Evaluation of DNA Methylation Changes by CRED–RA Analysis Following Prednisone Treatment of Endophyte, Fusarium oxysporum

Abstract

Endophytes that represent a sub-set of plant resident microbes are a reservoir of bioactive metabolites. Many of the secondary metabolite biosynthetic gene clusters of endophytes are silent under axenic culture conditions. Epigenetic reprogramming of such cryptic pathways is possible by use of small molecule modulators like prednisone. Methylation changes induced by prednisone, a hypomethylating epigenetic modulator were studied in endophytic Fusarium oxysporum. CRED–RA analysis following exposure to non-cytotoxic dose (300 µM) revealed prednisone as effective in inducing non-methylation and semi-methylation pattern while inhibiting full-methylation of the genome. Effectiveness of prednisone as a DNA methyl transferase inhibitor can be explored in future to study alterations in secondary metabolite gene expression profile in endophytic F. oxysporum.

Introduction

Biosynthetic potential of endophytes from medicinal plants represents an attractive source for discovery of new bioactive metabolites. Zingiber zerumbet commonly named pinecone or shampoo ginger is one such medicinal plant of research interest due its wide range of pharmaceutical activities [1]. Our earlier studies had characterized Z. zerumbet endophytic repertoire and identified the endophyte designated ZzEF8 as exhibiting inhibitory activity against Pythium myriotylum, an economically significant phytopathogen [2]. Metabolite analysis of ZzEF8 identified as F. oxysporum by ITS/16S rRNA analysis [2] detected umbelliferone as major metabolite responsible for anti-Pythium and cytotoxic effects (unpublished). Umbelliferones synthesized via phenylpropanoid pathway are phytoalexins produced by plants following infection with pathogenic fungi [3, 4]. While umbelliferone production by numerous plant species have been reported earlier, reports on production of umbelliferone from endophytes are scarce [5, 6].

Comparative genome analyses indicate Fusarium species to have significant genetic potential to produce wide range of secondary metabolites. Secondary metabolite biosynthetic genes in filamentous fungi are organized as gene clusters with many being either silent or expressed at very low levels under axenic culture conditions [7–9]. Earlier studies had used epigenetic modulators for pleiotropic activation of cryptic or silent secondary metabolite biosynthetic genes [10] resulting in the production and/or enhancement of bioactive compounds that would otherwise not be produced under normal growth conditions [11]. Use of prednisone, a small molecule epigenetic modulator for regulation of secondary metabolite gene cluster(s) in endophytes has been studied in Pestalotiopsis fici for production of polyketides like pestaloficiols T–W and macrodiolide ficiolides A–K [12] and Bacillus pumilus strain RI06-95 for lipopeptide antibiotics [13]. Prednisone, a corticosteroid, is a DNA hypomethylating agent regulating epigenetic modifications by inhibiting DNA methyltransferase (DMT) or histone methyltransferase (HMT) [14]. DNA methylation that adds methyl group to cytosine in CpG islands can be detected by various techniques [15–17] of which coupled restriction enzyme digestion and random amplification (CRED–RA) is a significant method examined by many researchers [18, 19]. The technique is a modification of Random Amplified Polymorphic DNA (RAPD) technique and uses methylation-sensitive enzymes. Methylation in eukaryotic DNA is largely confined to CpG doublet and restriction enzymes with CpG recognition sequences can distinguish between methylated and unmethylated states [20, 21]. Many studies are indicative of complicated pattern and divergent function of DNA methylation in fungi [22, 23] with wide variation in degree and function of DNA methylation among fungal species [24]. Present study was undertaken to evaluate changes in methylation pattern in endophytic F. oxysporum (ZzEF8) following treatment with DMT inhibitor, Prednisone by CRED–RA technique. Methylation sensitive restriction enzymes, Hpa II and Hae II with CpG recognition sequence were used for the analysis. Changes in metabolite accumulation in ZzEF8 following treatment with Prednisone were also evaluated by UV–vis spectroscopy analysis.

