Dissecting allele-specific fungicide resistance mechanisms by heterologous expression of the demethylase inhibitor target gene Cyp51 in a phytopathogen model

BACKGROUND: Fungicide resistance is a major concern both in agriculture and clinical disease control. Whilst several mechanisms of resistance have been elucidated, assigning phenotype to genotype is often difficult and reliant on correlations. Resistance to demethylase inhibitor (DMI) fungicides was recently reported in the economically important filamentous fungal barley (Hordeum vulgare) pathogen Pyrenophora teres f. teres (Ptt) in Australia. The target of DMI fungicides is encoded by the Cyp51 gene family; single allele of Cyp51B and two copies of the Cyp51A gene1. Five Cyp51A alleles (W1-A1, 9193-A1, KO103-A1, W1-A2 and 9193-A2) were identified in Ptt with KO103-A1 containing the mutation F489L (F495L) which correlates with resistance to various DMIs.1 RESULTS: We replaced the coding region of the native Cyp51B gene of the filamentous fungal Dothideomycete wheat pathogen Parastagonospora nodorum with each of the five Ptt Cyp51A alleles to compare the phenotypic effects of each allele in isolation. The native Cyp51B of P. nodorum could be functionally replaced by Cyp51-A1 but not Cyp51-A2. Transformants carrying KO103-A1 exhibited significantly higher gene expression than 9193-A1 and W1-A1, suggesting the mechanism of gene regulation lies within the coding sequence and is conserved between Ptt and P. nodorum. The EC50 values of the KO103-A1 transformants were significantly higher than any other transformants or wild type isolates for metconazole, prochloraz and tebuconazole but lower for epoxiconazole. CONCLUSION: This system permits the functional characterisation of fungicide target genes in an isogenic background that mimics the physiological environment of plant pathogens. We suggest the system will prove useful in dissecting the impact of genetic mutations on a spectrum of fungicides and permit the design of fungal strains for screening active ingredients that may control strains resistant to existing fungicides.