TY - JOUR
AU - Geunhwa, Jung,
AB - Abstract Cytochrome P450s have been shown to play a vital role in the xenobiotic detoxification system of Sclerotinia homoeocarpa, the causal agent of the turfgrass disease dollar spot. A previous study indicated that three CYP450s were validated to play a functional role in resistance against different fungicide classes including propiconazole and plant growth regulator, flurprimidol. In this study, we present these CYP450s possess the capability to modify the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been shown to persist in soils. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP561 and CYP65 overexpression strains when compared to the wild-type and CYP68 overexpression strain. Our GC-MS results show that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloro-isophthalonitrile is produced by CYP450s’ metabolism. These findings suggest fungal CYP450s can biotransform chlorothalonil for biodegradation or detoxification. biodegradation, Cytochrome P450, chlorothalonil, Sclerotinia homoeocarpa, dollar Spot, fungicide INTRODUCTION Cytochrome P450 monooxygenases (CYP450s) are common enzymes found in every living kingdom. CYP450s play a vital role in both primary and secondary metabolism and catalyze multiple physiological reactions. In ascomycete fungi, CYP450s are hugely diverse and many are involved in specialized processes that allow fungi to occupy specific niches (Deng, Carbone and Dean 2007; Chen et al.2014). Because of their potential to degrade xenobiotics, fungal CYP450s are of great interest in biotechnology and bioremediation industries (Durairaj, Hur and Yun 2016). Sclerotinia homoeocarpa (Salgado-Salazar et al. 2018), the causal agent of dollar spot disease, is the most economically important cool-season turfgrass pathogen. This sterile ascomycete fungus has developed cross-resistance and multiple resistance to the demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), succinate dehydrogenase inhibitor (SDHI) and dicarboximide fungicide classes, and plant growth regulators (PGRs) (Bishop et al.2008; Putman, Jung and Kaminski 2010; Allan-Perkins et al.2017; Ok et al.2011; Popko et al. 2018). Recently, Sang et al. (2018) has discovered and validated through RNA-sequencing and molecular genetics approaches that three CYP450s are involved in resistance to multiple fungicide classes mediated by xenobiotic detoxification. Dollar spot strains overexpressing each CYP450 were generated to study their metabolizing function in xenobiotic detoxification. CYP450 overexpressing strains were named by their respective CYP450 family and identified as CYP561, CYP65 and CYP68. Chlorothalonil (2,4,5,6-tetrychloroisophthalonitrile) (Fig. 1) is a broad-spectrum, non-systemic, contact fungicide commonly used for preventative control of foliar diseases of commercial crops and turfgrass (Hladik and Kuivila, 2008). Repeated sprays of chlorothalonil are subject to runoff and their presence in surface and groundwater has been of concern. Furthermore, chlorothalonil has been reported to photodegrade into several compounds in water and soil (Kwon and Armbrust 2006). The predominant microbial biotransformation product is 4-hydroxy-2,5,6-trichloroisophthalonitrile (Fig. 1), which has been shown to be 30 times more toxic to mice than chlorothalonil, as well as more mobile and persistent in soil (Cox 1997). Figure 1. View largeDownload slide Chemical structures of chlorothalonil and 4-hydroxy-2,5,6-trichloroisophthalonitrile. Figure 1. View largeDownload slide Chemical structures of chlorothalonil and 4-hydroxy-2,5,6-trichloroisophthalonitrile. Currently, only two microbial degradation pathways for chlorothalonil have been identified. A glutathione S-transferase in Ochrobactrum anthropic SH35B was able to act on the chlorine atoms of chlorothalonil, and Pseudomonas sp. CTN-3 utilizes a hydrolytic dehalogenase to substitute a hydroxyl on the 4-chlorine atom (Wang et al.2010). The present study is