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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 stress in Klebsormidium nitens, afreshwater algal species possessing a NOS and established as a model to study plants adaptationto land. We applied the Biotin Switch method followed by mass spectrometry analysis. Thismethod allows the purification of S-nitrosated proteins. We found 43 proteins with significantlyhigher S-nitrosation levels in salt response condition. Orthology analysis were performedagainst the model plant Arabidopsis thaliana, in order to determine the potential function ofthese proteins. Among them, we selected an interesting protein called INOSITOLPOLYPHOSPHATE MULTIKINASE 2 (IPK2), potentially involved in cell signaling andstress response. We produced it successfully in Escherichia coli and measured its activity incollaboration with the Leibniz-Forschungsinstitut für Molekulare Pharmakologie. The Snitrosation of IPK2 was proved to occur on Cys125. We generated 2 mutants, IPK2(C125A)and IPK2(C125S), and will observe the conservation of their activity before proceeding to themeasurement after S-nitrosation of the wild type and mutated proteins. Structure analysis werealso caried in silico with alphafold, giving insights of potential NO effects. The identificationand functional analysis of S-nitrosated proteins in K. nitens, this project will provide a betterunderstanding of the functions of NO in unicellular green algae with NOS.

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 fromsubcellular structures or protein complexes, and brings them to the proteasome to facilitate their recycling or degradation. Therefore, Cdc48/VCP is involved in numerous cellular pathways such as membranes associated degradation, cell cycle regulation, genome stability, vesicular trafficking, autophagy and apoptosis. The role of Cdc48 in response to biotic stresses has been investigated. However, nuclear function(s) of Cdc48 in plant immunity remain unclear and poorly understood.In this project, we aimed to characterize the nuclear function(s) of the Cdc48 protein during the plant immune response triggered by cryptogein (an elicitor produced by Phytophthora cryptogea) in tobacco cells. We analyzed the nuclear dynamic of Cdc48 under normal and elicitation conditions through Fluorescence Correlation Spectroscopy analysis (FCS). Thiscomparison has shown that during plant immune response, (i) the nuclear mobility of Cdc48 increased and (ii) Cdc48 interacted more quickly with more proteins. Then, nuclear partners interacting with the endogenous nuclear Cdc48 were identified through immunoprecipitation and mass spectrometry analysis.To summarize, this work provides new information regarding the role of Cdc48 and its dynamic in the nucleus during plant immunity.

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 salt stress in Klebsormidium nitens, a freshwater algal species possessing a NOS and established as a model to study plants adaptation to land. We applied the Biotin Switch method followed by mass spectrometry analysis. This method allows the purification of S-nitrosated proteins. We found 43 proteins with significantly higher S-nitrosation levels in salt response condition. Orthology analysis were performed against the model plant Arabidopsis thaliana, in order to determine the potential function of these proteins. Among them, three were selected for being potentially involved in cell signaling. We aim to produce them in bacteria, in order to measure their activity and observe the impact of the S-nitrosation on it.

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 salt stress in Klebsormidium nitens, a freshwater algal species possessing a NOS and established as a model to study plants adaptation to land. We applied the Biotin Switch method followed by mass spectrometry analysis. This method allows the purification of S-nitrosated proteins. We found 43 proteins with significantly higher S-nitrosation levels in salt response condition. Orthology analysis were performed against the model plant Arabidopsis thaliana, therefore we established a classification of these proteins according to their localization, molecular function and biological process they are involved in. This is done with the PANTHER classification system, based on Gene Ontology terms (GO terms). Then, from this list, we selected an interesting protein called INOSITOL POLYPHOSPHATE MULTIKINASE 2a/b (IPK2a/b), potentially involved in cell signaling and stress response. We produced it in Escherichia coli and are currently measuring its activity. This protein will then be Snitrosated in vitro and we will determine if its activity is impaired. Through the identification and functional analysis of Snitrosated proteins in K. nitens, this project will provide a better understanding of the functions of NO in unicellular green algae with NOS

