Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells.

International audience; Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin-mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP-tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein-induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense-associ…

Perception of elicitins by receptor complex in solanaceae

Extensive infections of crop plants by plant pathogens have repeatedly resulted in catastrophic harvest failures causing major economic and social problems worldwide. Successful plant defence against pathogens is based on early microbe recognition and fast activation of downstream immune signalling (e.g. phosphorylations, production of reactive oxygen species, transcriptomic reprogramming). Plant surface pattern-recognition receptors (PRRs) act as initial detectors that can recognise microbial-associated molecular patterns (MAMPs) to induce the basal resistance response called MAMP-triggered immunity (MTI). Despite the increasing number of PRRs discovered in the past 20 years, for most of t…

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

Abstract Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which w…

Reticulon-like proteins in Arabidopsis thaliana: structural organization and ER localization

International audience; Reticulons are proteins that have been found predominantly associated with the endoplasmic reticulum in yeast and mammalian cells. While their functions are still poorly understood, recent findings suggest that they participate in the shaping of the tubular endoplamic reticulum (ER). Although reticulon-like proteins have been identified in plants, very little is known about their cellular localization and functions. Here, we characterized the reticulon-like protein family of Arabidopsis thaliana. Three subfamilies can be distinguished on the basis of structural organization and sequence homology. We investigated the subcellular localization of two members of the larg…

Induction des mécanismes de défense des plantes : Les acquis du modèle tabac/cryptogéine

Cryptogein is a small protein secreted by the oomycete Phytophthora cryptogea, able to trigger on tobacco plants both the developmenl of an hypersensitive response and the induction of resistance against a wide range of pathogens. The deciphering of molecular events associated with this process, using tobacco as a model plant, gave significant informations about the set up of defense mechanisms. The role of plasma membrane dynamics in such a signal transduction has been evidenced. ln particular; the presence of plasma membrane microdomains concentrating signaling proteins, and the modification of the proteic content of these domains upon cryptogein treatment have been demonstrated. Moreover…

Utilisation de la technique du FRAP pour évaluer la fluidité de la membrane plasmique de cellules végétales en suspension

Rôle de la dynamique de la membrane plasmique dans la mise en place des réactions de défense chez le tabac

The binding of cryptogein, a plant defense elicitor secreted by an oomycete, to an unknown receptor triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco cells. One of these early events is the transient production of Reactive Oxygen Species (ROS) by an NADPH oxidase, NtrbohD. We were thus interested to analyze the dynamic of the plasma membrane during these early stages. We determined that internalization of the endocytotic marker FM4-64 is stimulated a few minutes after addition of cryptogein to tobacco BY-2 cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein since a lipid transfer protein, which bin…

Liquid ordered domains of tobacco cell plasma membrane : in vivo visualization, spatial distribution and modulation upon elicitation

SPE IPM; International audience; The ability of lipids in complex mixtures to form liquid-ordered, sterol-rich phases, together with the association of specific proteins with sterol-enriched biochemical fractions extracted from biological membranes, gave raise to the "lipid rafts" hypothesis. This suggests the presence within biological membranes of small-sized, ordered domains, of particular lipid and protein composition, which could serve as signal transduction platforms, particularly during plant-microorganisms interactions. However, the precise organization of living cell plasma membrane is still poorly described and the spatial distribution of segregating liquid-disordered and liquid-o…

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells

International audience; The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of …

Analyse des interactions trophiques entre les plantes et les microorganismes par des biosenseurs de sucres

Dynamics of sugar transports in tobacco-microbe interactions

Dynamic changes in the subcellular distribution of the tobacco ROS-producing enzyme RBOHD in response to the oomycete elicitor cryptogein.

Highlight text The oomycete elicitor cryptogein triggers the relocation of RBOHD from intracellular compartments to the plasma membrane in tobacco cells. This suggests that intracellular trafficking is a potential determinant of RBOHD activity.

Ratiometric fluorescence live imaging analysis of membrane lipid order in Arabidopsis mitotic cells using a lipid order-sensitive probe

SPE Pôle IPM; International audience; Eukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence …

Study of lipidic membrane component effect on the membrane order level by using GUVs

National audience

Rôle des microdomaines du plasmalemme dans la réponse de la cellule végétale au stress biotique

National audience

The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells  

Abstract The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. To…

Exocytosis and endocytosis act together to modify the plasma membrane order in cryptogein-elicited tobacco cells

SPEIPMUBINRA; Exocytosis and endocytosis act together to modify the plasma membrane order in cryptogein-elicited tobacco cells. 19. European Network for Plant Endomembrane Research meeting

Characterization of membrane structure modifications induced during plant defense signaling

SPEIPMUBINRADOCT; Plasma membrane was long considered as a simple separation between internal and external cellular environment. But more, it is a dynamic interface, an exchange platform. Now, recent researches highlight its role in plant defense responses to biotic and abiotic stresses. We use the tobaccocryptogein model to study the early steps of plant cell responses to pathogen detection. The signaling cascade includes an oxidative burst triggering an increase of plasma membrane order. The level of membrane order informs us about both packing and arrangement of membrane components, this parameter being evaluated by spectrofluorimetric approaches using a lipidic environment-sensitive pro…

Évaluation de la fluidité de la membrane plasmique avec la sonde fluorescente di-4-ANEPPDHQ: Adaptation de la technique de recouvrement de fluorescence après photoblanchiment (FRAP) sur cellules végétales en suspension

National audience; Au cours de l’interaction entre plantes et microorganismes, la membrane plasmique joue un rôle essentiel dans l’organisation précoce des mécanismes de réactions de défense mises en place par la plante. L’équipe Dynamique membranaire et réponse aux stress biotiques, de l’UMR Plante-Microbe-Environnement, s’intéresse aux modifications des propriétés du plasmalemme des cellules végétales dans ce type d’interactions. La fluidité membranaire est une de ces propriétés. La technique de retour de fluorescence après photoblanchiment (FRAP), réalisée à l’aide d’un microscope confocal à balayage laser, a été adaptée à l’étude des variations de la fluidité de la membrane plasmique de…

Asymétrie de la membrane plasmique végétale : caractérisation et rôle dans la signalisation immunitaire des plantes

National audience; La membrane plasmique (MP) forme une barrière sélective entre la cellule et le milieu extérieur.C’est un senseur des modifications environnementales et une plateforme orchestrant latransduction du signal permettant de déployer une réponse adaptée. Elle joue donc un rôleessentiel dans la physiologie cellulaire. Décrypter les bases moléculaires de l’organisation dela MP apparaît donc nécessaire pour comprendre comment les plantes s’adaptent auxmodifications environnementales. La MP est une bicouche de lipides à laquelle des protéinessont associées, et les données accumulées sur ce modèle ont révélé son organisation complexeet dynamique et le rôle essentiel joué par les lipi…

Starvation-induced expression of autophagy-related genes in Arabidopsis

Background information. Autophagy is a catabolic process for degradation of cytoplasmic components in the vacuolar apparatus. A genome-wide survey recently showed evolutionary conservation among autophagy genes in yeast, mammals and plants. To elucidate the molecular and subcellular machinery responsible for the sequestration and subsequent digestion of intracellular material in plants, we utilized a combination of morphological and molecular methods (confocal laser-scanning microscopy, transmission electron microscopy and real-time PCR respectively). Results. Autophagy in Arabidopsis thaliana suspension-cultured cells was induced by carbon starvation, which triggered an immediate arrest of…