Stock cultures of endophytic F. oxysporum used for the present experiments were inoculated in potato dextrose agar (PDA) plates at pH 6.5 and temperature 25 ± 3 °C. Fermentation experiments were carried out by inoculating fungal discs (5 mm diameter) from actively growing regions of PDA plates in absence (control) and presence (300 µM) of epigenetic modifier, prednisone. To exclude the possibility of Prednisone solvent viz., Dimethy sulfoxide (DMSO) as interfering with methylation status, experiments were also carried out with DMSO in equal amount. The flasks were incubated for 5 days in dark at 25 ± 3 °C, following which genomic DNA was extracted using cetyl tri-methyl ammonium bromide (CTAB) extraction buffer. Briefly for genomic DNA isolation, 100 mg mycelial mat finely ground with liquid nitrogen was suspended in CTAB buffer (5 μL/mg) and incubated at 65 °C for 1 h. Equal volume chloroform: isoamyl alcohol (24:1) was added, incubated in ice for 15 min and centrifuged at 12,000 rpm for 15 min at 4 °C. Supernatant after RNase A (Promega) treatment was precipitated using one-fourth volume 3 M sodium acetate and double volume ice-cold isopropanol. DNA pellet was washed with 70% (v/v) ethanol, suspended in TE buffer (1X), analyzed qualitatively by agarose gel electrophoresis (0.8% w/v) and quantitatively with Nanodrop Spectrophotometer (NanoDrop 2000C Spectrophotometer, Thermo Scientific, USA). For metabolite extraction, the liquid nitrogen ground mycelial mat was extracted with dichloromethane (DCM) (1 g/5 mL), defatted thrice using chloroform: methanol (2:1) and analyzed by UV–visible spectroscopy.

For CRED–RA analysis, genomic DNA from control and prednisone treated mycelia were digested with Hae II (NEB) and Hpa II (Thermofischer) enzymes as per manufacturer’s instructions. Reactions were carried out in final 10 μL volume containing 1X buffer, DNA (500 ng) and restriction enzyme (5 U). Reaction mixture was incubated at 37 °C for 1 h for Hpa II and 15 min for Hae II. At the end of incubation period, reactions were thermally inactivated at 80 °C for 20 min. The digested DNA was used as template for RAPD-PCR analysis using eleven primers (OPA1, OPA2, OPB8, OPD4, OPD7, OPD18, OPH17, OPH18, OPW1, OPY6, and OPY15 (Table 1). Reactions were performed in 20 µL reaction volume consisting of 1X buffer with 1.5 mM MgCl₂, 200 µM dNTPs, each primer at 10 µM, 1 U Taq DNA Polymerase (GeNei) and 20 ng genomic DNA. Thermal cycling was carried out in a MJ Mini™ thermal cycler (BioRad) with initial denaturation step of 95 °C for 5 min, followed by 35 amplification cycles of 94 °C for 60 s, 40 °C for 60 s and 72 °C for 1 min and a final extension step at 72 °C for 5 min. Amplification products were analyzed on 1.2% (w/v) agarose gel. Polymorphisms observed in CRED–RA were based on the appearance of new bands or disappearance of the normal bands compared to the profile of control sample [12]. Polymorphism (%) was calculated as: (a/n) × 100 where “a” indicates number of polymorphic bands in prednisone treated sample and “n” is the number of total bands in untreated control. Genomic template stability (GTS) values were calculated and expressed in percentage as: (100 − a/n) × 100. Depending on the methylation status of cytosines, four methylation types were identified for Hpa II and Hae II that have different restriction abilities. Accordingly for Hpa II and Hae II, band profile obtained were scored as presence (1) or absence (0) as suggested by Wang et al. [13] that is indicative of cytosine methylation status. All experiments were performed in triplicate and repeated thrice.

Table 1.

Details of RAPD primers used in the study showing the polymorphism rates (%) and GTS rates (%)

Primer	Sequence (5′ to 3′)	Polymorphism (%)	Genomic template stability (GTS) (%)
OPA1*	CAGGCCCTTC	14.2	85.8
OPA2*	TGCCGAGCTG	18.1	81.8
OPB8*	GTCCACACGG	66.6	33.3
OPD4*	GTAGGCCTCA	9.09	90.9
OPD7	CGGTAGTGGT	0	–
OPD18*	ACGAGAGGCA	25.0	75.0
OPH17*	CACTCTCCTC	0	–
OPH18*	GAATCGGCCA	14.2	85.8
OPW1*	CTCAGTGTCC	25.0	75.0
OPY6*	AAGGCTCACC	25.0	75.0
OPY15*	AGTCGCCCTT	50.0	50.0

Primers used for CRED–RA analysis marked with asterisk (*)