the first report of a CYP450 directly modifying chlorothalonil. Our High Performance Liquid Chromatography (HPLC) results suggest that CYP561 has the highest rate of chlorothalonil biotransformation over CYP65 and CYP68. We also show that 4-hydroxy-2,5,6-trichloroisophthalonitrile was formed in cultures incubated with chlorothalonil, which is the primary transformation intermediate found in chlorothalonil treated soils (Van Scoy and Tjeerderma 2014). MATERIALS AND METHODS CYP450 overexpression strains Overexpression of CYP561, CYP65 and CYP68 genes were previously described in Sang et al. (2018). Briefly, a pYHN3-ptrpC-CYP561, -CYP65 and -CYP68 plasmid was transformed into protoplasts of the sensitive S. homoeocarpa isolate HRS10 to generate CYP561, CYP65 and CYP68 overexpression strains, respectively. CYP561, CYP65 and CYP68 overexpression strains were confirmed by quantitative PCR analysis, and each gene was expressed 2190-, 277- and 97024-fold greater than the wild type, respectively. HPLC analysis Potato dextrose broth (25 mL) with and without mycelia (approximately 1 g) of strain HRS10 and CYP561, CYP65 and CYP68 overexpression strains were supplemented with chlorothalonil (10 μg ml−1, Sigma, 99%) and cultured at 25°C (100 rpm) in biological triplicates. The sample was prepared at each time point (0, 12, 24, 36, 48 and 72 h) by a methanol extraction method (Im et al.2016). Biomass was weighed before and after the 72 h, but no significant growth was observed in any of the replicates. An Agilent 1200 Series HPLC equipped with a diode array detector (DAD) was used for the detection and quantification of chlorothalonil. Separation was performed on an Agilent Eclipse XDB C18 column (4.6 mm × 150 mm, 5 micron) using a 12-min linear gradient of de-ionized water (50% to 90%) at a flow rate of 1.5 ml min−1. The DAD was set at 254 nm to provide the real-time chromatogram, and the UV/Vis spectra from 190 to 400 nm were recorded for detection of transformation products. GC-MS analysis A reference standard of 4-hydroxy-2,5,6-trichloroisoph-thalonitrile (Sigma, 99%) was used for identification of the biotransformed metabolite. Mycelia (2 g) of HRS10 were supplemented with 10 μg ml−1 of chlorothalonil and cultured at 25°C (100 rpm). After 7 days, mycelium was removed and the analytes were extracted through SAX/PSA cartridges using acetonitrile/toluene (75:25 v/v), as described by Duca et al. (2014). The analytes were derivatized to trimethyl silyl derivatives by BSTFA:TMCS (99:1 v/v) in a 1:1 ratio, and heated to 70°C for 1 h, as described by Hladik and Kuivila (2008). Injections were made into an Agilent 6890 GC with 5973 MS detector. The column was a J&W DB-5ms column with 30 m × 250 um, 0.25 um film thickness. 1 μL sample injections used a 10:1 split, and He carrier gas flow was set to 1 mL min−1. The injector and MS source temperatures were held at 300°C and 250°C, respectively. The oven was held at 90°C, held for 3 min, then ramped up to 90–185°C at 10°C min−1 with a 4 min hold time, followed by ramp up to 185–300°C at 20°C min−1 with a 2 min final hold time. Mass spectra were required in EI mode. RESULTS Biotransformation rate of chlorothalonil by CYP561, CYP65 and CYP68 overexpression strains HPLC was used to show rate of chlorothalonil biotransformation over time by the overexpression strains (Fig. 2). The peak for chlorothalonil was identified using a signal at 254 nm, and the retention time was 7.5 min. Figure 2. View largeDownload slide Biotransformation of chlorothalonil over time by CYP450 overexpression strains and DMI sensitive isolate HRS10 by measurement of peak area. Error bars represent standard error. Figure 2. View largeDownload slide Biotransformation of chlorothalonil over time by CYP450 overexpression strains and DMI sensitive isolate HRS10 by measurement of peak area. Error bars represent standard error. CYP68 did not appear to modify chlorothalonil differently than the wild type, however, CYP561 modified