Studies on anilinopyrimidine resistance and mode of action in Botrytis cinerea suggest a mitochondrial target

Anilinopyrimidine (AP) fungicides are used worldwide for the control of different ascomycetes, including the grey mould pathogen Botrytis cinerea. Currently, the mode of action of APs is proposed to be inhibition of methionine biosynthesis (http://www.frac.info/). However, although they were introduced more than 20 years ago, neither the mechanism(s) of resistance nor their molecular target have been determined. We combined different approaches to discover and validate mutations resulting in loss of sensitivity towards APs in B. cinerea.

UV mutagenesis followed by fungicide selection was used to generate resistant mutants in the lab. Resistance mechanisms were determined by next generation sequencing and validated by reverse genetics. This led to the identification of eight different nuclear genes in which specific non-synonymous nucleotide polymorphisms were independently conferring resistance. One of these genes, a homologue of the mitochondrial ABC transporter Mdl1 in yeast, was also found to be responsible for loss of sensitivity in a proportion of field samples displaying the AniR1 phenotype. However, a majority of AniR1 samples did not carry one of the mutations identified by UV screening.

A mapping population from a cross between an AniR1 isolate and a sensitive reference was generated, and the resistance locus determined by bulked segregant analysis. Resistance was linked to a region on chromosome 10, which was further delimited to the causal mutation by an iterative reverse genetic procedure. The resistance gene is homologous to the mitochondrial NADH kinase Pos5 of yeast. Phenotyping of deletion and overexpression mutants, as well as enzymatic tests suggested that APs act by perturbation of Bc-Pos5 activity. Mitochondrial and cell biology explorations showed a very elusive phenotype and complex interplay between the molecular target and the regulation of cell cycle progression. Further studies will be required to better understand the components involved and ascertain Bc-Pos5 as the primary target in Botrytis.