RESUMEN
Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.
Asunto(s)
Membrana Celular , Microdominios de Membrana , Plantas , Terminología como Asunto , Microdominios de Membrana/metabolismo , Membrana Celular/metabolismoRESUMEN
Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.
RESUMEN
Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfatidilserinas/metabolismo , Transducción de Señal/fisiología , Arabidopsis/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Membrana Celular/metabolismo , Plantas/metabolismoRESUMEN
Biological membranes are highly dynamic, in particular due to the constant exchange of vesicles between the different compartments of the cell. In addition, the dynamic nature of membranes is also caused by their inherently fluid properties, with the diffusion of both proteins and lipids within their leaflets. Lipid diffusion is particularly difficult to study in vivo but recent advances in optical microscopy and lipid visualization now enable the characterization of lipid lateral motion, and here we review these methods in plants. We then discuss the parameters that affect lipid diffusion in membranes and explore their consequences on the formation of membrane domains at different scales. Finally, we consider how controlled lipid diffusion affects membrane functions during cell signaling, development, and environmental interactions.
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Membrana Dobles de Lípidos , Membrana Dobles de Lípidos/metabolismo , Membrana Celular/metabolismo , DifusiónRESUMEN
The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides/metabolismo , Ligandos , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismoRESUMEN
Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.
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Proteínas Portadoras , Proteínas de Plantas , Proteínas Portadoras/metabolismo , Proteínas de Plantas/química , Membrana Celular/metabolismo , Plantas/metabolismo , Membrana Dobles de Lípidos/metabolismoRESUMEN
In Arabidopsis, the receptor-like kinase (RLK) FERONIA (FER) senses peptide ligands in the plasma membrane (PM), modulates plant growth and development, and integrates biotic and abiotic stress signaling for downstream adaptive responses. However, the molecular interplay of these diverse processes is largely unknown. Here, we show that FER, the receptor of Rapid Alkalinization Factor 1 (RALF1), physically interacts with C2 domain ABA-related (CAR) proteins to control the nano-organization of the PM. During this process, the RALF1-FER pathway upregulates CAR protein translation, and then more CAR proteins are recruited to the PM. This acts as a rapid feedforward loop that stabilizes the PM liquid-ordered phase. FER interacts with and phosphorylates CARs, thereby reducing their lipid-binding ability and breaking the feedback regulation at later time points. The formation of the flg22-induced FLS2-BAK1 immune complex, which depends on the integrity of FER-containing nanodomains, is impaired in fer and pentuple car14569 mutant. Together, we propose that the FER-CAR module controls the formation of PM nano-organization during RALF signaling through a self-contained amplifying loop including both positive and negative feedback.
Asunto(s)
Arabidopsis , Transducción de Señal , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Fosfotransferasas/metabolismo , Desarrollo de la Planta , Transducción de Señal/genética , Estrés Fisiológico/genética , Inmunidad de la Planta/genéticaRESUMEN
Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. Here, we identify the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with CELLULOSE SYNTHASE 1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Microtúbulos/metabolismo , Membrana Celular/metabolismo , Celulosa/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1's function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.
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Proteínas de Arabidopsis , Arabidopsis , Autofagosomas , Vacuolas , Arabidopsis/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Nitrógeno/metabolismo , Vacuolas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H+-ATPase proton pumping activity by inhibiting PM-associated PP2C.D phosphatases, thereby increasing the PM electrochemical potential, acidifying the apoplast, and stimulating cell expansion. Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/metabolismo , Lípidos , Monoéster Fosfórico Hidrolasas/genética , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Protones , ARN/metabolismoRESUMEN
Membrane lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal, and plant cells, where it regulates a wide range of cellular processes including endocytic trafficking. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the putative phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are enriched in a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with Src Homology 3 Domain Protein 2 (SH3P2), a protein involved in endocytic trafficking. In the absence of SAC9, SH3P2 localization is altered and the clathrin-mediated endocytosis rate is reduced. Together, our results highlight the importance of restricting PI(4,5)P2 at the plasma membrane and illustrate that one of the consequences of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking.