RAPD analysis of prednisone treated and untreated ZzEF8 revealed that except for OPD7 that failed to give any amplification (Lane 5: Fig. 1a i, ii), other ten primers yielded amplification (Fig. 1a) with polymorphism rates ranging from 9.09 to 66.6% (Table 1). Primers OPB8 (66.6%) and OPY15 (50%) that showed considerable polymorphic band patterns (Fig. 1) can be used as good indicators for detecting epigenetic modifications induced by prednisone treatment of endophytic F. oxysporum cultures. Calculation of GTS rates for the RAPD profiles reveal the highest rate for OPD4 with 90.9% (Table 1). The ten RAPD primers were used for CRED–RA analysis of both untreated (Fig. 1b iii) and prednisone treated ZzEF8 (Fig. 1b iv). Percentage ratio of methylation types (Type I/Type II/Type III/Type IV) were calculated by dividing the number of bands of each of the methylation type to the total number of bands obtained from all the methylation types (I + II + III + IV) and multiplied by 100 [22]. Analysis revealed a one-fold increase in Type I and Type II methylation in prednisone treated sample compared to control (Fig. 2). Type I categorized as non-methylation patterns is representative of non-methylated cytosine on double as well single strand of DNA [15] while Type II indicates semi-methylation pattern with the outer cytosine methylated on a single DNA strand (Table 2). In F. oxysporum, prednisone treatment decreased Type III and Type IV methylation by one-fold compared to control sample (Fig. 2). Type II represents inner cytosine methylated on double DNA starnd while Type IV indicates outer cytosine methylated on a DNA double strand. Spectral scan (190–400 nm) of ZzEF8 DCM metabolite extract revealed changes in metabolite profile following prednisone treatment (Fig. 3).

Fig. 1.

Representative CRED–RA profile generated from endophytic F. oxysorum (ZzEF8) following treatment with prednisone (300 µM). ZzEF8 DNA amplification profile with 10 RAPD primers before (a) and after (b) restriction enztme digestion. (i) and (iv): untreated control; (ii) and (v): prednisone treated, Hae II digested; (iii) and (vi): prednisone treated, Hpa II digested. Lane M: Mid-range DNA ladder (BioLit, SRL, India) and Lanes 1–11: amplification profile obtained using 11 RAPD primers viz., OPA1, OPA2, OPB8, OPD4, OPD7, OPD18, OPH17, OPH18, OPW1, OPY6 and OPY15

Fig. 2.

Average (%) rate of methylation types (I–IV) expressed in percentage obtained following CRED–RA analysis of prednisone (300 µM) treated ZzEF8. Percentage ratio of methylation types (Type I/Type II/Type III/Type IV) calculated by dividing number of bands of each methylation type to total number of bands obtained from all the methylation types (I + II + III + IV)

Table 2.

Methylation types of Hpa II and Hae II restriction enzymes according to their digestion abilities

Type	Methylation pattern		Restriction state		Score of band profile
	Hpa II	Hae II	Hpa II	Hae II	x	y	z
I Non-methylation	CCGGGGCC	GCGCCGCG	Digested	Digested	−/1	+/0	+/0
II Semi-methylation	CCGGGGCC	GCGCCGCG	Digested	Undigested	−/1	+/0	−/1
III Full-methylation	CCGGGGCC	GCGCCGCG	Undigested	Digested	−/1	−/1	+/0
IV Full-methylation	CCGGGGCC/CCGG GGCC	GCGCCGCG/GCGCCGCG	Undigested	Undigested	−/1	−/1	−/1

Score of band profile in the table indicates if PCR product is not digested by either enzyme (x), digested by Hpa II (y) and digested by Hae II (z). Scores “+” refer to digestion and “−” refer to undigestion while “1” refer to band presence and “0” refer to band absence

Fig. 3.

UV–visible spectral analysis (190–400 nm) of ZzEF8 DCM metabolite extract following treatment with Prednisone (300 µM). Arrow heads indicate the alterations observed in profile of major metabolite(s) following prednisone treatment

Various studies have reported the significance of DNA methylation as a dynamic epigenetic process contributing to fungal development [24]. Genomic cytosine methylation in fungi have been reported to vary widely from undetectable levels of ≤ 0.1% in Aspergillus flavus [25] to low but detectable levels of 0.2–8% like 0.22% in Magnaporthe oryzae [24], 0.38–0.42% in Metarhizium robertsii [23], 1.8% in Ganoderma sinense [26] and 6.4–7.7% in Cryphonectria parasitica [27] to markedly high levels of 36.9–39.6% in Tuber melanosporum [28]. Generally considered as a silencing epigenetic modification, DNA methylation is being reported in many studies using fungi as influencing gene transcription such as by regulating secondary metabolism [26]. Present analysis determining the methylation status through CRED–RA technique of F. oxysporum (ZzEF8) reveal prednisone as effective in inducing non-methylation and semi-methylation pattern while inhibiting full-methylation of the genome. This reveals its effectiveness as a good DNMT (DNA methyl transferase inhibitor) causing alteration in gene expression profile of biosynthetic gene clusters (BCGs) that can result in production of important metabolites that otherwise would not be produced and/or detected at low concentration under axenic culture conditions.