Biology subjects

Duggan, C.

Publications and source records attributed to Duggan, C..

12 recordsLinked to original sources

bioRxiv2026

Comparison of multiple cefiderocol susceptibility testing methods against genomic determinants of resistance in blaNDM carbapenemase producing Enterobacterales

BackgroundCefiderocol is a siderophore-conjugated cephalosporin antibiotic used to treat multi drug resistant Gram negative infections, including metallo-beta-lactamase producing Enterobacterales. Antimicrobial useage is guided by antimicrobial susceptibility testing (AST) which is hampered by differences between EUCAST and CLSI breakpoints, methodological challenges of AST, and lack of information on clinical outcome related to AST. ObjectivesThis study assessed the agreement between AST methods under EUCAST and CLSI breakpoints in a collection of 57 blaNDM producing Enterobacterales isolated from a UK hospital network. MethodsAll isolates, including Klebsiella pneumoniae, Enterobacter hormaechei, Escherichia coli and Citrobacter freundii, were whole-genome sequenced and tested with disk diffusion and MIC gradient test strip, and broth microdilution MICs were determined for a subset. Categorical agreement between methods was calculated using both EUCAST and CLSI breakpoints. Mutations and acquired resistance genes associated with cefiderocol resistance were identified and compared with AST results. ResultsThe disk diffusion method, based on EUCAST interpretation, classified 94.7% of isolates as cefiderocol resistant and 5.3% as susceptible, with 22.8% within the Area of Technical Uncertainty. The CLSI breakpoint classified one isolate as resistant (1.8%) and 5.26% intermediate. Category agreement of broth microdilution and disk diffusion for E. coli using EUCAST guidelines was 38.5%. Mutations associated with cefiderocol resistance were highly prevalent and varied between species. ConclusionsThe discordant EUCAST and CLSI breakpoint values provided have large impacts on the classification of isolates susceptibility to cefiderocol, which will impact global cefiderocol usage and surveillance of resistance, further complicated by poor agreement between AST methods.

microbiology↗

bioRxiv2023

Parallel, Continuous Monitoring and Quantification of Programmed Cell Death in Plant Tissue

The accurate quantification of hypersensitive response (HR) programmed cell death is imperative for understanding plant defense mechanisms and developing disease-resistant crop varieties. In this study, we report an accelerated phenotyping platform for the continuous-time, rapid and quantitative assessment of HR: Parallel Automated Spectroscopy Tool for Electrolyte Leakage (PASTEL). Compared to traditional HR assays, PASTEL significantly improves temporal resolution and has high sensitivity, facilitating the detection of microscopic levels of cell death. We validated PASTEL by transiently expressing the effector protein AVRblb2 in transgenic lines of the model plant Nicotiana benthamiana (expressing the corresponding resistance protein Rpi-blb2) to reliably induce HR. We were able to detect cell death at microscopic intensities, where leaf tissue appeared healthy to the naked eye one week after infiltration. PASTEL produces large amounts of frequency domain impedance data captured continuously (sub-seconds to minutes). Using this data, we developed a supervised machine learning models for classification of HR. We were able to classify input data (inclusive of our entire tested concentration range) as HR-positive or negative with 84.1% mean accuracy (F1 score = 0.75) at 1 hour and with 87.8% mean accuracy (F1 score = 0.81) at 22 hours. With PASTEL and the ML models produced in this work, it is possible to phenotype disease resistance in plants in hours instead of days to weeks.

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

An atypical NLR protein modulates the NRC immune receptor network

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical, but essential member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, transient RNA interference of NRCX in mature N. benthamiana leaves doesnt result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.

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↗

bioRxiv2021

Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post-activation during pathogen infection remains poorly known. Here we show that NRC4, from the NRC solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N-terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses. Significance statementPlant NLR immune receptors function as intracellular sensors of pathogen virulence factors known as effectors. In resting state, NLRs localize to subcellular sites where the effectors they sense operate. However, the extent to which NLRs alter their subcellular distribution during infection remains elusive. We describe dynamic changes in spatiotemporal localization of an NLR protein in infected plant cells. Specifically, the NLR protein accumulates at the newly synthesized plant-pathogen interface membrane, where the corresponding effectors are deployed. Following immune recognition, the activated receptor re-organizes to form punctate structures that target the cell periphery. We propose that NLRs are not necessarily stationary immune receptors, but instead may spread to other cellular membranes from the primary site of activation to boost immune responses.

plant biology↗

bioRxiv2020

The Irish potato famine pathogen subverts host vesicle trafficking to channel starvation-induced autophagy to the pathogen interface

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How and why adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phythophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway, while antagonizing antimicrobial autophagy. Here we show that PexRD54 induces autophagosome formation by bridging small GTPase Rab8a-decorated vesicles with autophagic compartments labelled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing that specific trafficking pathways underpin selective autophagy. We discovered that Rab8a contributes to basal immunity against P. infestans, but PexRD54 diverts a sub-population of Rab8a vesicles to lipid droplets that associate with autophagosomes. These are then diverted towards pathogen feeding structures that are accommodated within the host cells. We propose that PexRD54 mimics starvation-induced autophagy by channeling host endomembrane trafficking towards the pathogen interface possibly to acquire nutrients. This work reveals that effectors can interconnect independent host compartments to stimulate complex cellular processes that benefit the pathogen. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/000117v1_ufig1.gif" ALT="Figure 1"> View larger version (83K): org.highwire.dtl.DTLVardef@bfc9fforg.highwire.dtl.DTLVardef@166b5f8org.highwire.dtl.DTLVardef@34d581org.highwire.dtl.DTLVardef@ad397f_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2019

An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4--a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal 1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time.

plant biology↗

bioRxiv2019

Chloroplasts navigate towards the pathogen interface to counteract infection by the Irish potato famine pathogen

Upon immune activation, chloroplasts switch off photosynthesis, produce anti-microbial compounds, and associate with the nucleus through tubular extensions called stromules. Although it is well-established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplasts movement in response to pathogen attack. Here, we report that chloroplasts accumulate at the pathogen interface during infection by the Irish potato famine pathogen Phytophthora infestans, associating with the specialized membrane that engulfs the pathogen haustorium. Chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at the pathogen haustoria, suggesting this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and interconnecting chloroplasts, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector mediated suppression of BAK1 mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

plant biology↗

bioRxiv2017

Host autophagosomes are diverted to a plant-pathogen interface

Filamentous plant pathogens and symbionts invade their host cells but remain enveloped by host-derived membranes. The mechanisms underlying the biogenesis and functions of these host-microbe interfaces are poorly understood. Recently, we showed that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host protein ATG8CL to stimulate autophagosome formation and deplete the selective autophagy receptor Joka2 from ATG8CL complexes. Here, we show that during P. infestans infection, ATG8CL autophagosomes are diverted to the pathogen interface. Our findings are consistent with the view that the pathogen coopts host selective autophagy for its own benefit.

plant biology↗