Impact of mutations in the target-encoding CYP51 gene in Mycosphaerella graminicola populations developing resistance to triazole fungicides

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Several mechanisms are known to contribute to resistance to azoles. These include changes in the target site protein, as well as participation of other proteins, known as transporters, that are able to pump the fungicide out of the fungus. Therefore, to understand how changes in the target protein may affect azole sensitivity, the altered forms of the protein must be expressed and studied in isolation. The proposed project aims to determine the effect on sensitivity of mutations in the azole target that is a cytochrome P450 called CYP51 involved in sterol 14?-demethylation. The research will assess how such mutations affect interactions between the fungicide and the protein, as well as the activity of the enzyme itself, using several approaches. The mutant proteins will be expressed in another fungus, the yeast Saccharomyces cerevisiae, to see how sensitivity to different azoles is affected, and to make pure samples of the protein to measure enzyme activity, altered properties and inhibition. Many of the changes detected by DNA sequencing of the azole target gene have not been seen in clinical resistance and therefore require characterisation. Indeed, few of the mutations associated with resistance have been characterised at the level of purified protein even in the clinical setting. In particular the project will concentrate on a variety of mutations that have occurred quite recently in M. graminicola populations exposed to azole fungicides. We also intend to introduce further changes to the protein by targeted mutation to assess their effects on sensitivity and protein function.

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