Valérie Toquin

Fungal model systems and the elucidation of pathogenicity determinants

Highlights • History of seven fungal species used as models for studying development and pathogenicity.• Outline of central stages of their life cycle and their infection processes.• Molecular toolkits used to study different aspects of pathogenicity.• Insight gained from genome sequencing projects.• Current research trends and future challenges.

Site-directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B.cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L) , affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radiolabelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.

Novel Tools to Identify the Mode of Action of Fungicides as Exemplified with Fluopicolide

The expanding field of fungal genomics stimulates the development of genome wide functional tools and comparative analyses in plant pathogenic fungi. As a consequence, transcriptomic, proteomic and metabolomic studies coupled with high throughput forward and reverse genetics are now available in a significant number of fungal plant pathogens (e.g. Ustilago maydis, Magnaporthe grisea, Fusarium graminearum, Botrytis cinerea). Genomics together with classical biochemical tools and microscopy offer the possibility to accelerate the identification of the biochemical mode of action of novel fungicides. This knowledge is also required to discover efficiently novel antifungal compounds and to characterize and follow efficiently the emergence of resistance. The available genomic tools for plant pathogenic fungi will be reviewed as exemplified with the mode of action of fluopicolide, a novel fungicide active against Oomycetes. Biological studies performed with Phytophthora infestans and Plasmopara viticola showed that fluopicolide affects the release and motility of zoospores and the germination of cysts, as well as mycelial growth and sporulation. Biochemical studies showed that its mode of action differs from that of known anti-oomycetes compounds. Fluopicolide does not show cross-resistance to commercial fungicide classes such as phenylamides, strobilurins (QoIs) and carboxylic acid amides (CAAs). Cytological studies in P. infestans showed that fluopicolide specifically modifies the spatial and cellular distribution of proteins labelled by antibodies specific for animal cytoskeleton associated proteins spectrin. Treatments with fluopicolide induced a fast redistribution of spectrin-like protein(s) from the membrane to the cytoplasm in both hyphae and zoospores. Whereas animal spectrin(s) play an important role in membrane stability, they are poorly characterized in fungi and oomycetes. Cytoskeletal proteins such as actins, tubulins, integrins and spectrins provide structural stability to cells as they form a network sustaining the plasma membrane. Fluopicolide may interfere and destabilize this network leading to cell disorganization. This hypothesis is supported by the observation that treatments of zoospores lead to the relocalization of spectrin-like protein(s) into the cytoplasm within a few minutes followed immediately by cell swelling and burst. Preliminary data of gene expression profiling in P. sojae treated cells showed a differential expression (up and down regulation) of genes involved in vesicular transport. The link between golgi function, vesicle transport and cellular relocation of spectrin like proteins will be discussed.