Aphanomyces euteiches Cell Wall Fractions Containing Novel Glucan-Chitosaccharides Induce Defense Genes and Nuclear Calcium Oscillations in the Plant Host Medicago truncatula

[EN] N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to β-1,6-glucans, and contain a β-(1,3;1,4)-glucan backbone whose β-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.SIThis work is part of the “Laboratoire d’Excellence” (LABEX) entitled TULIP (ANR -10-LABX-41); it was funded by the Région Midi-Pyrénées, the CNRS (PhD grant INEE 36 to AN), and the French Agence Nationale de la Recherche (ANR-08-BLAN-0208-01 “Sympasignal”)

Inflammasome inhibition by the ubiquitination of NLRP3

La réponse immunitaire innée constitue la première barrière de défense de l’organisme contre les dangers. Elle est mise en place grâce à des récepteurs reconnaissant des motifs moléculaires de pathogènes ou de dommages qui entrainent l’inflammation permettant l’élimination des pathogènes et la réparation des lésions tissulaires. Parmi ces récepteurs, NLRP3 est activé en réponse à une perte d’intégrité ou de fonction cellulaire. Celles-ci peuvent avoir lieu à la suite de stimulus très variés comme une infection, mais aussi des cancers ou des agrégats protéiques ayant lieu dans certaines maladies. NLRP3 est impliqué dans le déclenchement et le développement de nombreuses maladies multifactorielles comme la maladie d’Alzheimer, l’athérosclérose ou le diabète de type II. De plus, des mutations « gains de fonction » de NLRP3 sont directement associées au syndrome CAPS, une maladie auto-inflammatoire héréditaire. Il est donc essentiel de comprendre les mécanismes précis de régulation de NLRP3. Actuellement, il est connu que l’activation de NLRP3 requiert deux signaux : un signal de priming et un signal d’activation qui aboutissent à la formation d’un complexe multiprotéique appelé inflammasome. L’inflammasome est composé du senseur NLRP3, des adaptateurs NEK7 et ASC et de l’effecteur pro-caspase-1. L’assemblage et l’activation de l’inflammasome mène à l’activation de la caspase-1 qui clive les cytokines inflammatoires et la protéine GSDMD qui forme des pores dans la membrane, menant à une mort cellulaire programmée appelée pyroptose. Les mécanismes connectant les signaux de priming puis d’activation à l’assemblage de l’inflammasome sont méconnus, mais de nombreuses études suggèrent un rôle important des modifications post-traductionnelles de NLRP3, notamment son ubiquitination. Par exemple, la déubiquitination de NLRP3 par BRCC3 est nécessaire à son activation. Les travaux présentés dans cette thèse se concentrent sur le rôle de l’ubiquitination de NLRP3 dans son activation. Une première partie n’a pas permis d’identifier de nouveaux rôles de la déubiquitinase BRCC3 dans l’immunité innée. Toutefois, une seconde étude a mis en évidence par spectrométrie de masse deux lysines porteuse d’ubiquitination, préalablement associées à des mutations présentes chez des patients CAPS. Afin d’étudier l’impact de ces ubiquitinations sur l’activation de l’inflammasome, nous avons générés par CRISPR/Cas9 des souris mutantes substituant la lysine par une arginine pour empêcher cette ubiquitination. Les macrophages primaires issus des deux lignées de souris Nlrp3 KR/KR traités avec le signal de priming LPS présentent une sécrétion des cytokines IL-1β et IL-18, ainsi qu’une perméabilisation membranaire en absence de signal d’activation. Cette sécrétion a lieu en aval de la protéine TRIF et nécessite l’ensemble des composants de l’inflammasome NEK7, NLRP3, ASC et caspase-1. Il s’agit d’une activation de l’inflammasome NLRP3 à un niveau plus faible qu’une activation classique mettant en jeu un signal de priming et un signal d’activation. De plus, elle n’entraine pas de formation de specks d’ASC. Enfin, les deux lignées de souris Nlrp3 KR/KR semblent plus sensibles au choc endotoxique provoqué par une injection de LPS. L’ensemble de ces résultats décrit l’identification de deux lysines ubiquitinées agissant comme points de contrôle inhibiteur de la voie d’activation de l’inflammasome NLRP3 et