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

Lead Research Organisation: Rothamsted Research
Department Name: Biological Chemistry & Crop Protection

Abstract

The emergence of resistance to antifungal compounds is an increasing problem for the management of fungal diseases in agriculture and medicine. One of the most important groups of fungicides is the triazoles, used for the treatment of fungal infections of both crop plants and humans. Unfortunately, when fungi are exposed to these chemicals, the treatment can select for strains of the fungus that are less sensitive to the chemical. Over time, resistant strains of the fungus can develop and the chemical no longer controls the disease. This has happened with the human pathogenic yeast Candida, and there is now evidence that some plant pathogenic fungi are also becoming less sensitive to triazoles. The aim of this project is to understand the mechanisms responsible for these changes in sensitivity to triazoles in Mycosphaerella graminicola, the causal agent of Septoria leaf blotch, the most important foliar disease of wheat in the UK. This pathogen has already adapted to many of the fungicides used to control it. Resistance to the strobilurin fungicides, first detected in 2002, is now widespread in UK and European M. graminicola populations, so control of the disease now relies heavily on triazoles. Over the past decade there has been a gradual change in the sensitivity of the fungus to these fungicides so that higher doses are now required to achieve disease control. There is a growing concern, amongst both the agricultural and agrochemical industries, that further shifts in triazole sensitivity will reduce our ability to manage this important disease. There are few new Septoria fungicides available, and the older chemicals used for Septoria control are more damaging to the environment. Several mechanisms are known to contribute to resistance to triazoles. 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 triazole 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 triazole target protein, a cytochrome P450 called CYP51 involved in sterol 14a-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 triazoles is affected, and to make pure samples of the protein to measure enzyme activity, altered properties and inhibition. In particular the project will concentrate on several mutations that have occurred quite recently in M. graminicola populations exposed to triazole fungicides. We also intend to introduce further changes identified in Candida to the protein by targeted mutation to assess their effects on sensitivity and protein function. The project will provide key information on the contribution of target site mutations to the current status of M. graminicola azole sensitivity in the field and the potential for future resistance development. Information gained from these studies will be used to design novel molecular diagnostics to detect target site changes and monitor the occurrence of such changes in field populations of the fungus. This will show how the fungus evolves in response to treatment by different triazoles and help to devise control strategies to maintain the effectiveness of this group of chemicals against M. graminicola. Results should also aid the design of more effective triazole compounds. Furthermore, determining the relationship between target site changes, fungicide sensitivity and enzyme activity will improve understanding of the molecular evolution of the CYP51 protein in plant pathogenic fungi in response to selection.

Technical Summary

Resistance to antifungal agents is an increasing challenge in the management of fungal diseases in agriculture and medicine. Human pathogens such as Candida have developed resistance to triazoles and the mechanisms involved have been partly characterised. Less is known about resistance in plant pathogens. Septoria leaf blotch, (Mycosphaerella graminicola), is the most important disease of wheat in the UK. Almost all wheat crops are treated with triazoles to control Septoria. Their efficacy in disease control has declined over the past 10 years due to the emergence of strains with reduced sensitivity. The project will determine the contribution of mutations in the CYP51 gene encoding the sterol 14a-demethylase target for triazoles to reduced sensitivity of M. graminicola isolates. Sequencing of the CYP51 genes of recent isolates of M. graminicola will identify which mutations are closely correlated with reduced sensitivity. Key mutations will be tracked by Pyrosequencing the CYP51 gene from M. graminicola DNA in samples from our historical wheat archive, and also introduced into a 'sensitive' CYP51 M. graminicola gene, both as individual changes and in combination, by site directed mutagenesis. The effects of these changes on sensitivity to different triazoles will be determined by expression in an S. cerevisiae cyp51 mutant. Heterologously expressed mutant CYP51 proteins will be solubilised, purified and evaluated for fungicide binding affinity, intrinsic enzyme activity, and other aspects of functionality affecting fitness. Mutations known to confer triazole resistance in Candida will be introduced by targeted mutagenesis. Bioassay of yeast transformants expressing these engineered CYP51 genes, and triazole binding and enzyme activity studies of the purified proteins will provide essential information on the evolution of M. graminicola CYP51 in response to triazoles and the risk of future resistance development.

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