The Protein Kinase A Pathway Regulates Zearalenone Production by Modulating Alternative ZEB2 Transcription

Abstract

Zearalenone (ZEA) is an estrogenic mycotoxin that is produced by several Fusarium species, including Fusarium graminearum. One of the ZEA biosynthetic genes, ZEB2, encodes two isoforms of Zeb2 by alternative transcription, forming an activator (Zeb2L-Zeb2L homooligomer) and an inhibitor (Zeb2L-Zeb2S heterodimer) that directly regulate the ZEA biosynthetic genes in F. graminearum. Cyclic AMP-dependent protein kinase A (PKA) signaling regulates secondary metabolic processes in several filamentous fungi. In this study, we investigated the effects of the PKA signaling pathway on ZEA biosynthesis. Through functional analyses of PKA catalytic and regulatory subunits (CPKs and PKR), we found that the PKA pathway negatively regulates ZEA production. Genetic and biochemical evidence further demonstrated that the PKA pathway specifically represses ZEB2L transcription and also takes part in posttranscriptional regulation of ZEB2L during ZEA production. Our findings reveal the intriguing mechanism that the PKA pathway regulates secondary metabolite production by reprograming alternative transcription.

Introduction parasiticus

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB), which is the most destructive and economically important disease of small grain cereals worldwide [6,20]. During the 1990s, an FHB outbreak in wheat and barley reached epidemic proportions in North America, causing an estimated $3 billion in losses [29]. In addition to severe yield losses, FHB disease often results in the contamination of grains by hazardous mycotoxins, trichothecenes and zearalenone (ZEA).

ZEA is a polyketide mycotoxin that is produced by several Fusarium species, including F. graminearum, F. culmorum, F. equiseti, and F. crookwellense [6,19,33]. In mammals, ZEA induces hyperestrogenic effects, which result in reproductive disorders in farm and laboratory animals and pose a serious risk to food safety and animal health [6]. Because effective strategies to manage the risk of mycotoxins are not currently available, understanding the molecular mechanisms of secondary metabolite production in F. graminearum would provide a solution for mycotoxin management.

ZEA biosynthetic genes are located in the gene cluster that is typical of fungal secondary metabolite biosynthesis [32]. The ZEA biosynthetic cluster genes PKS4, PKS13, ZEB1, and ZEB2 encode a reducing polyketide synthase, a nonreducing polyketide synthase, an isoamyl alcohol oxidase, and a basic leucine zipper (bZIP) transcription factor, respectively [14]. In particular, ZEB2 encodes two isoforms (Zeb2L and Zeb2S) that are produced via alternative promoter usage. Zeb2L directly activates the expression of other cluster genes, whereas Zeb2S inhibits Zeb2L function by forming Zeb2L-Zeb2S heterodimers [25].

The cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway is one of the major signal transduction pathways involved in various cellular processes in eukaryotes [23]. The recognition of environmental signals by membrane-bound G-protein-coupled receptors leads to the activation of adenylyl cyclase (Ac) via G-proteins or Ras proteins. Ac then converts adenosine triphosphate (ATP) into cAMP, and an elevated level of cAMP dissociates the PKA tetramer into PKA catalytic subunits (PkaCs) and PKA regulatory subunits (PkaRs). The released PkaCs are capable of phosphorylating downstream enzymes or transcription regulators. In this pathway, PKA downregulation occurs via a feedback mechanism involving a cAMP-scavenging phosphodiesterase that converts cAMP to 5’-AMP. Filamentous fungi also possess conserved PKA signaling genes; F. graminearum has two genes for PkaC (CPK1 and CPK2) and one gene for PkaR (PKR) [13].

Biochemical and genetic evidence revealed that the PKA pathway is closely involved in various developmental stages in filamentous fungi [13,27]. In particular, connections between the PKA pathway and secondary metabolite production have been well reported in Aspergillus species, including A. nidulans and A. parasiticus [26-28]. A recent study also showed that the PKA pathway regulates asexual and sexual development, deoxynivalenol production, and virulence in F. graminearum [13]. However, no information regarding the genetic requirement of the PKA pathway for ZEA biosynthesis or the identity of the signaling cascades involved in ZEB2 regulation in F. graminearum has been reported.

The objectives of this study were to investigate the role of the PKA pathway in ZEA biosynthesis and to determine how ZEB2 is regulated by the PKA pathway in F. graminearum. The results demonstrated that the activation of PKA signaling negatively regulates ZEA production by reprogramming alternative ZEB2 transcription. This study provides evidence for the fine-tuned mechanism of secondary metabolite production and new insight into the regulatory roles of the PKA signaling pathway in filamentous fungi.