chlorothalonil at a significantly faster rate than the wild-type over 72 h. CYP65 was shown to have a slightly faster rate of biotransformation, although not nearly as efficient as CYP561. Identification of the transformation product by GC-MS The chromatograms for the 4-hydroxy-2,5,6-trichloroisoph-thalonitrile reference standard and the analytes extracted from HRS10 supplemented with chlorothalonil are shown in Fig. 3. The retention times of the main peaks did slightly differ; 11.5 min for the reference standard and 10.5 min from the extracted sample. For improved sensitivity, the experimental sample was a splitless injection instead of a split injection, which likely accounts for the difference in retention time. Figure 3. View largeDownload slide Chromatograms of 4-hydroxy-2,5,6-trichloroisophthalonitrile (a) at ∼50 μg ml-1, and the extracted sample from DMI sensitive HRS10 (b). Figure 3. View largeDownload slide Chromatograms of 4-hydroxy-2,5,6-trichloroisophthalonitrile (a) at ∼50 μg ml-1, and the extracted sample from DMI sensitive HRS10 (b). A reference standard of chlorothalonil was correctly identified by the mass ion (265 MW) by the NIST/EPA/NIH MASS SPECTRAL LIBRARY (NIST 14) and NIST MASS SPECTRAL SEARCH PROGRAM Version 2.2 (Fig. 4). The mass ion for the silylated derivative of 4-hydroxy-2,5,6-trichloroisophthalonitrile (320 MW) was not found (Fig. 5). However, the molecular ion without a methyl group (M – 15) was identified, which corresponds well to the findings of Hladik and Kuivila (2008) in their GC-MS analysis of the metabolite. We concluded that S. homoeocarpa utilizes CYP450s, specifically CYP561 and CYP65, to biotransform chlorothalonil into the 4-hydroxy-2,5,6-trichloroisophthalonitrile metabolite. Figure 4. View largeDownload slide Full mass spectra between 60 and 280 m/z of chlorothalonil, structure identified through the NIST database. Figure 4. View largeDownload slide Full mass spectra between 60 and 280 m/z of chlorothalonil, structure identified through the NIST database. Figure 5. View largeDownload slide Full mass spectra between 0 and 600 m/z of the 4-hydroxy-2,5,6-trichloroisophthalonitrile reference standard (a) and extracted sample from DMI sensitive HRS10 (b). Figure 5. View largeDownload slide Full mass spectra between 0 and 600 m/z of the 4-hydroxy-2,5,6-trichloroisophthalonitrile reference standard (a) and extracted sample from DMI sensitive HRS10 (b). DISCUSSION Xenobiotic detoxification capabilities of CYP561, CYP65 and CYP68 CYP450s involved in Phase I of xenobiotic detoxification target compounds to increase water solubility, and allow for conjugation reactions to occur. In S. homoeocarpa, Sang et al. (2018) suggests CYP450s are coordinately regulated with Phase III efflux transporters under regulation of a fungal specific transcription factor (ShXDR1, S. homoeocarpa Xenobiotic Detoxification Regulator) to gain multidrug resistance. Here, we show CYP561 and CYP65 can oxidize chlorothalonil, although field resistance to chlorothalonil in S. homoeocarpa has not been discovered. In addition, all three CYP450 overexpression strains display a resistance phenotype to a DMI fungicide propiconazole and PGR flurprimidol, with varying in vitro insensitivities to a SDHI fungicide boscalid and dicarboximide fungicide iprodione. Our data supports the findings of Sang et al. (2018), who propose CYP561, CYP65 and CYP68 have different substrate specificities dependent on the fungicide class, and could possibly metabolize a wide-range of compounds. A novel chlorothalonil biotransformation pathway Chlorothalonil is a common fungicide primarily applied on peanuts, tomatoes and turfgrasses (US EPA 1999). The predominant metabolite found in chlorothalonil treated soils is 4-hydroxy-2,5,6-trichloroisophthalonitrile. The 4-hydroxy metabolite is more water soluble, persistent and toxic than the parent compound (Cox 1997). Due to the metabolites’ toxic effect and ability to pervade into water systems, more attention