Caractérisation fonctionnelle de lipide acyl-hydrolases (LAH) (Etude de l'implication de AtPLP2 dans la résistance aux agents pathogènes chez Arabidopsis thaliana)

La libération d acides gras à partir de lipides membranaires est régulée en réponse à différents stress. Elle est catalysée par des enzymes appelées lipide acyl-hydrolases (LAH) dont le rôle supposé est d être impliquées dans la résistance antimicrobienne et de fournir des précurseurs de dérivés d acides gras, les oxylipines. L exploration du génome de la plante Arabidopsis thaliana nous a permis de mettre en évidence plusieurs groupes structuraux de gènes codant pour des LAH putatives et de montrer que plusieurs membres sont induits en réponse à différents microbes. Nous avons donné la priorité à l étude fonctionnelle des patatines. Cette famille comprend 9 gènes, dont deux (AtPLP2 et AtPLP7) sont induits dans les feuilles infectées. L accumulation de AtPLP2 en réponse à Botrytis cinerea et Pseudomonas syringae est dépendante des signaux de défense, éthylène et acide jasmonique. L expression d une fusion AtPLP2-GFP et l analyse des propriétés biochimiques indiquent que cette protéine est une LAH cytoplasmique. Des plantes transgéniques modifiées dans l expression de AtPLP2 ont été testées pour leurs niveaux de résistance. De façon inattendue, AtPLP2 favorise la progression du champignon Botrytis et la susceptibilité vis-à-vis de bactéries avirulentes, alors que les plantes réprimées sont plus résistantes. Ces données indiquent que l activité lipolytique dépendante de AtPLP2 est recrutée par certains pathogènes pour faciliter la colonisation de la plante hôte. AtPLP2 semble également potentialiser la mort cellulaire lors de l infection par B. cinerea et réduire la capacité de la réaction d hypersensibilité à restreindre la multiplication de bactéries avirulentes. L étude systématique des LAH chez Arabidopsis a permis d identifier des candidats prometteurs pour une analyse fonctionnelle détaillée. Les outils générés, dont les mutants knock-out, seront au centre des études futures des phénomènes de mobilisation d acides gras dans les mécanismes de défense des plantes.Membrane lipid catabolism is regulated in response to several stresses. Enzymes responsible for lipid hydrolysis are named lipid acyl hydrolases (LAH). An important role anticipated for such enzymes is to be involved in antimicrobial resistance and to provide precursors for the biosynthesis of oxylipins that are regulatory fatty acid derivatives. Exploration of the Arabidopsis thaliana genome has revealed the existence of numerous structural families of potential LAH genes, with members being upregulated in response to biotic stress. We have given priority to the functional study of Arabidopsis LAH related to patatin. This family comprises 9 members, two of which (AtPLP2 and AtPLP7) being strongly upregulated in leaves challenged with pathogens. AtPLP2 protein accumulation in response to the fungus Botrytis cinerea or Pseudomonas syringae bacteria is dependent on jasmonic acid and ethylene signaling. Expression of a AtPLP2-GFP fusion and biochemical analysis of recombinant AtPLP2 indicates that AtPLP2 encodes a cytoplasmic LAH. Transgenic plants with altered levels of AtPLP2 protein were generated and assayed for pathogen resistance. Unexpectedly, AtPLP2 expression increases B. cinerea colonization and susceptibility to avirulent bacteria whereas silenced plants displayed enhanced resistance. Collectively, the data indicate that AtPLP2-encoded lipolytic activity is recruited by pathogens with different lifestyles to facilitate host colonization. Particularly, AtPLP2 potentiates plant cell death upon infection by B. cinerea and reduces the efficiency of the hypersensitive response known to normally restrict avirulent bacteria multiplication. This global Arabidopsis LAH study opened some perspectives in identifying several candidates genes for detailed functional studies. Tools like numerous LAH knock-out mutants obtained will be the basis of our future work to decipher fatty acid mobilisation processes during plant defense responses.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Cross Talk between Reactive Nitrogen and Oxygen Species during the Hypersensitive Disease Resistance Response

Review relativa ai ruoli di ossido nitrico e specie reattive dell'ossigeno nella morte cellulare ipersensibile delle piante, come forma estrema di resistenza a patogeni

Targeting the AtCWIN1 gene to explore the role of invertases in sucrose transport in roots and during Botrytis cinerea infection

Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H+ symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e. STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and AtVIN genes accompanied with an enhanced vacuolar invertase activity, suggesting that the AtCWIN1-independent pathway is efficient upon infection. In absence of AtCWIN1, we postulate that intracellular sucrose hydrolysis is sufficient to provide intracellular hexoses to maintain sugar homeostasis in host cells and to fuel plant defenses. Finally, we demonstrated that B. cinerea possesses its own functional sucrolytic machinery and hexose uptake system, and does not rely on the host apoplastic invertases

EST sequencing for fungal genes expressed in the arbuscular symbiosis

International audienc

Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responses

The three closely related Arabidopsis basic leucine zipper (bZIP) transcription factors TGA2, TGA5 and TGA6 are required for the establishment of the salicylic acid (SA)-dependent plant defense response systemic acquired resistance, which is effective against biotrophic pathogens. Here we show that the same transcription factors are essential for the activation of jasmonic acid (JA)- and ethylene (ET)-dependent defense mechanisms that counteract necrotrophic pathogens: the tga256 triple mutant is impaired in JA/ET-induced PDF1.2 and b-CHI expression, which correlates with a higher susceptibility against the necrotroph Botrytis cinerea. JA/ET induction of the trans-activators ERF1 and ORA59, which act upstream of PDF1.2, was slightly increased (ERF1) or unaffected (ORA59). PDF1.2 expression can be restored in the tga256 mutant by increased expression of ORA59, as observed in the tga256 jin1 quadruple mutant, which lacks the transcription factor JIN1/AtMYC2 that functions as a negative regulator of the JA/ET-dependent anti-fungal defense program. Whereas JA/ET-induced PDF1.2 expression is strongly suppressed by SA in wild-type plants, no negative effect of SA on PDF1.2 expression was observed in the tga256 jin1 quadruple mutant. These results imply that the antagonistic effects of TGA factors and JIN1/AtMYC2 on the JA/ET pathway are necessary to evoke the SA-mediated suppression of JA/ET-induced defense responses

