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. 2018 Jul 17;9(4):e00457-18.
doi: 10.1128/mBio.00457-18.

A Xenobiotic Detoxification Pathway Through Transcriptional Regulation in Filamentous Fungi

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Free PMC article

A Xenobiotic Detoxification Pathway Through Transcriptional Regulation in Filamentous Fungi

Hyunkyu Sang et al. mBio. .
Free PMC article

Abstract

Fungi are known to utilize transcriptional regulation of genes that encode efflux transporters to detoxify xenobiotics; however, to date it is unknown how fungi transcriptionally regulate and coordinate different phases of detoxification system (phase I, modification; phase II, conjugation; and phase III, secretion). Here we present evidence of an evolutionary convergence between the fungal and mammalian lineages, whereby xenobiotic detoxification genes (phase I coding for cytochrome P450 monooxygenases [CYP450s] and phase III coding for ATP-binding cassette [ABC] efflux transporters) are transcriptionally regulated by structurally unrelated proteins. Following next-generation RNA sequencing (RNA-seq) analyses of a filamentous fungus, Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrasses, a multidrug resistant (MDR) field strain was found to overexpress phase I and III genes, coding for CYP450s and ABC transporters for xenobiotic detoxification. Furthermore, there was confirmation of a gain-of-function mutation of the fungus-specific transcription factor S. homoeocarpa XDR1 (ShXDR1), which is responsible for constitutive and induced overexpression of the phase I and III genes, resulting in resistance to multiple classes of fungicidal chemicals. This fungal pathogen detoxifies xenobiotics through coordinated transcriptional control of CYP450s, biotransforming xenobiotics with different substrate specificities and ABC transporters, excreting a broad spectrum of xenobiotics or biotransformed metabolites. A Botrytis cinerea strain harboring the mutated ShXDR1 showed increased expression of phase I (BcCYP65) and III (BcatrD) genes, resulting in resistance to fungicides. This indicates the regulatory system is conserved in filamentous fungi. This molecular genetic mechanism for xenobiotic detoxification in fungi holds potential for facilitating discovery of new antifungal drugs and further studies of convergent and divergent evolution of xenobiotic detoxification in eukaryote lineages.IMPORTANCE Emerging multidrug resistance (MDR) in pathogenic filamentous fungi is a significant threat to human health and agricultural production. Understanding mechanisms of MDR is essential to combating fungal pathogens; however, there is still limited information on MDR mechanisms conferred by xenobiotic detoxification. Here, we report for the first time that overexpression of phase I drug-metabolizing monooxygenases (cytochrome P450s) and phase III ATP-binding cassette efflux transporters is regulated by a gain-of-function mutation in the fungus-specific transcription factor in the MDR strains of the filamentous plant-pathogenic fungus Sclerotinia homoeocarpa This study establishes a novel molecular mechanism of MDR through the xenobiotic detoxification pathway in filamentous fungi, which may facilitate the discovery of new antifungal drugs to control pathogenic fungi.

Keywords: ATP-binding cassette transporter; Sclerotinia homoeocarpa; cytochrome P450; fungus-specific transcription factor; multidrug resistance; xenobiotic detoxification.

