REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement

Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3’s phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses

S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signalling

SummaryPlant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signalling molecules. Receptor kinases are regulated by numerous post-translational modifications. Here, using the immune receptor kinases FLS2 and EFR, we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biophysical properties and behaviour within the membrane environment. We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following perception of its ligand flg22, in a BAK1 co-receptor dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signalling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR supressed elf18 triggered signalling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents and native membrane DIBMA nanodiscs indicates that S-acylation stabilises and promotes retention of activated receptor kinase complexes at the plasma membrane to increase signalling efficiency

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling

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

S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signaling

Plant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signaling molecules. Receptor kinases are regulated by numerous post-translational modifications.1,2,3 Here, using the immune receptor kinases FLS24 and EFR,5 we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biochemical properties and behavior within the membrane environment.6 We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following the perception of its ligand, flg22, in a BAK1 co-receptor and PUB12/13 ubiquitin ligase-dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signaling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR suppressed elf18-triggered signaling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents, and native membrane DIBMA nanodiscs indicates that S-acylation stabilizes, and promotes retention of, activated receptor kinase complexes at the plasma membrane to increase signaling efficiency

Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases

A membrane-bound ankyrin repeat protein confers race-specific leaf rust disease resistance in wheat

Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca2+-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding

Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity

Perception of biotic and abiotic stresses often leads to stomatal closure in plants 1,2. Rapid influx of calcium ions (Ca 2+) across the plasma membrane has an important role in this response, but the identity of the Ca 2+ channels involved has remained elusive 3,4. Here we report that the Arabidopsis thaliana Ca 2+-permeable channel OSCA1.3 controls stomatal closure during immune signalling. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phosphoproteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22, which is derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca 2+, and that BIK1-mediated phosphorylation on its N terminus increases this channel activity. Notably, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immune signalling, and OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid—a plant hormone associated with abiotic stresses. This study thus identifies a plant Ca 2+ channel and its activation mechanisms underlying stomatal closure during immune signalling, and suggests specificity in Ca 2+ influx mechanisms in response to different stresses

Function of Plant Plasma Membrane Nanodomains : Study of Group 1 REMORINs during Plant-Virus Interactions

L’organisation par compartimentalisation est une propriété générale des systèmes naturels coordonnant les évènements biologiques dans l’espace et le temps. Au cours des trente dernières années, il a pu être démontré qu’à l’échelle d’une membrane de nombreux sous-compartiments coexistent. Une telle organisation semble cruciale pour l’ensemble des activités biologiques cellulaires et par conséquent prépondérante pour le développement et la survie des organismes vivants. La membrane plasmique présente une grande diversité de compartiments, néanmoins leurs fonctions et les mécanismes moléculaires régissant leur organisation ne sont pas très bien compris. Afin d’apporter de nouvelles informations sur comment et pourquoi la membrane plasmique des plantes est souscompartimentée, nous étudions les nanodomaines des REMORINEs du groupe 1 au cours de l’infection de N. benthamiana par le Potato Virus X (PVX). En menant une approche multidisciplinaire, nous avons décortiqué un mécanisme moléculaire impliqué dans l’organisation en nanodomaine de REMORIN à la membrane plasmique. Via la génération de mutants nous présentons des liens fonctionnels entre l’organisation en domaine de REMORIN, son statut de phosphorylation, sa capacité à réguler la perméabilité des plasmodesmes et la propagation de cellule à cellule du PVX. Nous présentons également des éléments montrant qu’au cours de la perception de PVX par N. benthamiana, l’organisation de la membrane plasmique est cruciale pour la mise en place des mécanismes de défense de la plante. Cette étude met en avant pour la première fois chez les plantes l’importance de la régulation spatio-temporelle des protéines à la membrane plasmique dans l’identité et la fonction de domaines membranaires.Organization by compartmentalization is a general property of natural systems coordinating biological events in space and time. Over the past three decades, it has been demonstrated that multiple micrometric to nano-metric sub-compartments co-exist at a single membrane level. Such membrane organization seems critical for most all cell bioactivities and therefore critical for development and survival of potentially all living organisms. Plants respond to pathogens by activating highly regulated plasma membrane-bound signalling pathways. Plant plasma membrane (PM) displays a great diversity of compartments, but underlying functions and molecular mechanisms governing such organization are not well understood. To get insight in how and why plant PM is compartmentalized, we choose to study the plant PM nanodomain goup 1 REMORIN during the interaction between N. benthamiana and the Potato Virus X (PVX). Using a multidisciplinary approach we decipher a molecular mechanism involved in defining REMORIN PM domains localization. Making mutants we provide a functional link between REMORIN PM organization at single molecule level, its phosphostatus, regulation of plasmodesmata permeability and PVX cell-to-cell movement restriction. We then provide evidences that during N.benthamiana PVX sensing, PM organization appears critical for the modulation plant defence mechanisms and cell signaling. This study provides a unique mechanistic insight into how tight control of protein spatio-temporal organization at PM level is crucial to confer membrane domains identity and functionality