qui pourrait être utilisées comme future cibles thérapeutiques.Innate immune response is the organism’s first line of defence against dangers. A large array of immune receptors recognizing pathogen and damage molecular pattern initiate inflammation which allows pathogen clearance and healing of tissue injury. Among these receptors, NLRP3 is activated in response to the loss of cellular function and integrity. These cellular stresses can happen as a result of various stimuli such as infections, cancers or protein aggregates as observed in certain diseases. NLRP3 is involved in the outset and development of several multifactorial diseases such as Alzheimer’s disease, atherosclerosis, and type II diabetes. In addition, gain-of-function mutations in Innate immune response is the organism’s first line of defence against dangers. A large array of immune receptors recognizing pathogen and damage molecular pattern initiate inflammation which allows pathogen clearance and healing of tissue injury. Among these receptors, NLRP3 is activated in response to the loss of cellular function and integrity. These cellular stresses can happen as a result of various stimuli such as infections, cancers or protein aggregates as observed in certain diseases. NLRP3 is involved in the outset and development of several multifactorial diseases such as Alzheimer’s disease, atherosclerosis, and type II diabetes. In addition, gain-of-function mutations in NLRP3 are directly linked to CAPS syndrome, a hereditary auto-inflammatory disease. Thus, it is of utmost importance to understand the precise mechanism regulating NLRP3. Currently, it is known that NLRP3 activation requires two signals: a priming signal followed by an activation signal which result in the formation of a multiproteic complex called inflammasome. The inflammasome is composed of the sensor NLRP3, the adaptors NEK7 and ASC, and the effector pro-caspase-1. Inflammasome assembly and activation leads to caspase-1 activation which, in turn, cleaves pro-inflammatory cytokine as well as GSDMD. GSDMD forms pores in the membrane, leading to a lytic programmed cell death known as pyroptosis. The mechanisms connecting priming and activation signals to inflammasome assembly are poorly known, even if several studies suggest an important role for post-translational modification of NLRP3, especially its ubiquitination. For instance, NLRP3 deubiquitination by BRCC3 is necessary for its activation.The results presented in this thesis focus on the role of ubiquitination in NLRP3 activation. In a first part, we were not able to highlight novel roles for BRCC3 in innate immunity. However, in a second part, we identified through mass spectrometry two ubiquitinated lysines. These lysines were previously known as substitution observed in CAPS patients. In order to study the impact of these two ubiquitinations on inflammasome activation, we generated mutant mice replacing the lysines by arginines to prevent the ubiquitination of each residue. Primary macrophages from these two Nlrp3 KR/KR murine line treated with priming signal LPS show IL-1β and IL-18 secretion as well as membrane permeabilization, despite the absence of activation signal. This secretion happens downstream of TRIF pathway and requires all inflammasome components NEK7, NLRP3, ASC, and caspase-1. This inflammasome activation upon LPS happens at a lower level than classical activation involving a priming and an activation signal. Despite this activation, no ASC specks were found upon LPS in Nlrp3 KR/KR. Finally, the two Nlrp3 KR/KR murine line seem to be more sensitive to LPS-induced endotoxic shock. Altogether, these results describe the identification of two ubiquitinated lysine on NLRP3, playing a role as inhibitory checkpoint in NLRP3 inflammasome activation pathway which could be used as future therapeutic targets

Lipo-chitooligosaccharide signalling blocks a rapid pathogen-induced ROS burst without impeding immunity

International audienc

Statistical analysis of the grapevines mortality associated Esca or Eutypa dieback foliar expression.

