New insights about the role of the chaperon-like protein Cdc48, a target for nitric oxide in plant immunity

Functional characterization of the chaperon-like protein Cdc48 in cryptogein-induced immune response in tobacco. Communication orale et poster

International audience; Cdc48, a molecular chaperone conserved in diff erent kingdoms, is a member of the AAA+ family contributing to numerous processes in mammalian including proteins quality control and degradation, vesicular traffi cking, autophagy and immunity. The functions of Cdc48 plant orthologues are less understood. We previously reported that Cdc48 is regulated by S-nitrosylation in tobacco cells undergoing an immune response triggered by cryptogein, an elicitin produced by the oomycete Phytophthora cryptogea. Here, we investigated the function of NtCdc48 in cryptogein signaling and induced hypersensitive-like cell death. NtCdc48 was found to accumulate in elicited cells both at …

NO signaling in cryptogein-induced immune responses in tobacco

NO signaling in cryptogein-induced immune responses in tobacco

SPEIPM; International audience

Protein S-nitrosylation: specificity and identification strategies in plants

SPE Pôle IPM UB; International audience; The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identi…

Nuclear involvement of the Cell Division Cycle 48 protein during the plant immune response

The control of protein homeostasis, a balance between their synthesis and degradation, also called proteostasis, is essential for cell survival. Any imbalance of the proteome, for instance triggered by a stress, leads to an accumulation of misfolded proteins leading to proteotoxic stress that can induce cell death. The ubiquitin proteasome system (UPS) is a major actor in the selective degradation of misfolded proteins to preserve proteome balance.The chaperone-like Cdc48 is a member of the AAA+ ATPase enzyme family which isconserved in mammals (VCP), yeasts and plants (Cdc48: Cell Division Cycle 48/p97).Cdc48/VCP is a cytosolic and nuclear protein which segregates misfolded proteins fromsu…

Emerging functions of nitric oxide in plant immunity

SPEIPMUBAgrosupCNRS; The importance of nitric oxide (NO) in innate and adaptive immunity in mammals is well recognised. NO exerts antimicrobial properties against invaders but also displays immunoregulatory functions in which S-nitrosylation represents a signalling process of major importance. Over the last two decades, a growing body of evidence suggests that NO is also a major component of plant immunity. Our understanding of its role in plant defence has been enriched by the identification and functional analysis of S-nitrosylated proteins. The recent identification of new S-nitrosylated proteins including the chaperone-like enzyme cell division cycle 48 (CDC48), histone deacetylases (HD…

Study of the chaperone protein CDC48 and its involvement in plant immunity

CDC48 is a conserved chaperone protein belonging to the AAA+ ATPase family (ATPase associated with various activities). This protein uses binding and hydrolysis of ATP to generate forces to affect the transformation of polypeptide substrate in numerous cellular processes. Studies on mammalian CDC48 orthologue revealed that it recognizes ubiquitylated polypeptides, directly or via partners, leading to substrate degradation or recycling. In plants, functions of CDC48 is less understood. The aim of my thesis, is to decipher the role of CDC48 in plant defense response context. First, I have to characterize NtCDC48 in tobacco (Nicotiana tabacum) suspension cells elicited by cryptogein, an elicit…

Identification and functional characterization of S-nitrosated proteins from Klebsormidium nitens

The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitous component of cell signalling. A key signalling mechanism mediating NO effects is S-nitrosation, a post-translational modification by which NO can impact the target protein activities, subcellular localizations and capacities to form protein complexes. The identification of proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly, land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans, while a few algal species possess it, thus raising many interrogations. Therefore, we focused on the identification of S-nitrosated proteins during sa…

Identification and functional characterization of S-nitrosated proteins from Klebsormidium nitens, a model alga to study plant adaptation to land

The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitous component of cell signalling. A key signalling mechanism mediating NO effects is S-nitrosation, a post-translational modification by which NO can impact the target protein activities, subcellular localizations, and capacities to form protein complexes. The identification of proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly, land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans, while a few algal species possess it, thus raising many interrogations. Therefore, we focused on the identification of S-nitrosated proteins during s…

S-Nitrosation of Arabidopsis thaliana Protein Tyrosine Phosphatase 1 Prevents Its Irreversible Oxidation by Hydrogen Peroxide

Tyrosine-specific protein tyrosine phosphatases (Tyr-specific PTPases) are key signaling enzymes catalyzing the removal of the phosphate group from phosphorylated tyrosine residues on target proteins. This post-translational modification notably allows the regulation of mitogen-activated protein kinase (MAPK) cascades during defense reactions. Arabidopsis thaliana protein tyrosine phosphatase 1 (AtPTP1), the only Tyr-specific PTPase present in this plant, acts as a repressor of H2O2 production and regulates the activity of MPK3/MPK6 MAPKs by direct dephosphorylation. Here, we report that recombinant histidine (His)-AtPTP1 protein activity is directly inhibited by H2O2 and nitric oxide (NO) …

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Role of nitric oxide synthase (NOS) of Klebsormidium nitens: Identification and characterization of partners

National audience; Nitric oxide (NO) is an important cellular signaling molecule across kingdoms. During bioticor abiotic stresses, NO burst is detected in both plants and mammals although no sequenceencoding the well described mammalian NO synthase (NOS) is highlighted in plants. Comparedto terrestrial plants, some algae present transcripts encoding the NOS-like enzyme. Amongthem, Klebsormidium nitens the model alga to study the early transition step from aquatic algaeto land plants is found. As mechanisms governing NO synthesis and signaling in green lineageremain unclear, the study of NOS from K. nitens (KnNOS) through (i) the identification ofregulator proteins, (ii) the identification …

