A helper NLR, NRC4, accumulates at the plant-pathogen interface to provide disease resistance against the Irish potato famine pathogen Phytophthora infestans

Plants and animals both rely on nucleotide-binding, leucine-rich repeat proteins (NLRs) to provide disease resistance. Plant NLRs often co-operate as pairs or networks. A sensor NLR detects the pathogen and a helper NLR executes cell death and/or downstream signalling. NLRs are believed to localise to the cytoplasm, plasma membrane or nucleus. However, these localisation patterns were observed in the absence of infection, not during infection with relevant pathogens due to the HR triggered by the NLR making it impossible to perform microscopy. Many plant proteins re-locate within the cell upon infection by microbes. Therefore the localisation of NLRs during infection may be different to those without infection. Additionally, determining the localisation of NLRs can help us understand what function the NLR provides and how it detects effectors and elicits an immune response. Here I study the localisation of a solanaceous helper NLR, NRC4 during live cell imaging of Nicotiana benthamiana leaves with and without infection by the Irish potato famine pathogen, Phytophthora infestans. I use transient gene expression and confocal microscopy to show that during NRC4, accumulates from the cytoplasm to a membrane surrounding the pathogen finger-like appendages called haustoria. The extra-haustorial membrane is a newly formed plant membrane interface surrounding the pathogen haustoria. This reveals that NLRs can be motile and alter localisation during infection to accumulate at pathogen contact sites. I overcome the problem of NLRs triggering cell death when their cognate effector is present by either studying the helper NLR NRC4 in the absence of a sensor, or by disarming NRC4 by mutating the first alpha helix of the NLR’s cell death eliciting coiled-coil domain. When activated, we found that NRC4 forms puncta which associate with the EHM or in the absence of infection, the plasma membrane. These puncta may represent inflammasome-like ‘resistosomes’ or groups of resistosomes. A phylogenetically and functionally related NLR, ZAR1, was recently shown to form a resistosome and is proposed to execute cell death by possibly forming pores in the host membrane. Further evidence is required in the future to determine if NRC4 is forming resistosomes and whether these are eliciting cell death directly. Intriguingly, efforts to prevent NRC4 accumulation at the EHM, by mutation, truncation or chimeric NLR generation, led to loss of NRC4’s cell death function. As a whole, this thesis provides evidence that NRC4 accumulates to the host-pathogen interface to provide disease resistance against P. infestans. This thesis reveals that NLRs can alter their localisations upon infection and that they can even focally accumulate at the host-pathogen interface. Further understanding into how NRC4 focally accumulates during infection and exactly what it achieves by doing so may help us understand how resistance to fungi and oomycetes occurs. Ultimately, this may allow us to engineer disease resistant crops by, for instance, altering NRC4’s specificity to recognise other EHM localised effectors, without altering NRC4’s localisation.Open Acces

Modulation of plant autophagy during pathogen attack

In plants, the highly conserved catabolic process of autophagy has long been known as a means of maintaining cellular homeostasis and coping with abiotic stress conditions. Accumulating evidence has linked autophagy to immunity against invading pathogens, regulating plant cell death, and antimicrobial defences. In turn, it appears that phytopathogens have evolved ways not only to evade autophagic clearance but also to modulate and co-opt autophagy for their own benefit. In this review, we summarize and discuss the emerging discoveries concerning how pathogens modulate both host and self-autophagy machineries to colonize their host plants, delving into the arms race that determines the fate of interorganismal interaction.Fil: Leary, Alexandre Y. Imperial College London; Reino UnidoFil: Sanguankiattichai, Nattapong. University of Oxford; Reino UnidoFil: Duggan, Cian. Imperial College London; Reino UnidoFil: Tumtas, Yasin. Imperial College London; Reino UnidoFil: Pandey, Pooja. Imperial College London; Reino UnidoFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Salguero Linares, Jose. Imperial College London; Reino UnidoFil: Savage, Zachary D. Imperial College London; Reino UnidoFil: Yow, Rui Jin. Imperial College London; Reino UnidoFil: Bozkurt, Tolga O.. Imperial College London; Reino Unid

Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition

In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response

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 a1 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

Does cannabis use predict psychometric schizotypy via aberrant salience?

