title: Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola.
abstract: A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.
authors.affiliation: Syngenta Crop Protection Münchwilen AG, Stein, Switzerland. gabriel.scalliet@syngenta.com
authors.forename: Gabriel
authors.initials: G
authors.lastname: Scalliet
authors.forename: Judith
authors.initials: J
authors.lastname: Bowler
authors.forename: Torsten
authors.initials: T
authors.lastname: Luksch
authors.forename: Lucy
authors.initials: L
authors.lastname: Kirchhofer-Allan
authors.forename: Diana
authors.initials: D
authors.lastname: Steinhauer
authors.forename: Keith
authors.initials: K
authors.lastname: Ward
authors.forename: Michael
authors.initials: M
authors.lastname: Niklaus
authors.forename: Andreas
authors.initials: A
authors.lastname: Verras
authors.forename: Michael
authors.initials: M
authors.lastname: Csukai
authors.forename: Antoine
authors.initials: A
authors.lastname: Daina
authors.forename: Raymonde
authors.initials: R
authors.lastname: Fonné-Pfister
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001549:Benzamides / Q000494:pharmacology; D001665:Binding Sites; D001713:Biphenyl Compounds / Q000494:pharmacology; D002261:Carboxin / Q000494:pharmacology; D003198:Computer Simulation; D017124:Conserved Sequence; D025141:Drug Resistance, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D005659:Fungicides, Industrial / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D009636:Norbornanes / Q000494:pharmacology; D018384:Oxidative Stress; D010935:Plant Diseases / Q000382:microbiology*; D011485:Protein Binding; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics*; D014908:Triticum / Q000382:microbiology*
chemical_list: C558633:3-(difluoromethyl)-1-methyl-N-(1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl)pyrazole-4-carboxamide; D001549:Benzamides; D001713:Biphenyl Compounds; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D009636:Norbornanes; D011720:Pyrazoles; 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