Overexpression of the VvSWEET4 Transporter in Grapevine Hairy Roots Increases Sugar Transport and Contents and Enhances Resistance to Pythium irregulare, a Soilborne Pathogen

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Synthesis, phloem mobility and induced plant resistance of synthetic salicylic acid amino acid or glucose conjugates

International audienceBACKGROUND: The growing demand for food, combined with a strong social expectation for a diet produced with fewer conventional agrochemical inputs, has led to the development of new alternatives in plant protection worldwide. Among different possibilities, the stimulation of the plant innate immune system by chemicals represents a novel and promising way. The vectorization strategy of an active ingredient that we previously developed with fungicides can potentially extend to salicylic acid (SA) or its halogenated analogues.RESULTS: Using the click chemistry method, six new conjugates combining SA or two mono- or di-halogenated analogues with L-glutamic acid or β-D-glucose via a 1,2,3-triazole nucleus have been synthesized. Conjugate 8a, which is derived from SA and glutamic acid, showed high phloem mobility in the Ricinus model, similar to that of SA alone despite a much higher steric hindrance. In vivo bioassays of the six conjugates against two maize pathogenic fungi Bipolaris maydis and Fusarium graminearum revealed that, unlike SA, the amino acid conjugate 8a with good phloem mobility exerted a protective effect not only locally at the application site, but also in distant stem tissues after foliar application. Moreover, compounds 8a and 8b induced up-regulation of both defense-related genes ZmNPR1 and ZmPR1 similar to their parent compounds upon challenge inoculation with B. maydis.CONCLUSION: The vectorization of salicylic acid or its halogenated derivatives by coupling them with an α-amino acid can be a promising strategy to stimulate SA-mediated plant defenses responses against pathogens outside the application site

Comparison of the molecular responses of tolerant, susceptible and highly susceptible grapevine cultivars during interaction with the pathogenic fungus Eutypa lata

Eutypa lata is the causal agent of eutypa dieback, one of the most destructive grapevine trunk disease that causes severe economic losses in vineyards worldwide. This fungus causes brown sectorial necrosis in wood which affect the vegetative growth. Despite intense research efforts made in the past years, no cure currently exists for this disease. Host responses to eutypa dieback are difficult to address because E. lata is a wood pathogen that causes foliar symptoms several years after infection. With the aim to classify the level of susceptibility of grapevine cultivars to the foliar symptoms caused by E. lata, artificial inoculations of Merlot, Cabernet Sauvignon, and Ugni Blanc were conducted over 3 years. Merlot was the most tolerant cultivar, whereas Ugni Blanc and Cabernet Sauvignon exhibited higher and differential levels of susceptibility. We took advantage of their contrasting phenotypes to explore their defense responses, including the activation of pathogenesis-related (PR) genes, oxylipin and phenylpropanoid pathways and the accumulation of stilbenes. These analyses were carried out using the millicell system that enables the molecular dialogue between E. lata mycelium and grapevine leaves to take place without physical contact. Merlot responded to E. lata by inducing the expression of a large number of defense-related genes. On the contrary, Ugni Blanc failed to activate such defense responses despite being able to perceive the fungus. To gain insight into the role of carbon partitioning in E. lata infected grapevine, we monitored the expression of plant genes involved in sugar transport and cleavage, and measured invertase activities. Our results evidence a coordinated up-regulation of VvHT5 and VvcwINV genes, and a stimulation of the cell wall invertase activity in leaves of Merlot elicited by E. lata, but not in Ugni Blanc. Altogether, this study indicates that the degree of cultivar susceptibility is associated with the activation of host defense responses, including extracellular sucrolytic machinery and hexose uptake during the grapevine/E. lata interaction. Given the role of these activities in governing carbon allocation through the plant, we postulate that the availability of sugar resources for either the host or the fungus is crucial for the outcome of the interaction

The glutaredoxin ATGRXS13 is required to facilitate Botrytis cinerea infection of Arabidopsis thaliana plants.

International audienceBotrytis cinerea is a major pre- and post-harvest necrotrophic pathogen with a broad host range that causes substantial crop losses. The plant hormone jasmonic acid (JA) is involved in the basal resistance against this fungus. Despite basal resistance, virulent strains of B. cinerea can cause disease on Arabidopsis thaliana and virulent pathogens can interfere with the metabolism of the host in a way to facilitate infection of the plant. However, plant genes that are required by the pathogen for infection remain poorly described. To find such genes, we have compared the changes in gene expression induced in A. thaliana by JA with those induced after B. cinerea using genome-wide microarrays. We have identified genes that are repressed by JA but that are induced by B. cinerea. In this study, we describe one candidate gene, ATGRXS13, that encodes for a putative glutaredoxin and that exhibits such a crossed expression. In plants that are infected by this necrotrophic fungus, ATGRXS13 expression was negatively controlled by JA and TGA transcription factors but also through a JA-salicylic acid (SA) cross-talk mechanism as B. cinerea induced SA production that positively controlled ATGRXS13 expression. Furthermore, plants impaired in ATGRXS13 exhibited resistance to B. cinerea. Finally, we present a model whereby B. cinerea takes advantage of defence signalling pathways of the plant to help the colonization of its host