Biology subjects

Yuen, E. L. H.

Publications and source records attributed to Yuen, E. L. H..

13 recordsLinked to original sources

bioRxiv2026

A helper NLR monitors catalytic perturbation of a vesicle trafficking regulator targeted by pathogen effectors

Plants rely on vesicle trafficking for immunity, yet pathogens deploy effectors to reprogram these pathways. NLR immune receptors can monitor trafficking components, but how plants detect effector perturbation of the enzymatic regulators controlling these pathways remains unclear. Here, we show that TOPGAP, a Rab GTPase-activating protein and susceptibility factor, couples effector activity to immune signaling involving the helper NLR NRC4. PiE355 and TIKI, related effectors from two Phytophthora species, bind the N-terminal regulatory domain of TOPGAP and trigger cell death requiring TOPGAP and its C-terminal GAP activity. The TOPGAP catalytic domain alone is sufficient to activate NRC4-mediated cell death, which is abolished by catalytic inactivation and suppressed by the N-terminal regulatory domain that the effectors bind. These findings implicate deregulated TOPGAP activity in immune activation. We propose that NRC4-dependent surveillance enables plants to detect deregulation of a host enzyme that pathogens can co-opt to promote infection.

plant biology↗

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

View Tomo: Context-aware targeting and analysis in electron cryo-tomography

Electron cryo-tomography (cryoET) resolves cellular structure in three dimensions, yet region selection is still typically based on two-dimensional projection images. Here, we introduce View Tomo, a workflow for rapid acquisition of low-magnification tomograms that enables screening, targeting and analysis in 3D. View Tomo tilt series are acquired in minutes at low dose (~3 e-/[A]2), producing high-contrast tomograms that remain compatible with subsequent high-resolution structural determination. We implemented View Tomo using an automated acquisition and reconstruction pipeline for rapid alignment. Across multiple viral and cellular systems, view tomograms revealed membrane remodelling events, assembly intermediates and cellular organisation that are difficult to identify in projection images. These data enabled targeted high-resolution imaging and quantitative analysis of spatial relationships within cells. View Tomo therefore extends cryoET workflows by improving target selection, enabling analysis of mesoscale organisation, and facilitating integration with correlative imaging approaches.

cell biology↗

bioRxiv2026

On-lamella super-resolution cryo-CLEM for cryo-ET enabled by vacuum-free ultra-stable cryogenic fluorescence microscopy

Cryogenic correlative light and electron microscopy (cryo-CLEM) combines specific fluorescence labelling of proteins inside cells with structural information at the angstrom-level. The introduction of super-resolution fluorescence methods in the field of cryogenic fluorescence microscopy is a necessary step to bridge the large resolution gap between the different imaging modalities. However, there are many challenges hindering the full potential of cryogenic super-resolution correlative light and electron microscopy and seamless integration with structural cell biology. One of the main limiting factors is a lack of dedicated cryogenic fluorescence microscopy systems with sufficient mechanical stability to enable the collection of high-quality super-resolution data and full compatibility with vitrified specimens for cryo-electron tomography. Here, we address this by developing a vacuum-free ultra-stable cryogenic optical microscope (VULCROM). VULCROM is a dedicated super-resolution cryo-CLEM (cryo-SR-CLEM) setup that combines the stability of a vacuum-insulated cryostat with the flexibility and modularity of an open microscopy system. We demonstrate that VULCROM enables detailed investigations of single-molecule cryo-photo-physics across timescales spanning milliseconds to hours. We furthermore demonstrate its suitability for routine cryo-SR-CLEM with a resolution in the 10 nm range in distinct vitrified biological specimen types. We resolve the nanoscale architecture of YFP-labelled PML bodies within the nucleus of mammalian cells and the distribution of ATG9-eGFP in its cellular structural context in a cryo-lift-out lamella of N. benthamiana plant tissue. Owing to its vacuum-free design, VULCROM can be readily adapted for diverse correlative workflows and other cryo-light microscopy applications.

biophysics↗

bioRxiv2025

An effector from the potato late blight pathogen bridges ENTH-domain protein TOL9a to an activated helper NLR to suppress immunity

Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. Here, we show that an effector from the potato late blight pathogen Phytophthora infestans bridges the host protein NbTOL9a, a putative member of the host ESCRT pathway, to a helper NLR to suppress immunity. In this work, we solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that unlike other RXLR-LWY effectors, AVRcap1b has a novel L-shaped fold that defines a new structural family of effectors in the Phytophthora genus. Moreover, we defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that dont bind NbTOL9a, impairing immune suppression. This indicates that ENTH binding is required for full virulence activity of this effector. Lastly, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. This suggests that the effector functions as a bridge that interconnects NbNRC2 with the NbTOL9a pathway. These results illustrate an unprecedented pathogen mechanism to hijack helper NLR pathways and suppress immunity.

