RNA-seq analysis of AtrR-regulated gene expression in Aspergillus fumigatus cells in response to voriconazole.

Aspergillus fumigatus transcription factor AtrR is a critical determinant of the azole resistance phenotype of this organism. AtrR positively regulates expression of a range of genes involved in azole resistance including the ATP-binding cassette transporter-encoding locus abcG1 (cdr1B/abcC) as well as the gene that encodes the enzymatic target of azole drugs cyp51A. Homology searches of A. fumigatus AtrR against a range of fungal species identified highly conserved 25 amino acid region in the carboxy-terminus. To determine the contribution of this region to AtrR function, we prepared a mutant from of the atrR that lacked this region (?855-879). We compared the response of an isogenic wild-type strain to a strain expressing the ?855-879 AtrR derivative using RNA-seq, both in the absence and the presence of voriconazole challenge. The resulting data indicate that this 25 amino acid segment of AtrR is required for expression of some but not all AtrR target genes. Overall design: This experiment was designed to compare the voriconazole-induced transcriptome that was altered by loss of the transcription factor AtrR or the presence of a mutant form of AtrR that lacked a 25 amino acid domain from its C-terminus that is conserved across fungi. Introduction of this mutant form of AtrR required replacement of the native 3' untranslated region with an element from the cyc1 gene and was linked to a phleomycin resistance gene. To make a wild-type control for this new derivative of atrR, we introduced this same construct into the native atrR locus (atrR-phleoR). This allowed us to confidently compare the level of wild-type AtrR transcription driven in the presence of this atrR-phleoR derivative compared to the ?855-879 atrR-phleoR locus. We compared transcription of isogenic wild-type and atrRD strains to assess the voriconazole-induced transcription in the presence or absence of AtrR.