysis with Pseudomonas syringae revealed the activation of distinct secretion systems for pathogenesis and differences in regulatory programs used during plant colonization. These findings underscore the importance of studying bacteria in biologically relevant contexts and highlight the limitations of relying solely on model strains to infer regulatory responses.

microbiology↗

bioRxiv2025

The autoactivity of tomato helper NLR immune proteins of the NRC clade is unaltered in prf mutants of Nicotiana benthamiana

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) can function in networks of sensors and helpers to induce hypersensitive cell death and immunity against pathogens. The tomato sensor NLR Prf guards the Pto kinase from AvrPto and AvrPtoB effector perturbation and activates the downstream helpers NRC2 and NRC3. Prf is conserved across the Solanaceae and its ortholog in the model species Nicotiana benthamiana is also required for detection of AvrPto/AvrPtoB function on Pto. A recent study reported that cell death induction after transient expression of an autoactive mutant of tomato NRC3 is abolished upon RNAi silencing of Prf in N. benthamiana. Here we generated loss-of-function prf mutants in N. benthamiana and demonstrate that autoactive mutants of eight canonical tomato NRCs (NRC0, NRC1, NRC2, NRC3, NRC4a, NRC4b, NRC6, and NRC7) still induce hypersensitive cell death when expressed transiently in the prf mutant background. Autoactive tomato NRCs also triggered cell death when expressed in lettuce (Lactuca sativa), an Asteraceae plant that does not have a Prf ortholog. These results confirm a unidirectional dependency of sensors and helpers in the NRC network and underscore the value of the N. benthamiana and lettuce model systems for studying functional relationships between paired and networked NLRs.

plant biology↗

bioRxiv2025

A hydrophobic core in the coiled-coil domain essential for NRC resistosome function

The nucleotide-binding leucine-rich repeat protein (NLR) required for cell death (NRC) family represents a group of helper NLRs that are required by sensor NLRs to execute hypersensitive cell death during pathogen infection. NRCs contain an N-terminal coiled-coil (CC) domain essential for their function, yet our knowledge of how this domain contributes to NRC function remains limited. Here, we identified a novel hydrophobic feature within the CC domain that contributes to NRC-mediated immunity. We screened for conserved hydrophobic residues among NRCs and identified seven required for NRC4-mediated cell death. Structural analysis revealed that four of these residues form a hydrophobic core in the CC domain. This hydrophobic core is important for NRC4 subcellular localization, oligomerization, and phospholipid association, but not for NRC4 focal accumulation at the extrahaustorial membrane during Phytophthora infestans infection. Sequence analysis and functional assays revealed this core is highly conserved in NRCs and some singleton NLRs but has degenerated in NRC-dependent sensor NLRs. Our study identifies a novel hydrophobic feature in the CC domain of NRCs and reveals its contribution to NLR-mediated immunity.

plant biology↗

bioRxiv2024

Rhizobium rhizogenes A4-derived strains mediate hyper-efficient transient gene expression in Nicotiana benthamiana and other solanaceous plants

Agroinfiltration, a method utilizing agrobacteria to transfer DNA into plant cells, is widely used for transient gene expression in plants. Besides the commonly used Agrobacterium strains, Rhizobium rhizogenes can also introduce foreign DNA into host plants for gene expression. While many R. rhizogenes strains have been known for inducing hairy root symptoms, their use for transient expression has not been fully explored. Here, we showed that R. rhizogenes A4 outperformed all other tested agrobacterial strains in agroinfiltration experiments on leaves of Nicotiana benthamiana and other solanaceous plants. By conducting an agroinfiltration screening in N. benthamiana leaves using various agrobacterial strains carrying the RUBY reporter gene cassette, we discovered that A4 mediates the strongest and fastest transient expression. Utilizing the genomic information, we developed a collection of disarmed and modified strains derived from A4. By performing vacuum infiltration assays, we demonstrated that these A4-derived strains efficiently transiently transform 6-week-old N. benthamiana leaves, showing less sensitivity to the age of plants compared to the laboratory strain GV3101. Furthermore, we performed agroinfiltration using AS109, an A4-derived disarmed strain, on the leaves of tomato, pepper, and eggplant. Remarkably, AS109 mediated transient gene expression on tested solanaceous plants more effectively than all the tested commonly used agrobacterial strains. This discovery paves the way for establishing R. rhizogenes A4-derived strains as a new option for enhancing transient expression in N. benthamiana and facilitating the functional study of plant genes in other solanaceous species.

plant biology↗

bioRxiv2024

A helper NLR targets organellar membranes to trigger immunity

Upon activation, plant NLR immune receptors are known to assemble into oligomeric resistosomes that insert into the plasma membrane, forming Ca2+-permeable channels and triggering immunity. Here, we show that the RPW8-like coiled-coil (CCR-) NLR NRG1 primarily targets organelles instead of the plasma membrane. Unlike canonical CC-NLRs, activated NRG1 accumulates at the chloroplast envelope and channels stromal Ca2+ into the cytosol. AlphaFold modeling of the NRG1 resistosome reveals an unusually long N-terminal membrane-insertion structure that would span the double chloroplast membranes. Nanobody-mediated relocalization shows functional membrane specificity: chloroplast trapping abolishes activity of the canonical helper CC-NLR NRC4 but not NRG1. NRG1 orthologs, from non-flowering lineages to angiosperms, target chloroplasts, suggesting that organelle-centered defense dates back to at least [~]360 million years. We propose that coiled-coil NLR diversification has enabled compartment-specific immune signaling to capture diverse Ca2+ stores.

plant biology↗

bioRxiv2024

The curses of performing differential expression analysis using single-cell data

Differential expression analysis is pivotal in single-cell transcriptomics for unraveling cell-type- specific responses to stimuli. While numerous methods are available to identify differentially expressed genes in single-cell data, recent evaluations of both single-cell-specific methods and methods adapted from bulk studies have revealed significant shortcomings in performance. In this paper, we dissect the four major challenges in single-cell DE analysis: normalization, excessive zeros, donor effects, and cumulative biases. These "curses" underscore the limitations and conceptual pitfalls in existing workflows. In response, we introduce a novel paradigm addressing several of these issues.

bioinformatics↗

bioRxiv2024

Development of a tightly regulated copper-inducible transient gene expression system in Nicotiana benthamiana incorporating suicide exon and Cre recombinase

Chemical-inducible gene expression systems have been frequently used to regulate gene expression for functional genomics in various plant species. However, a convenient chemical-inducible system that can tightly regulate transgene expression in Nicotiana benthamiana is still missing. In this study, we developed a tightly regulated copper-inducible system that can be used to regulate transgene expression and perform cell death assays in N. benthamiana. We tested several chemical-inducible systems using Agrobacterium-mediated transient expression and found that the copper-inducible system showed the least concerns of leakiness issues. Using the MoClo-based synthetic biology approach, we optimized the design of the copper-inducible system and incorporated the use of the suicide exon HyP5SM/OsL5 and Cre/LoxP as additional regulatory elements to enhance the tightness of the regulation. This new design allowed us to tightly control the hypersensitive cell death induced by several tested NLRs and their matching AVRs, and it can also be easily applied to regulate the expression of other transgenes in transient expression assays. Our findings provide new approaches for both fundamental and translational studies in plant functional genomics.

plant biology↗