has been drawn to the environmental fate of chlorothalonil (Van Scoy and Tjeerderma 2014). Identification of the 4-hydroxy metabolite was successful by GC-MS analysis when the sensitive isolate HRS10 was cultured in media supplemented with chlorothalonil. Other microbial degradation pathways for chlorothalonil have been identified in bacteria, but as of this report, a fungus biotransforming chlorothalonil has not been reported. In addition, this is the first report of a CYP450 oxidizing chlorothalonil. Since the 4-hydroxy metabolite is the predominant metabolite found in soils, it is possible ascomycete and other fungal CYP450s are important to the microbial degradation of chlorothalonil. Whether or not CYP561 and CYP65 are unique in their ability to oxidize chlorothalonil when compared to other fungal ascomycetes is a major question. Searching for a close homolog to CYP561 or CYP65 in other ascomycete species may identify other fungi capable of biotransforming chlorothalonil. Based on this data, the hypothesized reaction mechanism of chlorothalonil biotransformation into the 4-hydroxy metabolite occurs via epoxidation on the 4-position chlorine, and is subsequently reduced to the hydroxyl group, possibly by a non-catalyzed reducing agent, such as glutathione. It is possible S. homoeocarpa initiates a conjugation reaction of the 4-hydroxy metabolite for increased efflux from the cell. Conjugation reactions covalently attach smaller polar groups to metabolized xenobiotics after CYP450 action, which produce easily excretable or inactive compounds (Jancova, Anzenbacher and Anzenbacherova 2010). Expression of one glutathione S-transferase (GST) in S. homoeocarpa is overexpressed in a DMI resistance isolate, as found by RNA-sequencing (Sang et al.2018). However, it is unknown whether or not Phase II enzymes play a significant role in S. homoeocarpa’s detoxification system. Investigation of these CYP450s revealed a novel mechanism of chlorothalonil biotransformation. This discovery may be useful in identifying the holistic picture of chlorothalonil's environmental fate. In addition, resistance to chlorothalonil has not been reported in S. homoeocarpa. However, MDR and chlorothalonil field resistance has been reported in other plant pathogenic fungi, such as Botrytis cinerea (Barak and Edgington 1984). It is possible orthologous CYP450s in B. cinerea are responsible for the MDR phenotype. FUNDING This work was supported by the National Institute of Food and Agriculture (NIFA), United States Department of Agriculture (USDA), the Massachusetts Agricultural Experiment Station, and the Stockbridge School of Agriculture at the University of Massachusetts Amherst, under the project number MAS00436. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the USDA or NIFA. Conflict of interest. None declared. REFERENCES Allan-Perkins E , Campbell-Nelson K , Popko JT et al. Investigating selection of demethylation inhibitor fungicide-insensitive Sclerotinia homoeocarpa isolates by boscalid, flurprimidol, and paclobutrazol . Crop Sci 2017 ; 57 : S-301 – S-309 . Google Scholar Crossref Search ADS Barak E , Edgington LV . Cross-resistance of Botrytis cinerea to captan, thiram, chlorothalonil and related fungicides . Canad J Plant Pathol 1984 ; 6 : 318 – 20 . Google Scholar Crossref Search ADS Bishop P , Sorochan J , Ownley BH et al. Resistance of Sclerotinia homoeocarpa to iprodione, propiconazole, and thiophanate-methyl in Tennessee and northern Mississippi . Crop Sci 2008 ; 48 : 1615 – 20 . Google Scholar Crossref Search ADS Chen W , Lee MK , Jefcoate C et al. 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TI - Chlorothalonil biotransformation by cytochrome P450 monooxygenases in Sclerotinia homoeocarpa
JO - FEMS Microbiology Letters
DO - 10.1093/femsle/fny214
DA - 2018-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/chlorothalonil-biotransformation-by-cytochrome-p450-monooxygenases-in-b4MqCWtCTp
VL - 365
IS - 19
DP - DeepDyve
ER -