Biology subjects

Hasegawa, K.

Publications and source records attributed to Hasegawa, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Epithelial QKI Protects Against Emphysema by Maintaining Mitochondrial Integrity

Single-cell transcriptomic profiling of chronic obstructive pulmonary disease (COPD) lungs identified QKI, an RNA-binding protein, as a candidate emphysema-associated gene, but its epithelial role in COPD pathobiology remains unclear. We show that QKI expression is reduced in human COPD lungs and that alveolar type 2 epithelial (AT2) cell QKI protein levels correlate strongly with spirometric indices and diffusing capacity (DLCO). Lung epithelium-specific QKI knockout mice (QKI{Delta}/{Delta}) developed spontaneous airspace enlargement with emphysema-like mechanics, and QKI-deficient AT2 cells showed impaired spheroid colony formation and increased apoptosis. Integrated transcriptomic and proteomic analyses of primary AT2 cells revealed a selective reduction in functional mitochondrial (respiratory-chain and metabolic) protein abundance despite relatively preserved transcript levels, consistent with mitochondrial transcriptome-proteome discordance. QKI loss increased mtDNA abundance and TOMM20 staining but decreased ATP5A, indicating accumulation of structurally increased but functionally dysfunctional mitochondria. In human epithelial cells, CRISPR-mediated QKI deficiency reduced oxidative respiration, increased glycolytic reliance, elevated mitochondrial ROS and membrane potential, and increased apoptosis; these phenotypes were partially rescued by QKI re-expression. These findings identify epithelial QKI as a regulator of mitochondrial integrity and stress tolerance in COPD.

cell biology↗

bioRxiv2026

Region-specific molecular regulatory programs define epithelial identity, progenitor states, and mucus homeostasis in human distal airways

Small distal airways differ from proximal large airways in structure, airflow dynamics, and epithelial composition, and represent a central site of muco-obstructive lung disease pathogenesis. However, due in part to their inaccessibility, the molecular mechanisms that establish regional epithelial identity and govern mucociliary defense in distal airway epithelia remain poorly defined. Here, we integrate transcriptomic, secretomic, and chromatin accessibility analyses of matched primary human large and small airway epithelial cultures to define region-specific regulatory networks. We identify distal airway-specific transcriptional and chromatin programs required for maintaining epithelial identity and mucus homeostasis. Loss of NKX2-1 impairs distal airway secretory cell (DASC) differentiation and shifts mucus properties toward a disease-associated state. Lineage-resolved organoid assays identify an NKX2-1-high distal airway basal cell population with hybrid basal-secretory features as a selective progenitor for DASCs. Collectively, these findings establish a molecular framework for distal airway epithelial biology and define mechanisms regulating region-specific mucociliary host defense.

Cell Biology↗

bioRxiv2026

Prion-like transmission of human tau strains in the mouse brain

Most neurodegenerative diseases are believed to spread through the brain by prion-like mechanisms, where filamentous protein assemblies self-propagate by templated seeding 1. Distinct conformations of amyloid filaments are thought to provide the physical basis for the strains that lead to different diseases 2. However, a central pillar of the prion hypothesis, that strains retain their structural identity upon transmission, has not been demonstrated. Here we show that the injection of tau filaments from the brains of individuals with Alzheimers disease or corticobasal degeneration into the brains of wildtype mice leads to the seeded assembly of amyloid filaments made of mouse tau with the same structures as those of the seeds. Thereby, we establish that, like prion strains, tau filaments propagate through templated seeding, and that the mouse is a suitable model to study the molecular mechanisms by which distinct tau folds drive disease-specific pathology in the brain.

neuroscience↗

bioRxiv2026

Exploiting HLA-II Promiscuity via Peptide Terminal Overhang Recognition for Pan-Allelic and Tumor-Selective AML Immunotherapy

