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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.

Fungi are the most prevalent plant pathogens, causing annually important damages. To infect and colonize their hosts, they secrete effectors including hydrolytic enzymes able to kill and macerate plant tissues. These secreted proteins are transported from the Endoplasmic Reticulum and the Golgi apparatus to the extracellular space through intracellular vesicles. In pathogenic fungi, intracellular vesicles were described but their biogenesis and their role in virulence remain unclear. In this study, we report the essential role of clathrin heavy chain (CHC) in the pathogenicity of Botrytis cinerea , the agent of gray mold disease. To investigate the importance of this protein involved in coat vesicles formation in eukaryotic cells, a T-DNA insertional mutant reduced in the expression of the CHC-encoding gene, and a mutant expressing a dominant-negative form of CHC were studied. Both mutants were strongly affected in pathogenicity. Characterization of the mutants revealed altered infection cushions and an important defect in protein secretion. This study demonstrates the essential role of clathrin in the infectious process of a plant pathogenic fungus and more particularly its role in virulence factors delivery., Competing Interests: M-PL was employed by the company Bayer SAS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Souibgui, Bruel, Choquer, de Vallée, Dieryckx, Dupuy, Latorse, Rascle and Poussereau.)

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciens- mediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted proteins in the infection process. In the twelve non-pathogenic mutants, the differentiation of infection cushions was also impaired, suggesting a link between the penetration structures and the secretion of proteins involved in the virulence of the pathogen., (Copyright © 2019 de Vallée, Bally, Bruel, Chandat, Choquer, Dieryckx, Dupuy, Kaiser, Latorse, Loisel, Mey, Morgant, Rascle, Schumacher, Simon, Souibgui, Viaud, Villalba and Poussereau.)