Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function

The recent decrease in the sensitivity of the Western European population of the wheat pathogenMycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in theCYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutatedM. graminicola CYP51 (MgCYP51) variants in aSaccharomyces cerevisiae mutant carrying a doxycycline-regulatabletetO7 -CYC promoter controlling nativeCYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein inS. cerevisiae . Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modernM. graminicola populations, have no effect on the capacity of theM. graminicola protein to function inS. cerevisiae . We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity ofMgCYP51 to complement theS. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of theM. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western EuropeanM. graminicola population emerged.