Esca and Eutypa dieback are two major wood diseases of grapevine in France. Their widespread distribution in vineyards leads to vine decline and to a loss in productivity. However, little is known either about the temporal dynamics of these diseases at plant level, and equally, the relationships between foliar expression of the diseases and vine death is relatively unknown too. To investigate this last question, we surveyed the vines of six vineyards cv. Cabernet Sauvignon in the Bordeaux region, by recording foliar symptoms, dead arms and dead plants from 2004 to 2010. In 2008, 2009 and 2010, approximately five percent of the asymptomatic vines died but the percentage of dead vines which had previously expressed esca foliar symptoms was higher, and varied between vineyards. A logistic regression model was used to determine the previous years of symptomatic expression associated with vine mortality. The mortality of esca is always associated with the foliar symptom expression of the year preceding vine death. One or two other earlier years of expression frequently represented additional risk factors. The Eutypa dieback symptom was also a risk factor of death, superior or equal to that of esca. The study of the internal necroses of vines expressing esca or Eutypa dieback is discussed in the light of these statistical results

Statistical analysis of grapevine mortality associated with esca or Eutypa dieback foliar expression

Esca and Eutypa dieback are two major wood diseases of grapevine in France. Their widespread distribution in vineyards leads to vine decline and to a loss in productivity. However, little is known either about the temporal dynamics of these diseases at plant level, and equally, the relationships between foliar expression of the diseases and vine death is relatively unknown too. To investigate this last question, the vines of six vineyards cv. Cabernet Sauvignon in the Bordeaux region were surveyed, by recording foliar symptoms, dead arms and dead plants from 2004 to 2010. In 2008, 2009 and 2010, approximately five percent of the asymptomatic vines died but the percentage of dead vines which had previously expressed esca foliar symptoms was higher, and varied between vineyards. A logistic regression model was used to determine the previous years of symptomatic expression associated with vine mortality. The mortality of esca is always associated with the foliar symptom expression of the year preceding vine death. One or two other earlier years of expression frequently represented additional risk factors. The Eutypa dieback symptom was also a risk factor of death, superior or equal to that of esca. The study of the internal necroses of vines expressing esca or Eutypa dieback is discussed in the light of these statistical results

Clinical Evaluation of Diode (980 nm) Laser-Assisted Nonsurgical Periodontal Pocket Therapy: A Randomized Comparative Clinical Trial and Bacteriological Study.

peer reviewedBackground: Mechanical debridement is the gold standard in the periodontitis therapy. However, it is suggested that adjunctive use of lasers can result in a more effective treatment outcome. Objective: Evaluate the efficiency of diode laser-assisted nonsurgical therapy of periodontitis as adjunctive to scaling and root planing (SRP). Methods: One hundred sixty vertical bone defects [pocket depth (PD) at baseline ≥6 mm] had been randomly allocated to receive SRP alone (group C) or SRP coupled to a diode laser (980 nm) protocol (group C+L): SRP, irrigation with hydrogen peroxide solution (3%), de-epithelization of the internal and external gingiva followed by blood stabilization, and coagulation by laser beam were made. Beam parameters: 10 μsec/pulse duration, 10 kHz, pick power of 10 W, average power of 1 W, and fiber diameter of 400 μm. Plaque index (PI), bleeding on probing, gingival recession (GR), clinical attachment level (CAL), and PD were measured at baseline, at 6 weeks, 12 weeks, 18 weeks, 6 months, and 12 months. Microbiological data were collected randomly from 26 pockets from both groups at baseline, 6 weeks, 12 weeks, and 6 months after treatment. Results: At all periods of follow-up, there was a significant difference between both groups in all clinical parameters except in GR. In group C+L, 76% of pockets had PD ≤3 mm after 12 months of follow-up and an average of PD = 1.77 ± 0.46 mm, while 56% of pockets in group control (C) had an average of PD = 5.00 ± 0.83 mm after 12 months of follow-up. Total bacteria count in group C + L was significantly lower compared to group C only at 12 weeks and 6 months of follow-up. Furthermore, there was high significant decrease in the number of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia at all the follow-up periods. Conclusions: As adjunctive to SRP, diode laser-assisted nonsurgical therapy of periodontitis has significantly improved clinical parameters of PI and POB and has significantly reduced the clinical attachment loss (CAL) and PD compared to the control group after 1 year of follow-up. A significant reduction in periodontal pathogens has been observed in group C + L only at 12 weeks and 6 months of follow-up