Antibodies targeting peptides presented by human leukocyte antigen (HLA) molecules expand the therapeutic landscape by enabling recognition of intracellular antigens. While most efforts have focused on allele-restricted peptides presented by HLA class I (HLA-I), HLA class II (HLA-II) epitopes remain underexplored despite their potential for promiscuous presentation. Acute myeloid leukemia (AML) is characterized by high expression of both HLA-II and the myeloid lineage antigen myeloperoxidase (MPO). Here, we identified an MPO-derived epitope (MPO100-132) that is promiscuously presented by multiple HLA-II molecules. We generated a MPO100-132-specific antibody (146D5) that recognizes the N-terminal overhang of this peptide independent of specific HLA contacts, enabling pan-allelic recognition. Engineered into bispecific T cell engagers (BiTEs), this antibody mediated robust cytotoxicity against primary AML samples across diverse HLA-II backgrounds. Crucially, 146D5-based BiTEs selectively spared normal myeloid cells, indicating that the MPO100-132 peptide, derived from the MPO propeptide, was functionally undetectable in normal myeloid cells, providing a significant safety window. In vivo, the MPO-targeting BiTE demonstrated potent antitumor activity and prolonged survival in AML xenograft models. Our findings identify peptide terminal overhangs as an actionable class of antibody targets and introduce a strategy to exploit HLA-II promiscuity for broadly applicable HLA-dependent but allele-agnostic immunotherapies.

immunology↗

bioRxiv2026

Seafloor video-acoustic monitoring in a Greenlandic glacial fjord records hyperbenthos, backward-swimming fish, and narwhals

Autonomous video-acoustic monitoring at the sea-floor can improve our understanding of poorly documented ecosystems and help interpret active or passive acoustic data, but it has been rarely carried out, particularly in the Arctic. This study deployed a video camera synchronized with a hydrophone, combined with red lights and other oceanographic instrumentation, on the bottom of a glacial fjord in Inglefield Bredning, northwest Greenland (to 260 m water depth). Through manual review and automatic analysis of high-frequency images (30 fps) and audios (96 kHz), the conditions and biodiversity near the sea-floor were quantified. The data revealed a highly turbulent environment with abundant suspended particles and fibers, with 88% of 478 detected organisms being Amphipoda, Copepoda, Hydrozoa, and Chaetognatha. Amongst the other observed animals were Decapoda, Liparidae, Pterotracheoidea, Ctenophora, and curious narwhals (Monodon monoceros). The number of marine snow particles was highly variable through time and could change up to twofold within several hours. The tide modulated the particle flow direction and speed. Overall, the results show that portable moorings with video recorders are an important tool for exploration of the Arctic seafloor.

ecology↗

bioRxiv2026

Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice

Understanding brain function requires tools that allow precise manipulation of receptor signaling in defined cell types within intact neural circuits. Optogenetics and conventional chemogenetic approaches primarily enable cell-type-specific control of neuronal excitability using engineered receptors or ion channels. However, direct and reversible inhibition of endogenous neurotransmitter receptors in defined cell types has remained technically inaccessible. Here, we introduce native receptor-targeted chemogenetics (NARCH), a chemogenetic strategy that integrates structure-guided receptor engineering with allosteric ligand design to achieve reversible and temporally precise inhibition of endogenous receptor signaling with cell-type specificity in vivo. By applying NARCH to metabotropic glutamate receptor 1 (mGlu1), we demonstrate that mGlu1 signaling in cerebellar Purkinje cells is required for stabilization of motor learning across training sessions in freely moving mice. NARCH thus establishes a receptor-level chemogenetic framework for causal analysis of neural circuits and behavior.

neuroscience↗

bioRxiv2026

CaMKIIα holoenzymes self-organize into worm-like mesoscale clusters

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in dendritic spines at concentrations comparable to those of cytoskeletal proteins and plays a central role in synaptic plasticity. During long-term potentiation (LTP), CaMKII further accumulates in spines. However, the mechanisms governing its higher-order organization remain poorly understood. Here, we use high-speed atomic force microscopy to visualize inter-holoenzyme interaction of CaMKII at mesoscopic scales (5-500 nm). Under freely diffusible conditions, CaMKII holoenzymes do not form stable clusters. In contrast, when spatially confined, they assemble into worm-like chain clusters mediated by kinase-domain interactions. These clusters expand upon activation, concomitant with the dissociation of the regulatory segment. Notably, the CaMKII P212L mutant associated with neurodevelopmental disorders, forms extensive clusters even in the basal state. Together, our findings demonstrate that CaMKII-CaMKII interactions drive mesoscale cluster formation and that precise regulation of cluster size and activation-dependent growth might be critical for synaptic signaling.

