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National audience; The Botrytis species complex responsible for grey mould disease on grapevine is composed of two species: Botrytis cinerea the major one (about 90%) and Botrytis pseudocinerea. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they do differ in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilide, B. pseudocinerea is naturally resistant. Enzyme assays showed that in B. pseudocinerea the fenhexamid target enzyme, the sterol 3-ketoreductase was less sensitive to fenhexamid. In addition, a synergic effect between fenhexamid and sterol 14Α- demethylation inhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase was observed in B. pseudocinerea. These results could suggest detoxification of fenhexamid by cytochromes P450. The cyp684 gene showing the strongest similarity to cyp51 among all B. cinerea cytochrome P450 genes was found strongly overexpressed in the presence of fenhexamid in B. pseudocinerea. In this work, we studied separately the effect of B. pseudocinerea erg27 polymorphism, erg27 encoding 3-ketoreductase, and of the recently identified cytochrome P450 gene, cyp684, on resistance to fenhexamid. The objective is to determine their respective implication in resistance. Experiments were conducted by exchange between erg27B.cinerea and erg27B.pseudocinerea in B. cinerea and by cyp684 deletion in B. pseudocinerea. In parallel, metabolization studies are conducted to identify metabolites and test their activity on Botrytis spp.

International audience; The Botrytis species complex responsible for grey mould disease on grapevine is composed of two species: Botrytis cinerea the major one (about 90%) and Botrytis pseudocinerea. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they do differ in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilide, B. pseudocinerea is naturally resistant. Enzyme assays showed that in B. pseudocinerea the fenhexamid target enzyme, the sterol 3-ketoreductase was less sensitive to fenhexamid. In addition, a strong synergism between fenhexamid and sterol 14α-demethylation inhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase was observed in B. pseudocinerea. These results could suggest detoxification of fenhexamid by cytochromes P450. The cyp684 gene showing the strongest similarity to cyp51 among all B. cinerea cytochrome P450 genes was found strongly overexpressed in the presence of fenhexamid in B. pseudocinerea. In this work, we studied separately the effect of B. pseudocinerea erg27 polymorphism, erg27 encoding 3-ketoreductase, and of the recently identified cytochrome P450 gene, cyp684, on resistance to fenhexamid, respectively by erg27 gene, and cyp684 inactivation. In parallel, metabolization studies are conducted to identify metabolites and test their activity on Botrytis spp.

International audience; Multidrug resistance (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR (or PDR) has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi, e.g., Botrytis cinerea, Oculimacula yallundae and Mycosphaerella graminicola are concerned by this phenomenon. In agriculture, the occurrence of MDR strains may threaten the efficacy of current fungicide treatments MDR strains were detected in septoria leaf blotch (M. graminicola) field populations since 2008. These strains are cross-resistant to fungicides with different modes of action. The identification of the molecular mechanism explaining the MDR phenotype in two isolated strains (MDR6 and MDR7) was the main goal of this study. Measuring the intracellular accumulation of a radiolabeled fungicide demonstrated increased fungicide efflux in both MDR strains in comparison to sensitive strains. RNA-sequencing led to the identification of several overexpressed transporter genes, out of which MgMFS1 encoding an MFS (major facilitator family) transporter had particularly abundant mRNA in both MDR strains. Crosses between both MDR strains showed that mdr6 and mdr7 loci are closely linked. We applied bulk-progeny sequencing to progeny of the crosses MDR6 x sensitive and MDR7 x sensitive in order to map the genomic regions co-segregating with the MDR phenotypes. SNP frequency analysis in sensitive and resistant bulks showed a clear co-segregation between phenotypes and the left arm of chromosome 7. This region harbors several genes including the MgMFS1 gene mentioned above with a 514 bp promoter insertion in both MDR strains. Gene disruption of MgMFS1 in the MDR6 strain abolished resistance to several fungicides and reduced the resistance to others, demonstrating the importance of MgMFS1 in the MDR phenotype in M. graminicola. Studies are underway to demonstrate if the promoter insertion by its own is sufficient or if an additional mutation is necessary to drive MgMFS1 overexpression and hence MDR in M. graminicola. Financial support: Arvalis Institut du Vegetal, BASF Agro SAS, Bayer SAS, DuPont de Nemours SAS, Syngenta Crop Protection AG. (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR (or PDR) has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi, e.g., Botrytis cinerea, Oculimacula yallundae and Mycosphaerella graminicola are concerned by this phenomenon. In agriculture, the occurrence of MDR strains may threaten the efficacy of current fungicide treatments MDR strains were detected in septoria leaf blotch (M. graminicola) field populations since 2008. These strains are cross-resistant to fungicides with different modes of action. The identification of the molecular mechanism explaining the MDR phenotype in two isolated strains (MDR6 and MDR7) was the main goal of this study. Measuring the intracellular accumulation of a radiolabeled fungicide demonstrated increased fungicide efflux in both MDR strains in comparison to sensitive strains. RNA-sequencing led to the identification of several overexpressed transporter genes, out of which MgMFS1 encoding an MFS (major facilitator family) transporter had particularly abundant mRNA in both MDR strains. Crosses between both MDR strains showed that mdr6 and mdr7 loci are closely linked. We applied bulk-progeny sequencing to progeny of the crosses MDR6 x sensitive and MDR7 x sensitive in order to map the genomic regions co-segregating with the MDR phenotypes. SNP frequency analysis in sensitive and resistant bulks showed a clear co-segregation between phenotypes and the left arm of chromosome 7. This region harbors several genes including the MgMFS1 gene mentioned above with a 514 bp promoter insertion in both MDR strains. Gene disruption of MgMFS1 in the MDR6 strain abolished resistance to several fungicides and reduced the resistance to others, demonstrating the importance of MgMFS1 in the MDR phenotype in M. graminicola. Studies are underway to demonstrate if the promoter insertion by its own is sufficient or if an additional mutation is necessary to drive MgMFS1 overexpression and hence MDR in M. graminicola. Financial support: Arvalis Institut du Vegetal, BASF Agro SAS, Bayer SAS, DuPont de Nemours SAS, Syngenta Crop Protection AG.