Figures

FIG 1
FIG 1
RNA-seq of the drug-sensitive strain HRS10 and multidrug-resistant (MDR) strain HRI11 in the absence and presence of the DMI fungicide propiconazole. (A) In vitro sensitivity of drug-sensitive and MDR strains to fungicides and plant growth regulator. Mean values from five MDR strains and five sensitive strains are shown. EC50 values for propiconazole, iprodione, and flurprimidol and EC95 (†) values for boscalid are shown for sensitive strains. The fold values of EC50 and EC95 values for sensitive strains are shown for MDR strains. Significant differences from mean values of sensitive strains are indicated by asterisks: *, P < 0.001. (B) Expression patterns for 208 genes constitutively overexpressed (log2 fold change of ≥1.5 and P < 0.05) in strain HRI11, compared to the genes in strain HRS10, before and after treatment with propiconazole. Red indicates higher relative expression, and green indicates lower relative expression. Three major groups of genes, A (n = 13), B (n = 57), and C (n = 138), were clustered based on the analysis of hierarchical clustering of gene expression patterns. (C) FPKM of 13 genes from group A in strains HRS10 and HRI11 before and after exposure to propiconazole.
FIG 2
FIG 2
Expression patterns of phase I CYP450s and phase III transporters in response to fungicides and plant growth regulator. (A) Relative expression (RE) of phase I genes (CYP561, CYP65, and CYP68) and phase III genes (ShPDR1 and ShatrD) before and after exposure to propiconazole (1 µg ml−1) for 10, 20, 40, and 60 min in strain HRS10. (B) RE of phase I and III genes before and after exposure to propiconazole (1 µg ml−1), boscalid (100 µg ml−1), flurprimidol (10 µg ml−1), and iprodione (10 µg ml−1) for 40 min in strains HRS10 and HRI11.
FIG 3
FIG 3
Phase I CYP450s are involved in xenobiotic detoxification and multidrug resistance. (A) Sensitivity of strain HRS10 and mutants overexpressing CYP561, CYP65, and CYP68 to fungicides and plant growth regulator. (B) Propiconazole biotransformation rate of strain HRS10 and mutants overexpressing CYP561, CYP65, and CYP68 by HPLC analysis.
FIG 4
FIG 4
Phase III transporters are involved in xenobiotic detoxification and multidrug resistance. (A) Sensitivity of strain HRS10 and mutants overexpressing ShPDR1 and ShatrD to fungicides and plant growth regulator. (B) Heterologous expression of ABC transporter ShPDR1 or ShatrD in a drug-hypersensitive yeast mutant (AD1–8). Sensitivity tests of the yeast strains were performed on Bacto yeast nitrogen base (YNB) agar medium lacking uracil, containing 2% galactose and amended with different classes of fungicides and plant growth regulator. The sensitivity assays of strains AD1–8-pYES2 and AD1–8:PDR1 with propiconazole, iprodione, boscalid, flurprimidol, and paclobutrazol were reported in previous studies (19, 49).
FIG 5
FIG 5
An activating mutation in a xenobiotic detoxification regulator 1 (ShXDR1) confers multidrug resistance by constitutive and induced overexpression of phase I and III genes. (A) Schematic diagram of the ShXDR1 transcription factor showing an amino acid substitution (methionine to threonine) in the MDR strains and alignment to the corresponding region of the mutation in sensitive (Sen) and MDR strains. (B) Sensitivity of strains HRS10, HRI11, HRI11(ΔShXDR1), and HRI11(ShXDR1M853) to fungicides and plant growth regulator. (C) Relative expression (RE) of phase I genes (CYP561, CYP65, and CYP68) and phase III genes (ShPDR1 and ShatrD) before and after exposure to propiconazole (1 µg ml−1) for 40 min in strains HRS10, HRI11, HRI11(ΔShXDR1), and HRI11(ShXDR1M853).
FIG 6
FIG 6
A mutation in a xenobiotic detoxification regulator 1 (ShXDR1) is a dominant gain-of-function mutation leading to multidrug resistance by constitutive and induced overexpression of phase I and III genes. (A) Sensitivity of strains HRS10, HRI11, and HRS10(+ShXDR1T853)-1 and -2 to fungicides and plant growth regulator. (B) Relative expression (RE) of phase I and III genes before and after exposure to propiconazole (1 µg ml−1) for 40 min in strains HRS10, HRI11, and HRS10(+ShXDR1T853)-1 and -2.
FIG 7
FIG 7
Detection of xenobiotic-responsive element (XRE) and interaction between ShXDR1 and the promoter region of the ShXDR1 target gene regulon. (A) Promoter deletion analysis of pShPDR1-YFP chimeric constructs. The promoter 5′ deletions in ShPDR1 are schematized with their corresponding plasmid constructs and S. homoeocarpa mutants. Relative expression (RE) of YFP is given for each mutant before and after exposure to propiconazole. The black region in −479 bp upstream of ShPDR1 indicates the potential promoter region containing the ShXDR1 binding motif. (B) Chromatin immunoprecipitation (ChIP) analysis of HA-tagged fusion ShXDR1 occupancy on promoters of ShPDR1 (−448 bp to −342 bp) and CYP561 (−493 bp to −387 bp) in the absence and presence of propiconazole. The presence of the promoter region of ShPDR1, CYP561, or Shact (as a negative control) sequence was assayed by quantitative PCR. The y axis depicts the fold enrichment over a mock immunoprecipitation control that lacks HA antibody. HRS10HA-P indicates HA-expressing strain HRS10 treated with propiconazole (1 µg ml−1).
FIG 8
FIG 8
Conserved detoxification systems through transcriptional regulation of phase I and III genes in Botrytis cinerea. (A) Sensitivity of Botrytis cinerea strains BC-001 and BC-001(+ShXDR1T853)-1 and -2 to fungicides and plant growth regulator. (B) Relative expression (RE) of phase I gene BcCYP65 and phase III gene BcatrD before and after exposure to propiconazole (1 µg ml−1) for 40 min in strains BC-001 and BC-001(+ShXDR1T853)-1 and -2.

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