neuroscience↗

bioRxiv2026

Reduced B cell activation in Germinal center reaction of the mouse model of Li-Fraumeni syndrome

The TP53 mutation associated with Li-Fraumeni syndrome in humans is known to exhibit p53 gain-of-function properties leading to early cancer onset. To explore whether these patients also have compromised immune responses due to p53 mutations, we investigated the humoral immune response using a Li-Fraumeni mouse model harboring a structural mutation, Trp53R172H (equivalent to codon 175 in humans). Trp53R172H mice were immunized with sheep red blood cells, and the Germinal center response was monitored. Our results revealed that SRBC-immunized Trp53R172H mice exhibit reduced B cell activation during the GC reaction. These suggest a selective role for p53 in promoting B cell activation early in the GC reaction prior to BCL6 upregulation. We propose that impaired B cell activation in Li-Fraumeni patients could contribute to immune deficiencies and heightened susceptibility to autoimmune disorders, potentially influencing the development of secondary cancers and impairing therapeutic responses to chemotherapies and immunotherapies. Key PointsO_LITrp52R172H mice exhibit reduced B cell activation during the Germinal center reaction C_LIO_LIRatio of activated B cells in DZ to LZ remain unchanged in Trp52R172H mice. C_LI

cancer biology↗

bioRxiv2025

Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

Adenosine Deaminase Acting on RNA 1 p110 (ADAR1p110), the constitutively expressed nuclear isoform of the RNA-editing enzyme ADAR1, plays well-established roles in adenosine-to-inosine RNA editing, but its functions during mitosis remain poorly defined. Here, we identify ADAR1p110 as a chromatin-associated factor essential for mitotic chromosome segregation and cell viability. Depletion of ADAR1 caused metaphase arrest, DNA damage accumulation, and apoptosis. Co-immunoprecipitation experiments revealed that ADAR1p110 physically interacts with Structural Maintenance of Chromosome 3 (SMC3), a core component of the cohesin complex. Genome-wide DNA immunoprecipitation sequencing (DIP-seq) showed that ADAR1p110 selectively binds centromeric -satellite DNA during mitosis, a finding validated by DNA immunoprecipitation quantitative polymerase chain reaction (DIP-qPCR). Complementary DNA-RNA immunoprecipitation sequencing (DRIP-seq) revealed that RNA:DNA hybrids (R-loops) are enriched in centromeric regions during mitosis and are further augmented by ADAR1 overexpression. We identified serine 614 (S614) as a key mitosis-specific phosphorylation site on ADAR1p110, and demonstrated that this post-translational modification is essential for its chromatin recruitment, stability, and mitotic function. Rescue experiments using phospho-mimetic mutants (S614D and 3xD) successfully restored mitotic progression in ADAR1-deficient cells, whereas non-phosphorylatable variants failed to do so. These results reveal that ADAR1p110 is phosphorylated in a cell cycle-dependent manner and functions at the intersection of post-transcriptional and post-translational regulation. By coordinating R-loop recognition at centromeres with chromosome cohesion, ADAR1p110 safeguards genome integrity during mitosis. Our findings uncover a previously uncharacterized mechanism through which a canonical RNA-editing enzyme contributes to chromosomal dynamics independent of its deaminase activity.

molecular biology↗

bioRxiv2025

Elucidating Chiral Myosin-Induced Actin Dynamics: From Single Filament Behavior to Collective Structures