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors

International audienc

Un duo grenoblois gagnant? L’intersection de l’Automatique et de l’Informatique: L’aventure du MAT 01

Conférences "hors les murs" co-organisées par l'UGA en parrallèle à l'exposition Histoire de savoir(s). L’université Grenoble Alpes (1339-2021) au musée de l'ancien évêché, du 19 mai 2021 au 6 mars 2022. Le programme est disponible sur le site de la direction de la culture et de la culture scientifique de l'UGA : l'ouvre-boîte. https://culture.univ-grenoble-alpes.fr/menu-principal/patrimoines/histoire-de-l-universite/histoire-de-l-universite-698992.kjsp?RH=1594815597121National audienc

NFP, a LysM protein controlling Nod factor perception, also intervenes in Medicago truncatula resistance to pathogens

International audiencePlant LysM proteins control the perception of microbial-derived N-acetylglucosamine compounds for the establishment of symbiosis or activation of plant immunity. This raises questions about how plants, and notably legumes, can differentiate friends and foes using similar molecular actors and whether any receptors can intervene in both symbiosis and resistance. To study this question, nfp and lyk3 LysM-receptor like kinase mutants of Medicago truncatula that are affected in the early steps of nodulation, were analysed following inoculation with Aphanomyces euteiches, a root oomycete. The role of NFP in this interaction was further analysed by overexpression of NFP and by transcriptome analyses. nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A.euteiches, whereas NFP overexpression increased resistance. Transcriptome analyses on A.euteiches inoculation showed that mutation in the NFP gene led to significant changes in the expression of c. 500 genes, notably involved in cell dynamic processes previously associated with resistance to pathogen penetration. nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M.truncatula immunity, suggesting an unsuspected role for NFP in the perception of pathogenic signals

Zeb1 represses TCR signaling, promotes the proliferation of T cell progenitors and is essential for NK1.1+ T cell development

International audienceT cell development proceeds under the influence of a network of transcription factors (TFs). The precise role of Zeb1, a member of this network, remains unclear. Here, we report that Zeb1 expression is induced early during T cell development in CD4 − CD8 − double-negative (DN) stage 2 (DN2). Zeb1 expression was further increased in the CD4 + CD8 + double-positive (DP) stage before decreasing in more mature T cell subsets. We performed an exhaustive characterization of T cells in Cellophane mice that bear Zeb1 hypomorphic mutations. The Zeb1 mutation profoundly affected all thymic subsets, especially DN2 and DP cells. Zeb1 promoted the survival and proliferation of both cell populations in a cell-intrinsic manner. In the periphery of Cellophane mice, the number of conventional T cells was near normal, but invariant NKT cells, NK1.1 + γδ T cells and Ly49 + CD8 T cells were virtually absent. This suggested that Zeb1 regulates the development of unconventional T cell types from DP progenitors. A transcriptomic analysis of WT and Cellophane DP cells revealed that Zeb1 regulated the expression of multiple genes involved in the cell cycle and TCR signaling, which possibly occurred in cooperation with Tcf1 and Heb. Indeed, Cellophane DP cells displayed stronger signaling than WT DP cells upon TCR engagement in terms of the calcium response, phosphorylation events, and expression of early genes. Thus, Zeb1 is a key regulator of the cell cycle and TCR signaling during thymic T cell development. We propose that thymocyte selection is perturbed in Zeb1-mutated mice in a way that does not allow the survival of unconventional T cell subsets