 

Materials and Methods

Fungal Strains and Culture Conditions

The F. graminearum wild-type strain Z-3639 [2] and the mutants derived from this strain were maintained according to the Fusarium Laboratory Manual [18] and were stored in 20% (v/v) glycerol at -70℃. The ZEA-deficient mutant, Δzeb2, was obtained from a previous study [14]. The heterothallic Δmat1 strain, which carries a MAT1-1-1 deletion, was used for outcrosses [15]. All strains used in this study are listed in Table 1. To isolate genomic DNA, the fungal strains were incubated in liquid complete medium (CM) for 5 days at 25℃ [18]. To analyze ZEA production, the strains were grown in liquid starch-glutamate (SG) medium or on rice medium as previously described [14].

Table 1.F. graminearum strains used in this study.

Nucleic Acid Manipulations, PCR Primers, and PCR Conditions

Genomic DNA was extracted using a cetyltrimethylammonium bromide procedure [18]. Restriction endonuclease digestion, gel electrophoresis, gel blotting, and Southern hybridizations with 32P-labeled probes were performed according to standard procedures [9]. The PCR primers used in this study (Table S1) were synthesized by the Bionics oligonucleotide synthetic facility (Seoul, Korea).

For quantitative real-time (qRT)-PCR analyses of ZEB2 in the F. graminearum strains, total RNA was prepared from fungal cultures that were grown in SG medium for 14 days using the Easy-Spin Total Extraction Kit (iNtRON Biotech, Seongnam, Korea). Total RNA (5 μg) was converted into cDNA with the SuperScript III First-Strand Synthesis System (Invitrogen, CA, USA) using oligo(dT)20 according to the manufacturer’s recommendations, and a 1:100 fraction (corresponding to 50 ng of reverse-transcribed RNA) was used for qRT-PCR. All reactions were performed in a volume of 20 μl using the iQ SYBR Green Master Mix (Bio-Rad, CA, USA) and a CFX96 Real-Time System (Bio-Rad) according to the manufacturer’s instructions. The thermal profile was 95℃ for 2 min, followed by 40 cycles of 95℃ for 10 sec and 58℃ for 30 sec. Three technical replications were conducted for all qRT-PCRs, using two biological replicates. The Ct values were normalized to the endogenous housekeeping gene cyclophilin (CYP1) for each sample.

Genetic Manipulation of F. graminearum Mutants

For targeted gene deletion, a double-joint (DJ)-PCR strategy was used [31]. Briefly, the 5’ and 3’ flanking regions of each target gene were amplified by PCR using the primer pairs Gene-5F/Gene-5R and Gene-3F/Gene-3R from the wild-type strain. A geneticin-resistance cassette (GEN) was amplified from pII99 [22]. These three amplicons (i.e., 5’ region, 3’ region, and GEN) were then mixed at a 1:1:2 molar ratio and fused in a second round of DJ-PCR. Fused constructs were amplified using the nested primers Gene-5N and Gene-3N.

To constitutively express the ZEB2L gene in CM, the ZEB2L gene fused with FLAG was cloned into the SpeI-HindIII site of pGZT001, which contained the ZEB2 terminator [25]. The strong promoter (PEF1α) was amplified from pSKGEN [16] using the primers PEFp-5 ApaI and PEFp-3 SpeI and was inserted into the vector. Fungal transformation was performed as previously described [11].

Outcrosses

For outcrosses, mycelia of the female strain, which was grown on carrot agar for 5 days, were gently removed with a glass spreader in the presence of a sterilized 2.5% Tween 60 solution and were spermatized with 1 ml of a conidia suspension (1 × 106 conidia/ml) for each male strain. After sexual induction, all of the cultures were incubated under near-UV light (wavelength, 365 nm; HKiv Import & Export Co., Ltd., China). Dozens of ascospores were randomly isolated from each outcross, and the genotype of each progeny was determined by antibiotic resistance and PCR screening using the combination of GEN-5F/GEN-with 5F primers (Table S1).