The myosin superfamily comprises over 70 classes, each with multiple subclasses, and shows substantial diversity in properties such as velocity, ATPase activity, duty ratio, and directionality. This functional diversity enables the specialized roles of each myosin in various organisms, organs, and cell types. Recent studies have revealed that certain myosins induce chiral curved motions of actin filaments. However, this newly identified property remains largely unexplored. Here, we investigated this chiral motion in vitro using Chara corallina myosin XI (CcXI), which drives fast counterclockwise (CCW) movement of actin filaments. This chiral motion arises from asymmetric displacement at the filaments leading tip, and its curvature depends on the surface density of myosin. Surprisingly, at near-physiological actin concentrations, actin filaments exhibiting chiral curved motion undergo collective motion, spontaneously forming a novel structure--termed the actin chiral ring (ACR)--that exhibits persistent CCW rotation. ACRs display remarkable stability, continuing to rotate at their formation site until ATP is depleted, while maintaining their structure even after rotation ceases. This stability is unprecedented among reported collective motions of cytoskeletal proteins driven by various motors. Our findings demonstrate that myosins with chiral activity can autonomously organize actin filaments into stable, chiral structures through collective motion, providing new insights into actin self-organization by unconventional myosins. This study advances our understanding of the diverse functional roles of unconventional myosins and introduces a new paradigm for cytoskeletal organization. Significance StatementMyosins are motor proteins that move along actin filaments and support various intracellular functions. Recently, some myosins have been found to drive actin filaments along chiral curved paths, but the mechanisms and significance of this behavior remain poorly understood. Here, we analyzed this activity and discovered that it not only drives chiral curved motion of single actin filaments, but also organizes them into stable, unidirectionally rotating ring structures through collective motion. These rings, termed actin chiral rings (ACRs), spontaneously emerge at near-physiological actin concentrations. Our findings uncover a previously unrecognized organizing principle of actin self-assembly driven by myosin with chiral activity and provide a new framework for understanding how cytoskeletal chirality is made.

cell biology↗

bioRxiv2024

Widespread Distribution of α-Synuclein Oligomers in LRRK2-related Parkinson's Disease

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinsons disease (PD). While the clinical features of LRRK2-PD patients resemble those of typical PD, there are significant differences in the pathological findings. The pathological hallmark of definite PD is the presence of -synuclein (SYN)-positive Lewy-related pathology; however, approximately half of LRRK2-PD cases do not have Lewy-related pathology. Lewy-related pathology is a late-stage SYN aggregation that can be visualized with hematoxylin and eosin stains or conventional immunohistochemistry (IHC). Increasing evidence has indicated that SYN oligomers, which represent the early-stage of SYN aggregation, may have neurotoxicity. Visualization of SYN oligomers requires specialized staining techniques, such as SYN-proximity ligation assay (PLA). The distribution and severity of SYN oligomers in the human brain of LRRK2-PD patients remain unknown. In this study, we performed phosphorylated SYN-IHC and SYN-PLA staining on postmortem brain sections of patients with three pathogenic LRRK2 mutants: p.G2019S (n=5), p.I2020T (n=5), and p.R1441C (n=4). The severity of Lewy-related pathology and SYN oligomers were assessed semi-quantitatively in the brainstem, limbic lobe, basal ganglia, and cerebral cortex. SYN oligomers were detected in LRRK2-PD cases even in cases without Lewy-related pathology; a negative correlation was observed between Lewy-related pathology and SYN oligomers (r=-0.26 [-0.39, -0.12]; P<0.0001). Our findings suggest that SYN oligomers may represent a common pathological feature of LRRK2-PD. Notably, patients harboring p.G2019S and p.I2020T had significantly higher levels of SYN oligomers in those without Lewy-related pathology compared to those with Lewy-related pathology. These cases also had a trend toward shorter disease duration. These results imply that in LRRK2-PD, SYN oligomers may initially accumulate in the brain but do not progress to form Lewy-related pathology. The present study suggests that targeting SYN oligomers may be a therapeutic strategy for LRRK2-PD even if there is no Lewy-related pathology.

neuroscience↗

bioRxiv2024

Inflammatory molecules in the glomerular endothelium in mild IgA-Nephropathy identified by single-cell and spatial transcriptome