Cannabis can induce acute psychotic symptoms in healthy individuals and exacerbate pre-existing psychotic symptoms in patients with schizophrenia. Inappropriate salience allocation is hypothesised to be central to the association between dopamine dysregulation and psychotic symptoms. This study examined whether cannabis use is associated with self-reported salience dysfunction and schizotypal symptoms in a non-clinical population. 910 University students completed the following questionnaire battery: the cannabis experience questionnaire modified version (CEQmv); schizotypal personality questionnaire (SPQ); community assessment of psychic experience (CAPE); aberrant salience inventory (ASI). Mediation analysis was used to test whether aberrant salience mediated the relationship between cannabis use and schizotypal traits. Both frequent cannabis consumption during the previous year and ASI score predicted variation across selected positive and disorganised SPQ subscales. However, for the SPQ subscales ‘ideas of reference’ and ‘odd beliefs’, mediation analysis revealed that with the addition of ASI score as a mediating variable, current cannabis use no longer predicted scores on these subscales. Similarly, cannabis use frequency predicted higher total SPQ as well as specific Positive and Disorganised subscale scores, but ASI score as a mediating variable removed the significant predictive relationship between frequent cannabis use and ‘odd beliefs’, ‘ideas of reference’, ‘unusual perceptual experiences’, ‘odd speech’, and total SPQ scores. In summary, cannabis use was associated with increased psychometric schizotypy and aberrant salience. Using self-report measures in a non-clinical population, the cannabis-related increase in selected positive and disorganised SPQ subscale scores was shown to be, at least in part, mediated by disturbance in salience processing mechanisms

An oomycete effector 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 adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization

Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial 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 chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that 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 facilitating chloroplast interactions, 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.Fil: Savage, Zachary. Imperial College London; Reino UnidoFil: Duggan, Cian. Imperial College London; Reino UnidoFil: Toufexi, Alexia. Imperial College London; Reino UnidoFil: Pandey, Pooja. Imperial College London; Reino UnidoFil: Liang, Yuxi. Imperial College London; Reino UnidoFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Yuen, Lok Him. Imperial College London; Reino UnidoFil: Gaboriau, David C. A.. Imperial College London; Reino UnidoFil: Leary, Alexandre Y.. Imperial College London; Reino UnidoFil: Tumtas, Yasin. Imperial College London; Reino UnidoFil: Khandare, Virendrasinh. Imperial College London; Reino UnidoFil: Ward, Andrew D.. Science and Technology Facilities Council; Reino UnidoFil: Botchway, Stanley W.. Science and Technology Facilities Council; Reino UnidoFil: Bateman, Benji C.. Science and Technology Facilities Council; Reino UnidoFil: Pan, Indranil. Alan Turing Institute; Reino Unido. Imperial College London; Reino UnidoFil: Schattat, Martin. Martin Luther Universitat Halle-Wittenberg; AlemaniaFil: Sparkes, Imogen. University of Bristol; Reino UnidoFil: Bozkurt, Osman Tolga. Imperial College London; Reino Unid

An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana

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 (NLR-REQUIRED FOR CELL DEATH) 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 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 in N. benthamiana 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, spot gene silencing of NRCX in mature N. benthamiana leaves doesn't 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

Resurrection of plant disease resistance proteins via helper NLR bioengineering

Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (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. An 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 potential strategy for resurrecting disease resistance in crop genomes

An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana

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 (NLR-REQUIRED FOR CELL DEATH) 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 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 in N. benthamiana 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, spot gene silencing of NRCX in mature N. benthamiana leaves doesn’t 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. Author summary Plants have an effective immune system to fight off diverse pathogens such as fungi, oomycetes, bacteria, viruses, nematodes and insects. In the first layer of their immune system, receptor proteins act to detect pathogens and activate the defense response. Plant genomes encode very large and diverse repertoires of immune receptors, some of which function in pairs or as complex receptor networks. However, the immune system can come at a cost for plants and inappropriate receptor activation results in growth suppression and autoimmunity. Here, we show that an atypical immune receptor gene functions as a modulator of the immune receptor network. This type of receptor gene evolved to maintain homeostasis of the immune system and balance fitness trade-offs between growth and immunity. Further understanding how plants regulate their immune receptor system should help guide breeding disease resistant crops with limited fitness penalties