Mycotoxin Analysis

ZEA extraction was performed as previously documented [14]. The F. graminearum strains were incubated on rice grains (30 g) or in 25 ml of SG medium at 25℃. Rice cultures were harvested 21 days after inoculation. The fungal mycelia that were grown in SG medium were harvested via filtration through Whatman No. 2 filter paper for 1-14 days, and the filtrates were used for ZEA extraction. The F. graminearum cultures were homogenized to a fine powder using a mortar and pestle. For ZEA detection, a filtrate extracted from the powder sample or the SG medium that was filtered with ethyl acetate was concentrated to dryness and dissolved with methanol. The methanol aliquot of each sample was subjected to chromatography on silica gel-coated TLC plates (Kiesel Gel 60; Merck, Germany) and separated with the developing solvent (chloroform-methanol, 9:1 (v/v)). Photographs for the detection of ZEA were obtained after the plates were exposed to UV radiation (254-364 nm).

Immunoprecipitation and Western Blot Analysis

To detect Zeb2L in the F. graminearum cultures, the filtered fungal mycelia were homogenized to a fine powder under liquid nitrogen using a mortar and pestle. The ground fungal cultures (50 mg) were sonicated in 250 μl of lysis buffer (25 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5% glycerol; 1% NP-40; 1 mM EDTA; and protease inhibitors), and the lysates were incubated with magnetic beads conjugated to a mouse anti-Flag antibody (Sigma-Aldrich) at 4℃ overnight. The incubated magnetic beads were washed five times with lysis buffer before they were resolved using SDS-PAGE (12.5%). After membrane transfer, the nitrocellulose membranes were first blotted with rabbit anti-Flag (Abcam, UK; 1:4,000) primary antibody and then with horseradish peroxidase-linked secondary antibodies (GE Healthcare, UK; 1:4,000). The target proteins were detected with the ECL Select Western Blotting Detection Reagent (GE Healthcare) according to the manufacturer’s instructions.

 

Results

Deletion of Genes Related to the PKA Signaling Pathway in F. graminearum

To investigate the role of the PKA signaling pathway in ZEA biosynthesis, we deleted three genes involved in the PKA pathway from the F. graminearum genome (Fig. 1). Two PKA catalytic subunits (CPK1 and CPK2) and one PKA regulatory subunit (PKR) were previously identified in F. graminearum [13]. In F. graminearum, the cpk1 deletion mutant had multiple effects on vegetative growth, conidiation, ascospore maturation, pathogenicity, and trichothecene production, whereas the cpk2 deletion mutant had no detectable phenotype. The phenotypes of our cpk1 and cpk2 deletion mutants were largely consistent with the observations of a previous study [13].

Fig. 1.Deletion of F. graminearum PKA genes. Schematic drawing of the (A) CPK1 (FGSG_07251), (B) CPK2 (FGSG_08729), and (C) PKR (FGSG_09908) gene replacement constructs and mutants. Southern blot analyses of the wild-type strain (lane 1) and each deletion mutant (lane 2) are shown to the right. DNA samples were digested with EcoRV (E), PstI (P), or BamHI (B). The upstream or downstream flanking sequence (probe) was used for hybridization.

Involvement of PKA Pathway Components in ZEA Production

To investigate the effects of PKA pathway components in ZEA biosynthesis, the ZEA production of the cpk1, cpk2, and pkr deletion mutants was evaluated via thin-layer chromatography (TLC) analysis of extracts of their rice cultures. The F. graminearum wild-type strain Z-3639 accumulated clearly detectable amounts of ZEA (Fig. 2). Compared with the wild-type strain, the cpk1 deletion mutants showed increased ZEA production. In contrast, the ZEA level produced by the pkr deletion mutants was below the detection limit. Deletion of CPK2 did not markedly affect ZEA production. These results suggest that activation of the PKA pathway represses ZEA production and the CPK1 and PKR genes of the PKA pathway are involved in regulating ZEA biosynthesis.

Fig. 2.Effects of PKA signaling-related genes on ZEA biosynthesis. The name of each fungal strain is indicated above the corresponding line. Samples were harvested after incubation of the F. graminearum strains on rice medium for 21 days. ZEA production was detected by TLC analysis. The TLC plate was developed with chloroform-methanol (9:1 (v/v)). ZEA, zearalenone standard; WT, wild-type strain.

Effects of the PKA Pathway on ZEB2 Expression and ZEA Production

To determine whether ZEB2 is under transcriptional control by the PKA pathway, we compared ZEA accumulation and ZEB2 transcript expression profiles between the wild-type, cpk1, and pkr strains during ZEA biosynthesis. ZEA was initially detected at 3 days after incubation in the cpk1 strains, whereas the wild-type strain and the pkr strains began to accumulate ZEA at 5 days and 9 days after inoculation, respectively (Fig. 3A).