Immunoglobulin A nephropathy (IgA-N) is a primary glomerulonephritis characterized by mesangial cell proliferation and expansion. It still leads to end-stage renal disease over the long course of the disease in many patients. Although glomerular endothelial cells (GE) have been implicated in the pathogenesis of IgA-N, their role remains poorly understood. We conducted single-cell and spatial transcriptomic analysis using human specimens with mild IgA-N compared with normal. We observed the significantly higher expression of several inflammation-associated molecules which have been reported and not reported to be involved in IgA-N pathogenesis, respectively. Furthermore, we show that several important molecules were also highly expressed in GE of IgA-N at protein level by tissue immunostaining. Our analysis suggests that the characteristic pathways identified in GE that involve these molecules are associated with the progression of IgA-N. These findings offer important insights on both the pathogenesis of IgA-N and its treatment at an early stage.

bioinformatics↗

bioRxiv2024

Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations

RationaleHyper IgE syndrome (STAT3-HIES), also known as Jobs syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. ObjectivesTo identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. MethodsSTAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1{beta}) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main ResultsMucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1{beta} expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1{beta} exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a {gamma}-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. ConclusionsSTAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

cell biology↗

bioRxiv2024

Structural and Dynamical Analyses of Apo and Cap-binding eIF4E: An in silico Study

Molecular dynamics (MD) simulations were utilized to investigate the dynamic behavior of eukaryotic translation initiation factor 4E (eIF4E) in its apo (ligand-free) and Cap-bound (complexed with mRNA Cap) forms. Specifically, we focused on the rearrangement of critical eIF4E residues, including W102, E103, W56, and key charged residues within the alpha helix, such as lysine and arginine. Three independent 1 {micro}s MD simulations were performed for four eIF4E structures based on an available Capbound eIF4E crystal structure (PDB: 4TPW) to sample protein conformations and explore metastable states. Metastable states were successfully sampled within the 1 {micro}s trajectories for all four structures; however, 2D-root-mean-square deviation (RMSD) and free-energy landscape (FEL) analyses indicate that additional trajectory data may be needed to fully explore the conformational space. Notably, the clearest state transitions were observed in the Cap-A simulations. Our data suggests that previously reported allosteric site eIF4E inhibitor 4EGI-1 stabilized the 7mGTP-eIF4E interaction by binding to a site dorsal from the eIF4G binding site, facilitating stable protein states even after ligand removal and promoting cleaner state transitions in simulations. Root-mean-square fluctuation (RMSF) and distance analyses aligned consistently with experimental results, affirming the validity of our MD simulation system. Observations on apo and Cap eIF4E structures revealed structural changes in one of the alpha helices (i.e., H3), particularly the transition from alpha helix to loop upon Cap binding. This transition was also observed during the cap to apo transition, where residues E105-R109 underwent structural rearrangements from loop to alpha helix. These findings support the strategy of designing agents targeting critical Cap-binding residues (W56, W102, E103) and H3 lysine residues (K106, K108) to disrupt the 7mGTP-eIF4E interaction, potentially informing future drug design efforts targeting this oncogenic protein. Future work will focus on studying the apo to Cap transition using available eIF4E apo structures, such as PDB entry 2GPQ, to deepen our understanding of eIF4Es metastable states during Cap binding.

biochemistry↗

bioRxiv2024

An AI-Driven Framework for Discovery of BACE1 Inhibitors for Alzheimer's Disease

Alzheimers Disease (AD) is a progressive neurodegenerative disorder that affects over 51 million individuals globally. The {beta}-secretase (BACE1) enzyme is responsible for the production of amyloid beta (A{beta}) plaques in the brain. The accumulation of A{beta} plaques leads to neuronal death and the impairment of cognitive abilities, both of which are fundamental symptoms of AD. Thus, BACE1 has emerged as a promising therapeutic target for AD. Previous BACE1 inhibitors have faced various issues related to molecular size and blood-brain barrier permeability, preventing any of them from maturing into FDA-approved AD drugs. In this work, a generative AI framework is developed as the first AI application to the de novo generation of BACE1 inhibitors. Through a simple, robust, and accurate molecular representation, a Wasserstein Generative Adversarial Network with Gradient Penalty (WGAN-GP), and a Genetic Algorithm (GA), the framework generates and optimizes over 1,000,000 candidate inhibitors that improve upon the bioactive and pharmacological properties of current BACE1 inhibitors. Then, the molecular docking simulation models the candidate inhibitors and identifies 14 candidate drugs that exhibit stronger binding interactions to the BACE1 active site than previous candidate BACE1 drugs from clinical trials. Overall, the framework successfully discovers BACE1 inhibitors and candidate AD drugs, accelerating the developmental process for a novel AD treatment.