Asunto(s)
Arabidopsis , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis , Endosomas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.
Asunto(s)
Arabidopsis , Autofagosomas , Arabidopsis/genética , Arabidopsis/metabolismo , Autofagosomas/metabolismo , Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
Phosphatidylinositol 4-phosphate (PI4P) is an anionic phospholipid which has been described as a master regulator of the Golgi apparatus in eukaryotic cells. However, recent evidence suggests that PI4P mainly accumulates at the plasma membrane in all plant cells analyzed so far. In addition, many functions that are typically attributed to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) in animal and yeast cells are also supported by PI4P in plants. For example, PI4P is the key anionic lipid that powers the strong electrostatic properties of the plasma membrane. Phosphatidylinositol 4-phosphate is also required for the establishment of stable membrane contacts between the endoplasmic reticulum and the plasma membrane, for exocytosis and to support signaling pathways. Thus, we propose that PI4P has a prominent role in specifying the identity of the plasma membrane and in supporting some of its key functions and should be considered a hallmark lipid of this compartment.
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Fosfatos de Fosfatidilinositol , Fosfatidilinositoles , Animales , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismoRESUMEN
ROP GTPases coordinate the complex morphogenesis of leaf pavement cells. Four new studies reveal how ROP activity is regulated by both activating and inhibitory proteins to orchestrate cell lobe formation in response to local extracellular cues.
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Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Forma de la Célula , GTP Fosfohidrolasas/metabolismo , Morfogénesis , Hojas de la Planta/metabolismoRESUMEN
Phosphoinositides (PIs) have critical roles in various cellular, physiological, developmental, pathological, and infectious processes. They are signaling phospholipids that can affect every aspect of membrane biology, including protein function (e.g., recruitment and activity), membrane physicochemical properties (e.g., curvature, surface charges, and packing), and the generation of secondary messengers. PIs act at precise locations within the cell in a dose-dependent manner, and their local concentration can vary drastically during signaling and trafficking. Thus, techniques able to manipulate PI amounts acutely and with subcellular accuracy are paramount to understanding the role of these lipids in vivo. Here, we review these methods and emphasize approaches recently developed to perturb PI levels in multicellular organisms.
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Fosfatidilinositoles , Transducción de Señal , Humanos , Fosfatidilinositoles/metabolismoRESUMEN
At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.
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Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador , Células Vegetales , Orgánulos/fisiologíaRESUMEN
In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.
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Membrana Celular/metabolismo , Pared Celular/metabolismo , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Plantas/metabolismo , Orgánulos/metabolismo , Células Vegetales/metabolismoRESUMEN
A small family composed of BRI1 KINASE INHIBITOR1 (BKI1) and MEMBRANE-ASSOCIATED KINASE REGULATORS (MAKRs) has recently captured the attention of plant biologists, due to their involvement in developmental processes downstream of hormones and Receptor-Like Kinases (RLK) signalling. BKI1/MAKRs are intrinsically disordered proteins (so-called unstructured proteins) and as such lack specific domains. Instead, they are defined by the presence of two conserved linear motifs involved in the interaction with lipids and proteins, respectively. Here, we first relate the discovery of the MAKR gene family. Then, we review the individual function of characterized family members and discuss their shared and specific modes of action. Finally, we explore and summarize the structural, comparative and functional genomics data available on this gene family. Together, this review aims at building a comprehensive reference about BKI1/MAKR protein function in plants.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrínsecamente Desordenadas , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brasinoesteroides , Regulación de la Expresión Génica de las Plantas , Hormonas/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Desarrollo de la Planta/genética , Plantas/genética , Plantas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/genéticaRESUMEN
Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regiones de Fijación a la Matriz , Antígenos de Histocompatibilidad Menor/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.