LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici

Increased drug efflux compromises the efficacy of a large panel of treatments in the clinic against cancer or bacterial, fungal, and viral diseases, and in agriculture due to the emergence of multidrug-resistant pathogenic fungi. Until recently, to demonstrate increased drug efflux, the use of labeled drugs or fluorescent dyes was necessary. With the increasing sensitivity of detection devices, direct assessment of drug efflux has become realistic. Here, we describe a medium-throughput method to assess the intracellular drug concentration in the plant pathogenic fungus Zymoseptoria tritici cultivated in the presence of a sublethal fungicide concentration. As a model fungicide, we used the succinate-dehydrogenase inhibitor boscalid. The boscalid concentration was assessed in the different culture fractions using mass spectrometry linked to liquid chromatography (LC-MS/MS). The ratio between the intracellular and total boscalid amount was used as an inversed proxy for the efflux activity. Using isogenic mutant strains known for their differential efflux capacities, we validated the negative correlation between the intracellular boscalid concentration and efflux activity. In addition, intra-cellular fungicide accumulation explains the susceptibility of the tested strains to boscalid. This assay may be useful in lead development when a new molecule displays good inhibitory activity against its isolated target protein but fails to control the target organism

A new mode of action for fluopicolide: modification of the cellular localization of a spectrin-like protein

Fluopicolide does not show any cross resistance to other commercial oomycete fungicides. lt controls fungi resistant to phenylamides, strobilurins and dimethomorph, suggesting that fluopicolide has a novel mode of action. Zoospores swell and lyse rapidly upon treatment with fluopicolide. Irnmunolocalization studies showed that a cytoskeleton-associated protein, spectrin-like protein, was delocalized after fluopicolide treatment. This happens in both zoospores and hyphae of P. infestans. Spectrins play a role in membrane stability in mammalian cells, but are poorly characterized in fungi. Here they might have an important role in maintaining membrane integrity as well. Western blot experiments indicated the presence of spectrin-like protein in a crude extract of P. infestans and purification of the relevant proteins is ongoing. The possibility that the observed cellular delocalization of spectrin-like protein is the result of an effect on another, related target cannot be excluded yet. The role of spectrin-like proteins in oomycete development and the target of fluopicolide need further investigation.Le fluopicolide ne présente pas de résistance croisée avec les autres fongicides commerciaux actifs sur oomycètes. Il contrôle les champignons résistants aux phénylamines, aux strobilurines et au diméthomorphe, ce qui sous-entend que le fluopicolide possède un nouveau mode d'action. Les zoospores gonflent et sont lysées rapidement après traitement au fluopicolide. Les études d'immunolocalisation ont montré qu'une protéine associée au cytosquelette, protéine de type spectrine, était délocalisée après traitement au fluopicolide. Ceci se produit aussi bien au niveau des zoospores que des hyphes de P. infestans. Les spectrines jouent un rôle dans la stabilité de la membrane des cellules mammifères mais ne sont que peu caractérisées chez les champignons. Dans ce cas, elles pourraient également jouer un rôle important au niveau du maintient de l'intégrité de la membrane. Des expériences dites de «Western blot» indiquent la présence de protéines de type spectrine dans un extrait brut de P. infestans et la purification des protéines pertinentes est en cours. La possibilité que la délocalisation cellulaire observée de la protéine de type spectrine soit le résultat d'un effet sur une autre cible similaire ne peut pas être exclue à l'heure actuelle. Le rôle des protéines de type spectrine dans le développement des oomycètes ainsi que la cible du fluopicolide nécessitent de plus amples investigations

Creation of a collection of Botrytis cinerea T-DNA transformants for pathogenic development and plant defence studies

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A Similar Secretome Disturbance as a Hallmark of Non-pathogenic Botrytis cinerea ATMT-Mutants?

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