RNA-seq analysis of Cercospora beticola DMI-resistant and -sensitive strains in response to tetraconazole

The hemi-biotrophic fungus Cercospora beticola causes Cercospora leaf spot (CLS) of sugarbeet. CLS management measures rely on the application of sterol demethylation inhibitor (DMI) fungicides. Reduced sensitivity to DMIs has been reported recently in sugarbeet growing regions worldwide. We have shown previously that CbCyp51, which encodes the DMI target enzyme sterol P450 14a-demethylase in C. beticola, is over-expressed in DMI-resistant isolates. After exposure to tetraconazole, DMI-resistant isolates respond with further induction of CbCyp51. However, no CbCyp51 promoter or gene mutations were associated with DMI resistance. To gain additional insight and identify other mechanisms involved with DMI-resistance, we used RNA-seq to identify genes differentially expressed in DMI-resistant and -sensitive C. beticola isolates upon exposure to tetraconazole or the control treatment in vitro. Interestingly, both the resistant and sensitive isolates responded with a marked induction of 15 of the 22 genes in the ergosterol biosynthesis pathway in response to tetraconazole. All 15 genes were induced to similar expression levels between the two isolates except for CbCyp51 and CbErg3, which were ~16- and 2-fold higher in the resistant isolate when compared to the sensitive isolate, respectively. In total, 110 genes were uniquely differentially-expressed in the DMI-resistant isolate. Genes previously implicated with fungicide resistance such as ABC and MFS transporters were identified. The most highly induced gene in the DMI-resistant isolate encodes a protein with multiple transmembrane regions known to provide DMI resistance in yeast. This protein is not known to provide efflux activity, but may bind directly to the fungicide. Further characterization of this gene and a detailed pathway analysis will be presented.