title: 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.
abstract: 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.
authors.affiliation: INRA, UR 1290 BIOGER-CPP, Avenue Lucien Brétignières, F-78850, Thiverval-Grignon, France.
authors.forename: Anaïs
authors.initials: A
authors.lastname: Lalève
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Gamet
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Walker
authors.forename: Danièle
authors.initials: D
authors.lastname: Debieu
authors.forename: Valérie
authors.initials: V
authors.lastname: Toquin
authors.forename: Sabine
authors.initials: S
authors.lastname: Fillinger
mesh_terms: D019943:Amino Acid Substitution; D001549:Benzamides; D001713:Biphenyl Compounds; D020171:Botrytis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D002261:Carboxin; D025141:Drug Resistance, Fungal / Q000235:genetics; D004791:Enzyme Inhibitors; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005659:Fungicides, Industrial; D016297:Mutagenesis, Site-Directed; D009536:Niacinamide / Q000031:analogs & derivatives; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011725:Pyridines; D013329:Structure-Activity Relationship; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001549:Benzamides; D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D021122:Protein Subunits; D011725:Pyridines; D009536:Niacinamide; C550088:2-chloro-N-(4-chlorobiphenyl-2-yl)nicotinamide; D002261:Carboxin; D013385:Succinate Dehydrogenase; C572868:N-(2-(3-chloro-5-(trifluoromethyl)-2-pyridyl)ethyl)-alpha,alpha,alpha-trifluoro-o-toluamide