Olivier Fernandez

Rhamnolipids (RLs) are bacterial glycolipids with potential applications in the biocontrol of plant pathogens. Although RLs are known to activate plant immune responses, the underlying signaling mechanisms remain poorly understood. Calcium-dependent protein kinases (CPKs) are a large family of kinases involved in various functions in plants including signaling of the plant immunity. Here, we investigated the contribution of At CPK5 and At CPK6 to RL-triggered immunity in Arabidopsis. RL treatment induced the expression of both AtCPK5 and AtCPK6 genes in Arabidopsis leaves. Functional analyses revealed that RL-induced responses, including reactive oxygen species production and the expression of defense-related genes ( AtWRKY46 , AtFRK1 and AtPR1 ), were enhanced in cpk5/6 mutants compared to wild-type plants. The cpk5 mutant exhibited intermediate responses, whereas cpk6 alone had little effect, except on AtFRK1 expression, indicating a predominant role for At CPK5 in regulating RL-triggered signaling. However, cpk5/6 mutations did not affect RL-induced electrolyte leakage or RL-mediated resistance to Pseudomonas syringae pv. tomato DC3000. Together, these results suggest that At CPK5 and At CPK6 negatively modulate RL-triggered immune signaling, while additional components contribute to the regulation of downstream defense responses.

L’objectif de mes travaux de thèse était d’étudier le métabolisme du tréhalose chez la vigne (Vitis vinifera L. cv. Chardonnay) en réponse à l’exposition à deux stress : le froid" chilling " et l’infection par le champignon pathogène Botrytis cinerea.Pour cela, nous avons tout d’abord optimisé un dosage du tréhalose par fluorimétrie qui nous a permis de caractériser le métabolisme du tréhalose en réponse à ces deux stress.Le métabolisme du tréhalose est activé différemment par le froid dans les organes dela vigne. Les gènes VvTPPA, codant une enzyme de synthèse du tréhalose, et VvTRE, codantla tréhalase, l’enzyme de dégradation, sont respectivement induits et réprimés dans les feuilleset leur expression est corrélée à une augmentation de la concentration en tréhalose. En outre,aucune synthèse de tréhalose n’est mesurée dans les tiges et il n’est pas détectable dans les racines. Enfin, la synthèse de T6P, son précurseur, est précoce dans les feuilles (3 heures aprèsl’exposition au froid). Nos résultats s’accordent avec le modèle actuel faisant du T6P une molécule-signal, corrélée à la concentration en saccharose. Ils excluent, pour le tréhalose, une participation significative à l’osmorégulation en réponse au froid chez la vigne. Par ailleurs,nous avons utilisé des plants de vigne bactérisés par Burkholderia phytofirmans, une bactérie endophyte induisant une tolérance au froid chez cette plante. Nous y avons observé une synthèse de T6P et de tréhalose et nous pensons que cette synthèse pourrait constituer une composante importante de la tolérance induite au froid.Lors de l’infection de feuilles de vitroplants de vigne par B. cinerea, nous avons observé (i) une forte augmentation de la quantité de tréhalose, (ii) l’induction de l’expression du gène VvTRE et (iii) l’augmentation de l’activité tréhalase. En revanche, aucune augmentation de la concentration en T6P n’a été détectée. Nous pensons donc que la synthèsede tréhalose in planta n’est pas favorisée durant l’infection et que le tréhalose détecté est probablement d’origine fongique. Nos résultats sont compatibles avec l’hypothèse selon laquelle l’induction du gène codant la tréhalase et l’augmentation concomitante de son activité interviendraient lors des interactions plantes-agents pathogènes pour éviter toute perturbation du rôle de molécule-signal joué par le T6P.Au final, le métabolisme du tréhalose participe à la réponse aux stress environnementaux chez la vigne et son étude mériterait d’être approfondie, notamment en ce qui concerne les stress biotiques.

Rhamnolipids (RLs) are a class of glycolipids naturally synthesized by bacteria potentially used in the biocontrol of plant pathogenic microorganisms in agriculture. While RLs trigger an immune response in plants, little is known about the signaling mechanisms involved after RL perception. Calcium Dependent Protein Kinases (CDPKs/CPKs) are a large family of kinases involved in various functions in plants including the regulation of the resistance response to phytopathogens. Here, we investigated the role of AtCPK5 and AtCPK6 in RL-triggered immunity in Arabidopsis thaliana (hereafter referred to as Arabidopsis). Gene expression analysis showed that RLs up-regulate the expression of both genes in Arabidopsis leaves. Using a functional approach, we demonstrated that signaling events exemplified by reactive oxygen species (ROS) production and defense gene expression including AtWRKY46 , AtFRK1 and AtPR1 are increased in cpk5/6 mutant plants compared to wild-type (WT) plants. Moreover, cpk5 mutants displayed intermediary responses while cpk6 mutation alone did not change the immune profile activated by RLs, except for AtFRK1 . Those results suggest that AtCPK5 and AtCPK6 regulate RL-triggered defenses, with AtCPK5 likely playing a more important role. Yet, the cpk5/6 mutations did not affect RL-triggered electrolyte leakage nor enhanced RL-mediated resistance to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our results therefore show that additional signaling components regulate AtCPK5 and AtCPK6 -mediated plant immune responses triggered by RLs.