The Botrytis species complex responsible for the grey mould disease found on grapevines is composed of two species: Botrytis cinerea, to a large extent (roughly 90%), and Botrytis pseudocinera. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they do differ in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilide fungicide, B. pseudocinerea is naturally resistant. Because a strong synergism was found on B. pseudocinerea between fenhexamid and sterol 14α-demethylation inhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase, it was hypothetized that the detoxification of fenhexamid by a cytochrome P450 monooxygenase similar to Cyp51 is involved in the resistance B. pseudocinerea. To test this, we sought the gene overexpressed in the presence of fenhexamid with the highest similarity to cyp51. Taking into account the Cyp P450 classification based on homology and phylogenetic criteria, this gene, whose function remains unknown, belongs to the Cyp684 family. It was then deleted in a B. pseudocinerea strain. cyp684 knock out mutants exhibit a loss of fenhexamid resistance and synergism between DMIs and fenhexamid, showing that the Cyp684 cytochrome P450 protein is responsible for B. pseudocinerea’s natural resistance to fenhexamid and is involved in fenhexamid detoxification. Although cyp684 is also present in B. cinerea, which is sensitive to fenhexamid, polymorphism was observed between the two species: in B. pseudocinerea the cyp684 promoter shows a deletion of 25 bp; an insertion of 10 bp is observed in the 3’ UTR of the gene. In addition, cyp684 expression is induced after exposure to fenhexamid in B. pseudocinerea suggesting that one of these gene modifications might be responsible for cyp684 overexpression. We are currently conducting experiments to correlate gene structure and resistance in different Botrytis species. The Cyp684 protein will be expressed in a heterologous host to easily identify the produced metabolites.

Multidrug resistance (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi are concerned by this phenomenon. In this study, we confirmed a drug efflux based resistance mechanism in two isolates of the septoria leaf blotch agent Mycosphaerella graminicola. Using next generation sequencing approaches, we could demonstrate that the MgMFS1 gene encoding a membrane transporter of the major-facilitatorsuperfamily was strongly overexpressed in both isolates. The mutation(s) responsible for the MDR phenotype was/were mapped by a bulk-progeny-sequencing approach to a 38.5 kb genomic fragment on chromosome VII, covering also the MgMFS1 gene. Inactivation of the MgMFS1 gene the MDR6 strain abolished the MDR phenotype showing the involvement of this transporter in drug resistance. However fine mapping is necessary to identify the mdr mutation(s) leading to MgMFS1 overexpression.

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Antifungal compounds such as ergosterol biosynthesis inhibitors are widely used to control crop diseases. Among them, one of the most recent, the hydroxyanilide fenhexamid, is efficient principally against Botrytis cinerea, the major causal agent of grey mould. Fenhexamid is a new type of ergosterol biosynthesis inhibitor affecting the sterol C4 demethylation processes due to its specific interaction with one of the four proteins of the enzymatic complex, the 3-ketoreductase. Our regular monitoring conducted on French vineyards allowed the identification of the first isolates of B. cinerea with acquired resistance. Two types of resistant isolates named HydR3- and HydR3+ were distinguished by their resistance level. This acquired resistance is due to point mutations in the erg27 gene leading to target modifications. These modifications induce a reduced in affinity of fenhexamid towards its target, the 3-ketoreductase. Because of their high resistant level, the HydR3+ strains have to be considered relative to the risk of resistance phenomenom occurrence in vineyards. Fitness studies conducted in vitro on isogenic mutants showed altered "overwintering" capacities of HydR3+ mutants suggesting that they probably do not impact fenhexamid's field efficacy. While B. cinerea's acquired resistance could be explained only by target modifications, as in most cases of fungicide resistance, the situation is different for the related species Botrytis pseudocinerea naturally resistant to fenhexamid. We show that erg27 polymorphism only slightly contributes to resistance whereas fenhexamid detoxification by a cytochrome P450 named cyp68.4 is the major mechanism responsible for the resistance. This is the first case of a functional validation of fungicide detoxification involved in resistance.