Role of nitric oxide synthases from Klebsormidium nitens: structural characterization and identification of protein partners

Nitric oxide (NO) is an important cellular signalling molecule regulating various physiological processes, in both animals andplants. In animals, NO synthesis is mainly catalysed by NO synthase (NOS) enzymes. During biotic or abiotic stresses, NOSlike activities that are sensitive to mammalian NOS inhibitors have been detected in plants, although no sequences encodingthe well described mammalian NOS are highlighted in land plants. Interestingly, we identified NOS-like sequences in 20 algaespecies. Among them, NOSs are found in Klebsormidium nitens the model alga to study the early transition step from aquaticalgae to land plants.As mechanisms governing NO synthesis and signalling in green l…

NO Signalling in Plant Immunity

The importance of nitric oxide (NO) in innate and adaptive immunity in mammals is well recognised. NO exerts antimicrobial properties against invaders but also displays immunoregulatory functions in which S-nitrosylation represents a signalling process of major importance. Over the last two decades, a growing body of evidence suggests that NO is also a major component of plant immunity. Our understanding of its role in plant defence has been enriched by the identification and functional analysis of S-nitrosylated proteins. The recent identification of new S-nitrosylated proteins including the chaperone-like enzyme cell division cycle 48 (CDC48), histone deacetylases (HDACs) and calmodulin (…

NO signaling in tobacco elicited by the MAMP cryptogein

SPEPôle IPM; International audience; During the past years, nitric oxide (NO) has been shown to be a major cell signaling messenger in plants. Its importance has been highlighted during plant responses to pathogen attack or MAMPs (microbe associated molecular patterns) and during induced resistance or priming phenomenon. The major focus of our research is to understand how nitric oxide can modulate the activity of protein involved in plant defense. We identified several proteins undergoing S-nitrosylation, a redox-based post-translational modification of proteins, in tobacco cells elicited by cryptogein, a 10 kDa protein produced by the oomycete Phytophthora cryptogea and inducing immune re…

Functional characterization of the chaperon-like protein Cdc48 in cryptogein-induced immune response in tobacco

SPEIPMUBINRASUPDATDOCT; Cdc48, a molecular chaperone conserved in different kingdoms, is a member of the AAA+ family contributing to numerous processes in mammals including proteins quality control and degradation, vesicular trafficking, autophagy and immunity. The functions of Cdc48 plant orthologues are less understood. We previously reported that Cdc48 is regulated by S-nitrosylation in tobacco cells undergoing an immune response triggered by cryptogein, an elicitin produced by the oomycete Phytophthora cryptogea. Here, we inv estigated the function of NtCdc48 in cryptogein signalling and induced hypersensitive-like cell death. NtCdc48 was found to accumulate in elicited cells at both th…

Role of nitric oxide synthases from klebsormidium nitens: first structural characterization and partners identification

Objectives: Nitric oxide (NO) is an important cellular signaling molecule regulating various physiologicalprocesses, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase(NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have beenmeasured, although no sequences encoding mammalian NOSs have been found in land plants.Interestingly, we identified NOS-like sequences in 20 algae species. These latter include thefilamentous charophyte green algae Klebsormidium nitens, a biological model to study the earlytransition step from aquatic algae to land plants. In order to understand the mechanisms governingNO synthesis and signaling in …

Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms

In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the sig…

Identification and functional characterization of S-nitrosated proteins from Klebsormidium nitens in response to salt stress

The small gaseous molecule nitric oxide (NO) is well established as a major ubiquitouscomponent of cell signalling. A key signalling mechanism mediating NO effects is Snitrosation, a post-translational modification by which NO can impact the target proteinactivities, subcellular localizations, and capacities to form protein complexes. The identificationof proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly,land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans,while a few algal species possess it, thus raising many interrogations. Therefore, we focused onthe identification of S-nitrosated proteins during salt str…

Nitric oxide production and signalling in algae

International audience

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants

Implication et régulation du système ubiquitine-protéasome (UPS) dans les interactions plantes-microorganismes bénéfiques et pathogènes chez le tabac

Le contrôle de la régulation entre la synthèse et la dégradation des protéines, ouhoméostasie, est indispensable à la survie cellulaire. Or, des situations de stress biotiques ouabiotiques dérégulent l'homéostasie des protéines. En particulier, lors d'une attaque par desmicro-organismes pathogènes chez les végétaux, les acteurs du système de dégradation desprotéines associé à l’ubiquitine et au protéasome (UPS) sont fortement mobilisés. Cependantleur fonction dans ces processus est encore mal connue. Par ailleurs, l’implication du systèmeUPS, ainsi que sa régulation, dans l’établissement d’interactions bénéfiques n’ont été que trèspeu appréhendées. Nous proposons donc d’étudier et de compar…

NO signaling in plant immunity: A tale of messengers

International audience; Nitric oxide (NO) is a free radical gas involved in a myriad of plant physiological processes including immune responses. How NO mediates its biological effects in plant facing microbial pathogen attack is an unresolved question. Insights into the molecular mechanisms by which it propagates signals reveal the contribution of this simple gas in complex signaling pathways shared with reactive oxygen species (ROS) and the second messenger Ca2+. Understanding of the subtle cross-talks operating between these signals was greatly improved by the recent identification and the functional analysis of proteins regulated through S-nitrosylation, a major NO-dependent post-transl…

The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analyzed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the amount of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ub…