Skip to main content
ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Sugarbeet and Potato Research » Research » Publications at this Location » Publication #321863
Title: RNA-Sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction

Author
item Bolton, Melvin
item EBERT, MALAIKA - North Dakota State University
item FAINO, LUIGI - Wageningen University
item RIVERA-VARAS, VIVIANA - North Dakota State University
item DEJONGE, RONNIE - Ghent University
item VAN DE PEER, YVES - Ghent University
item THOMMA, BART - Wageningen University
item SECOR, GARY - North Dakota State University

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/21/2016
Publication Date: 7/1/2016
Citation: Bolton, M.D., Ebert, M.K., Faino, L., Rivera-Varas, V., de Jonge, R., Van de Peer, Y., Thomma, B.P.H.J., Secor, G.A. 2016. RNA-Sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction. Fungal Genetics and Biology. 92:1-13.

Interpretive Summary: Cercospora beticola causes Cercospora leaf spot of sugarbeet. Cercospora leaf spot management measures include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. DMI fungicides target the CYP51 protein. Previous work has shown that expression of Cyp51 is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by identifying all the expressed genes in two C. beticola strains that are either DMI-sensitive or -resistant. The ergosterol pathway produces an important cell membrane component called ergosterol in fungal cells. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. In attempts to functionally characterize genes with a putative role in DMI-resistance, we generated several gene knock-out mutants checked their sensitivity to fungicide. However, regardless of the gene chosen for replacement or whether the transformation event resulted in a site-directed or ectopic mutant, fungicide resistance increased in all mutants, suggesting that the transformation process alone increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.

Technical Abstract: Cercospora beticola causes Cercospora leaf spot of sugarbeet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. In attempts to functionally characterize genes with a putative role in DMI-resistance, we generated several gene knock-out mutants using PEG-mediated transformation of protoplasts and assessed EC50 values compared to the progenitor wild-type strain. However, regardless of the gene chosen for replacement or whether the transformation event resulted in a site-directed or ectopic mutant, average EC50 values of all mutants were significantly higher than the wild-type strain, suggesting that the transformation process alone increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.