biochemistry↗

bioRxiv2024

Nobiletin, a Polymethoxyflavonoid, Activates the Desuccinylase Activity of SIRT5 and Prevents the Development of Heart Failure

Nobiletin is a natural compound useful for the prevention and treatment of several diseases. However, the precise role of nobiletin in heart failure is unclear. Nobiletin treatment prevents pressure overload- and myocardial infarction-induced heart failure. Using affinity purification of biotinylated nobiletin from rat heart cell lysates, we identified sirtuin 5 (SIRT5) as a novel nobiletin-binding protein. Nobiletin enhanced the desuccinylase activity of SIRT5 in vitro. Compared to wild-type mice, SIRT5-overexpressing transgenic mice resisted pressure overload-induced systolic dysfunction. Conversely, SIRT5 knockout disrupted the nobiletin-mediated therapeutic effects on heart failure in mice. SIRT5 desuccinylated p300 at lysine 1568 and reduced the histone acetyltransferase (HAT) activity of p300. The desuccinylated p300 mutant suppressed the phenylephrine-induced cardiomyocyte hypertrophic responses. These findings suggest that nobiletin prevents heart failure development through SIRT5-dependent inhibition of p300-HAT activity. Nobiletin, a nontoxic dietary compound, is a potential therapeutic agent for heart failure in humans.

molecular biology↗

bioRxiv2023

A simplified disease resistance assay using YFP-expressing Potato Virus X in N. benthamiana reveals a cell death-independent immune function of RBA1

R (resistance) proteins, such as intracellular NLRs (nucleotide-binding leucine-rich repeat receptors), are integral components of the plant innate immune system (van Wersch et al., 2020). Host responses following R protein activation include the generation of reactive oxygen species, sustained increases in cytosolic Ca2+, transcriptional reprogramming and, typically, rapid host cell death at sites of pathogen infection, which together ultimately lead to pathogen growth restriction (Wang et al., 2023). To assess the activity of R proteins, agroinfiltration-mediated transient gene expression assays have been widely used in Nicotiana species (e.g., N. benthamiana). In these transient assays, host cell death is often chosen as an indicator of R protein activity from the host responses mentioned above, in part because of the ease of experimentation. However, the extent to which host cell death is a proxy for disease resistance signaling has long been debated, as host cell death and pathogen growth restriction can be uncoupled in several cases (Bendahmane et al., 1999; Coll et al., 2010; Heidrich et al., 2011, Maekawa et al., 2023). To assess the disease resistance activity of R proteins, bacterial growth assays have been employed in combination with transient R gene expression in N. benthamiana (Sun et al., 2021). Bacterial growth assays, however, require multiple experimental procedures, including agroinfiltration, pathogen infection and bacterial counts, which hinders high-throughput studies of R gene-mediated disease resistance. Here, we report a simple plate reader-based assay to assess R gene-mediated disease resistance activity against PVX (Potato virus X) that expresses YFP (PVX-YFP). Unlike bacterial pathogens, PVX proliferation in N. benthamiana is not restricted by the intrinsic activity of the EDS1 signaling pathway as previously shown by virus-induced NbEDS1 gene silencing (Peart et al., 2002) and as we consistently show in this study using a Nbeds1 gene knockout mutant. This feature would increase the sensitivity of the assay, allowing it to capture a weak-to-moderate disease resistance activity of R proteins, as the contribution of basal immunity to PVX via the NbEDS1 pathway is negligible. Using this assay, we show that a non-cell death-inducing mutant of the R protein of RBA1 (Response to HopBA1), which lacks 2',3'-cAMP/cGMP synthetase activity but retains NADase activity, confers PVX resistance in an EDS1 signaling pathway-dependent manner.

plant biology↗

bioRxiv2023

Rapid propagation of Ca2+ waves and electrical signals in a liverwort Marchantia polymorpha

In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm/s, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels (TPCs) were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue.

plant biology↗