Oral - Session 1 : Interactions Moléculaires; absent

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International audience; Telomere sequences have been found to enhance transformation frequencies of fungal plasmids. Such plasmids do not integrate into the genome; they may be replicated independently. We constructed two derivatives of the pFAC1 plasmid, which transform B. cinerea at high frequencies (1). Our plasmids pFACR5 and pFB2N both give rise to transformation frequencies 500-1.000 times higher than integrative plasmids. They are replicated under selective pressure but unstable without selection. We analyzed conformation and copy number of both plasmids in B. cinerea transformants. Integration of a fenhexamid resistance allele into the pFB2N vector induces fungicide resistance in the transformants showing that dominant alleles can be selected from replicative plasmids (2). Genomic libraries in the pFB2N vector were used to transform the B05.10 strain. Transformants analysis showed a ratio of recombinant plasmids and an average insert size comparable to the initial E. coli library. The replicative plasmids are found at roughly 1 copy/genome. After the construction of a 3-4X genome coverage B05.10 transformant pool, harboring the replicative library generated from a fenhexamid resistant strain, we screened the transformants for fenhexamid resistance. Results will be presented. Perspectives and limitations of the system will be discussed

International audience

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National audience; Due to the development of resistant srains, the practical control of Botrytis cinerea remains a problem. Little is known about its respiratory chain and this paper describes the preparation and the properties of B cinerea mitochondria with the aim of testing new antifungal compounds. NADH, succinate and α-ketoglutarate as substrates provided good oxygen consumption. Respiratory control ratios were high and showed that electron transport was well coupled to oxidative phosphorylation. The phosphorylation efficiencies (ADP/O) obtained with all 3 substrates were compatible with theoretical values. The addition of cytochrome c did not greatly enhance the oxygen consumption due to ascorbate: the quality of the outer mitochondrial membrane was good enough to prevent externally added cytochrome c from interacting easily with the respiratory chain. These observations indicate that the mitochondria isolated sustain a high level of structural and functional integrity. This study showed also that they were devoid of the alternative pathway resistant to antimycin A and cyanide, but that they exhibited a rotenone-insensitive respiratory route. The action and the effective doses of oligomycin, carbonylcyanide m-chlorophenylhydrazone (CCCP) and carboxyatractyloside on B cinerea mitochondria were similar to those observed with plant mitochondria. At 10 μM, carboxin completely inhibited succinate oxidation in mitochondria from B cinerea but failed to have any effect on NADH and α-ketoglutarate oxidation. This toxicity level was comparable to that reported by other authors with members of the fungal class Basidiomycetes.; Du fait de l’apparition de phénomènes de résistance aux produits anti-Botrytis, la lutte contre la pourriture grise demeure un problème. La chaîne respiratoire de B cinerea, dont on connaît peu de chose, pourrait constituer une cible intéressante pour découvrir des produits de remplacement. Cet article décrit une méthode simple pour préparer des mitochondries de B cinerea et précise leurs caractéristiques. Le NADH, le succinate et l’α-cétoglutarate sont des substrats qui stimulent fortement la consommation d’oxygène. Les contrôles respiratoires sont élevés et prouvent que le transport des électrons est bien couplé à la phosphorylation oxydative. Les rapports ADP/O obtenus avec les 3 substrats sont compatibles avec les valeurs théoriques. L’addition de cytochrome c n’augmente pas notablement la consommation en oxygène provoquée par l’ascorbate : la qualité de la membrane externe des mitochondries est suffisante pour empêcher le cytochrome c de réagir sur la chaîne respiratoire. Ces observations montrent que les mitochondries isolées présentent une bonne intégrité tant sur le plan structural que fonctionnel. Cette étude montre aussi qu’elles ne possèdent pas une voie alternative résistante à l’antimycine A et au cyanure mais présentent une voie insensible à la roténone. Les effets et les niveaux d’activité de l’oligomycine, du carbonylcyanide m-chlorophénylhydrazone (CCCP) et du carboxyatractyloside sur les mitochondries de B cinerea ont été similaires à ceux généralement observés avec les mitochondries végétales. A 10 μmol.l-1, la carboxine inhibe complètement l’oxydation du succinate par les mitochondries de B cinerea mais n’a aucun effet sur les oxydations du NADH et de l’α-cétoglutarate. Ce niveau de toxicité est comparable à celui relevé par d’autres auteurs avec des champignons appartenant à la classe des Basidiomycètes.

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