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↗

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

An oomycete effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant- derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TBC1D15L, to remodel the host-pathogen interface. The effector, PiE354, hijacks TBC1D15L as a susceptibility factor to usurp its GAP activity on Rab8a--a key Rab GTPase crucial for defense-related secretion. By hijacking TBC1D15L, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co- opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

plant biology↗

bioRxiv2023

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 accumulates in vivo as a homodimer that converts into a higher order oligomer upon activation by its upstream virus disease resistance protein Rx. The Cryo-EM structure of NRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

plant biology↗

bioRxiv2023

A RabGAP-Rab GTPase pair regulates plant autophagy and immunity

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as an important membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL from Nicotiana benthamiana, but not its mutated form deficient in ATG8 binding, reduced autophagic flux in N. benthamiana and Arabidopsis. Furthermore, Rab3GAPL-knockout mutants of the liverwort Marchantia polymorpha exhibited enhanced autophagic flux under both normal and heat stress conditions, suggesting that Rab3GAPLs negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

plant biology↗

bioRxiv2022

Resurrection of plant disease resistance proteins via helper NLR bioengineering

Parasites counteract host immunity by suppressing helper NLR proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. A single amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a novel strategy for resurrecting disease resistance in crop genomes. One sentence summaryA helper NLR is mutated to evade inhibition by a parasite effector.

plant biology↗

bioRxiv2022

Sensor NLR immune proteins activate oligomerization of their NRC helper

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptors are important components of plant and metazoan innate immunity that can function as individual units or as pairs or networks. Upon activation, NLRs form multiprotein complexes termed resistosomes or inflammasomes. Whereas metazoan paired NLRs, such as NAIP/NLRC4, activate into hetero-complexes, the molecular mechanisms underpinning activation of plant paired NLRs, especially whether they associate in resistosome hetero-complexes is unknown. In asterid plant species, the NLR required for cell death (NRC) immune receptor network is composed of multiple resistance protein sensors and downstream helpers that confer immunity against diverse plant pathogens. Here, we show that pathogen effector-activation of the NLR proteins Rx (confers virus resistance) and Bs2 (confers bacterial resistance) leads to oligomerization of the helper NLR NRC2. Activated Rx does not oligomerize or enter into a stable complex with the NRC2 oligomer and remains cytoplasmic. In contrast, activated NRC2 oligomers accumulate in membrane-associated puncta. We propose an activation-and-release model for NLRs in the NRC immune receptor network. This points to a distinct activation model compared to mammalian paired NLRs.

plant biology↗

bioRxiv2021

Chloroplast movement and positioning protein CHUP1 is required for focal immunity against Phytophthora infestans

Communication between cellular organelles is essential for mounting effective innate immune responses to eliminate pathogens. In plants, the transport of cellular organelles to pathogen penetration sites and their assembly around the host membrane delineating plant-pathogen interface are well-documented. However, whether organelles associate with these specialized plant-pathogen membrane interfaces and the extent to which this process contributes to immunity remain unknown. Here, we discovered defense-related membrane contact sites (MCS) comprising a membrane tethering complex between chloroplasts and the extrahaustorial membrane (EHM) surrounding the pathogen haustorium. The assembly of this membrane tethering complex relies on the association between the chloroplast outer envelope protein CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), and its plasma membrane-associated partner, KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1). Our biochemical assays revealed that CHUP1 and KAC1 interact, while infection cell biology demonstrated their co-accumulation in foci where chloroplasts contact the EHM. Genetic depletion of CHUP1 or KAC1 reduces the deposition of callose--a cell wall material typically deployed to fortify pathogen penetration resistance--around the haustorium, without affecting other core immune processes. Our findings suggest that the chloroplast-EHM attachment complex positively regulates plant focal immunity, revealing the key components and their potential roles in the targeted deposition of defense components at the pathogen interface. These results advance our understanding of organelle-mediated immune responses and highlight the significance of MCS in plant-pathogen interactions.

plant biology↗