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses(Carvalho & Gomes, 2011). However, few studies have explored the effects of decreased plant endogenous defensin transcripts on these stress responses.When over-expressed in Arabidopsis thaliana, some of the seven members of thePlant Defensin type 1 family (PDF1) are recognised to improve plant responses tonecrotrophic pathogens(De Conincket al.,2010)and increase seedling tolerance to excess zinc (Zn;Mirouzeet al., 2006). We carried out an extensive physiological and biochemical comparative characterisation of i) novel transgenic amiRNA lines silenced for the five most similarAtPDF1s, and ii) a double null mutant for the two most distant AtPDF1s. Silencing of fiveAtPDF1transcripts was specifically associated with increased aboveground dry mass production in mature plants under Zn excess conditions, and with increased plant tolerance to different pathogens–one fungus, one oomycete and one bacterium.These unexpected results challenge the current paradigm describing the role ofPDFs in the plant response to stresses.Additional roles of plant defensins are discussed, which open onto new perspectives for their functions(Nguyen et al., submitted).

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 green lineage we initiated the functional characterization of K. nitensNOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response toabiotic stresses.Methods: KnNOSs nucleotide sequences were verified by RACE-PCR and sequencing, and their mRNAlevel were monitored by RT-qPCR and protein abundance by western blot. Protein partners werestudied, firstly in sillico using the BioGrid database and human NOS interaction data, and secondly invivo by immunoprecipitation experiments followed by mass spectrometry analysis.Results: Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possessesclassical mammalian NOS architecture consisting of oxygenase and reductase domains with somespecificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seemto be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditionstested, whereas KnNOS2 appeared to be transcriptionally regulated during stress. In parallel studies,we also built the in silico protein–protein interaction network of human NOSs. Interestingly, genesencoding orthologs of several of these candidates were found in K. nitens genome. Some of theseconserved partners are known to be involved in mammalian NOSs regulation and represent interestingcandidates for further investigation.Conclusions: Overall these findings open the way for a deeper characterization of KnNOSs and itsprotein partners and will facilitate further investigation of NO signaling in green lineage.Relevant references:Chatelain, P., Astier, J., Wendehenne, D., Rosnoblet, C., and Jeandroz, S. (2021). Identification of Partner Proteinsof the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in EukaryoticPhotosynthetic Organisms. Front. Plant Sci. 12, 3068. doi: 10.3389/fpls.2021.797451.Jeandroz, S., Wipf, D., Stuehr, D. J., Lamattina, L., Melkonian, M., Tian, Z., et al. (2016). Occurrence, structure,and evolution of nitric oxide synthase–like proteins in the plant kingdom. Sci. Signal. 9, re2–re2. doi:10.1126/scisignal.aad4403.

International audience; Both plants and animals are endowed with sophisticated innate immune systems to combat microbial attack. In these multicellular eukaryotes, innate immunity implies the presence of cell surface receptors and intracellular receptors able to detect danger signal referred as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Membrane-associated pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), receptor-like kinases (RLKs), and receptor-like proteins (RLPs) are employed by these organisms for sensing different invasion patterns before triggering antimicrobial defenses that can be associated with a form of regulated cell death. Intracellularly, animals nucleotide-binding and oligomerization domain (NOD)-like receptors or plants nucleotide-binding domain (NBD)-containing leucine rich repeats (NLRs) immune receptors likely detect effectors injected into the host cell by the pathogen to hijack the immune signaling cascade. Interestingly, during the co-evolution between the hosts and their invaders, key cross-kingdom cell death-signaling macromolecular NLR-complexes have been selected, such as the inflammasome in mammals and the recently discovered resistosome in plants. In both cases, a regulated cell death located at the site of infection constitutes a very effective mean for blocking the pathogen spread and protecting the whole organism from invasion. This review aims to describe the immune mechanisms in animals and plants, mainly focusing on cell death signaling pathways, in order to highlight recent advances that could be used on one side or the other to identify the missing signaling elements between the perception of the invasion pattern by immune receptors, the induction of defenses or the transmission of danger signals to other cells. Although knowledge of plant immunity is less advanced, these organisms have certain advantages allowing easier identification of signaling events, regulators and executors of cell death, which could then be exploited directly for crop protection purposes or by analogy for medical research.