Neofusicoccum parvum is a wood-colonizing fungus that threatens grape production. Its ability to colonize the host may involve the secretion of effector molecules, such as the phytotoxic metabolite (R)-mellein. However, the role of (R)-mellein in disease development and the factors influencing its production are poorly understood. Additionally, how this pathogen alters its metabolome during host interaction remains unclear. Here, we compared the metabolomes and (R)-mellein production of two wild-type N. parvum strains (Bt67 and NpB) and a UV mutant (UV9) cultured with grapevine wood in bioreactors. We found that wild-type strains reduced (R)-mellein production but increased the diversity of secreted metabolites, whereas the UV mutant showed minimal changes when grown with the host tissue. In particular, grapevine wood promoted the production of auxin-related compounds, tyramines, and phenylethanoids. Our findings suggest that N. parvum adapts its metabolite profile in response to host biomass, producing compounds with phytotoxic and antibiotic properties. Finally, (R)-mellein was purified using Centrifugal Partition Chromatography and preparative HPLC, which will aid future research into its role in disease.

(1) Background: Grapevine trunk diseases (GTDs) have become a global threat to vineyards worldwide. These diseases share three main common features. First, they are caused by multiple pathogenic micro-organisms. Second, these pathogens often maintain a long latent phase, which makes any research in pathology and symptomatology challenging. Third, a consensus is raising to pinpoint combined abiotic stresses as a key factor contributing to disease symptom expression. (2) Methods: We analyzed the impact of combined abiotic stresses in grapevine cuttings artificially infected by two fungi involved in Botryosphaeria dieback (one of the major GTDs), Neofusicoccum parvum and Diplodia seriata. Fungal-infected and control plants were subjected to single or combined abiotic stresses (heat stress, drought stress or both). Disease intensity was monitored thanks to the measurement of necrosis area size. (3) Results and conclusions: Overall, our results suggest that combined stresses might have a stronger impact on disease intensity upon infection by the less virulent pathogen Diplodia seriata. This conclusion is discussed through the impact on plant physiology using metabolomic and transcriptomic analyses of leaves sampled for the different conditions.

Two Neofusicoccumparvum isolates and a UV mutant were characterized for their phytotoxin production in vitro, their pathogenicity on grapevine, and their genome sequenced. The isolate Np-Bt67 produced high level of (-)-terremutin, but almost no (R)-mellein, and it was the most aggressive on grapevine, triggering apoplexy. Similar symptoms were not induced by purified (-)-terremutin. The isolate Bourgogne S-116 (Np-B) produced 3-fold less (-)-terremutin and high amounts of (R)-mellein, but it was less aggressive on grapevine than Np-Bt67. The UV9 mutant obtained from Np-B (NpB-UV9) no longer produced (-)-terremutin but overproduced (R)-mellein by 2.5-fold, and it was as pathogenic as its parent. NpB-UV9 differed from its parent by simple mutations in two genes (transcription factor UCR-NP2_6692, regulatory protein UCR-NP2_9007), not located neither near (R)-mellein, nor (-)-terremutin biosynthetic genes, but likely involved in the control of (-)-terremutin biosynthesis. Grapevine immunity was disturbed upon challenge with these pathogens or purified phytotoxins, leading to an upregulation of SA-dependent defenses, while (-)-terremutin interfered with host JA/ET-dependent defenses. Our results suggest that neither (-)-terremutin nor (R)-mellein alone is essential for the pathogenicity of N. parvum on grapevine, since isolate/mutant non-producing these toxins in vitro is pathogenic. However, these phytotoxins could play a quantitative role in the infection process.

Copper-based preparations have been used for more than 100 years in viticulture to control downy mildew caused by Plasmopara viticola. LC2017, and a new low-copper-based formulation, has been developed to control grapevine trunk diseases (GTDs). Previous greenhouse studies showed the potential of LC2017 to control GTDs by both fungistatic and plant defense elicitor effects. Here, we further characterize the effects of LC2017 in the field determining its impact on: (i) incidence of Esca, (ii) the vine microbiome, (iii) the vine physiology and (iv) enological parameters of juices. We observed a progressive decrease of cumulate Esca incidence in treated vines over the years with annual fluctuation related to the known erratic emergence of GTD symptoms. Neither harmful effects of LC2017 on the vine microbiota, nor on vine physiology were observed (at both transcriptomic and metabolomic levels). Similarly, no impact of LC2017 was observed on the enological properties of berries except for sugar content in juice from esca-diseased vines. The most important result concerns the transcriptomic profiles: that of diseased and LC2017 treated vines differs from that of disease untreated ones, showing a treatment effect. Moreover, the transcriptomic profile of diseased and LC2017-treated vines is similar to that of untreated asymptomatic vines, suggesting control of the disease.

As obligatory parasites, plant viruses alter host cellular metabolism. There is a lack of information on the variability of virus-induced metabolic responses among genetically diverse plants in a natural context with daily changing conditions. To decipher the metabolic landscape of plant-virus interactions in a natural setting, twenty-six and ten accessions of Arabidopsis thaliana were inoculated with Turnip mosaic virus (TuMV), in two field experiments over 2 years. The accessions were measured for viral accumulation, above-ground biomass, targeted and untargeted metabolic profiles. The phenotypes of the accessions ranged from susceptibility to resistance. Susceptible and resistant accessions were shown to have different metabolic routes after inoculation. Susceptible genotypes accumulate primary and secondary metabolites upon infection, at the cost of hindered growth. Twenty-one metabolic signatures significantly accumulated in resistant accessions whereas they maintained their growth as mock-inoculated plants without biomass penalty. Metabolic content was demonstrated to discriminate and be highly predictive of the susceptibility of inoculated Arabidopsis. This study is the first to describe the metabolic landscape of plant-virus interactions in a natural setting and its predictive link to susceptibility. It provides new insights on plant-virus interactions. In this undomesticated species and in ecologically realistic conditions, growth and resistance are in a permanent conversation.