Fig. 3.ZEB2 transcription and ZEA induction in the wild-type strain and in the cpk1 and pkr deletion mutants. (A) ZEA production by the fungal strains. The time course of ZEA production was monitored by TLC analysis. (B) Schematic representation of the ZEB2 gene and transcript structures. ZEB2 comprises two introns (dashed lines) and produces two transcripts, ZEB2L and ZEB2S. Locations of primers used for qRT-PCR are denoted as “Pair 1” and “Pair 2”. The image was modified from a previous study [25]. (C) Transcript expression levels of the ZEB2 (ZEB2L and ZEB2S) and (D) ZEB2L genes in the wild-type and mutant strains, as assayed by qRT-PCR analysis. RNAs were extracted from fungal cultures grown in SG medium for 1-14 days at 25℃. The relative expression levels of ZEB2 and ZEB2L were calculated using the CYP1 gene as the internal control. The expression of ZEB2 and ZEB2L of the wild-type strain on day 1 was arbitrarily set to 1. Two biological replicates for each sample were used for qRT-PCR analysis, and three technical replicates were analyzed for each biological replicate.

ZEB2 produces ZEB2L and ZEB2S, and Zeb2L functions as a direct activator of the ZEA biosynthetic cluster genes [25]. We designed primers specific to ZEB2L and to both ZEB2L and ZEB2S (Fig. 3B). In the wild-type strain, the transcript levels of ZEB2 (ZEB2L and ZEB2S) gradually increased until day 10 and then decreased thereafter (Fig. 3C). Although the transcript expression pattern of the cpk1 strains was similar to that of the wild type, transcript levels in the cpk1 strains peaked at 6 and 10 days of incubation; at 5-6 days of incubation, the transcript level in the cpk1 strain was higher than that in the wild type, whereas at 10 days of incubation, the level in the cpk1 strain was lower than that in the wild type.

We further quantified the ZEB2L transcripts using ZEB2L-specific primers (Fig. 3D). In the wild-type and pkr strains, the pattern of ZEB2L transcription was largely similar to that of the total ZEB2 transcripts (Fig. 3D). In the cpk1 strain, total ZEB2 transcript levels peaked twice during ZEA biosynthesis, at day 6 and day 10, whereas the transcript level of ZEB2L showed one main peak at day 6, suggesting that the ratio of ZEB2L/ZEB2S of the cpk1 strain was markedly altered compared with that in the wild type (Figs. 3C and 3D).

Posttranscriptional Regulation of ZEB2L

We also determined whether ZEB2L is under posttranscriptional regulation in a PKA-dependent manner. A ZEB2L-FLAG construct under the control of the elongation factor 1α promoter (PEF1α) was introduced into the zeb2 deletion mutant, generating the FAZ111 strain (Table 1). Through subsequent outcrosses, we generated the FAZ116 and FAZ117 strains, which overexpress ZEB2L-FLAG on a cpk1 and pkr deletion mutant background, respectively (Table 1). Both the FAZ116 and FAZ117 strains produced Zeb2L under the control of the strong promoter (PEF1α), but their ZEA production differed (Fig. 4). Compared with the FAZ111 strain, the FAZ116 strain, which had the cpk1 background, produced more Zeb2L protein, whereas the FAZ117 strain, which had the pkr background, produced less Zeb2L (Fig. 4A). The ZEA production was consistent with the amounts of Zeb2L in deletion mutants FAZ116 and FAZ117 (Fig. 4B).

Fig. 4.Production of Zeb2L and ZEA by F. graminearum strains constituvely expressing ZEB2L. (A) Zeb2L protein expression in the fungal strains. Flag-tagged Zeb2L was constitutively expressed in the fungal strains and purified by immunoprecipitation with magnetic beads conjugated to a mouse anti-Flag antibody. Western blot analysis with a rabbit anti-Flag antibody was performed to verify the purified Zeb2L protein. Coomassie blue staining was used as a loading control. (B) ZEA production of the fungal strains in CM. ZEA production of each fungal strain in CM was detected by TLC analysis. Two independent transformants for each construct were used in these experiments.

 

Discussion

The G-protein, cAMP, and PKA signaling cascades are highly conserved in fungi, but they exhibit functional divergence among fungal species due to specialized downstream regulators [17]. In S. cerevisiae, the PKA pathway plays important roles in carbon metabolism, cell cycle progression, and pseudo hyphal growth [3]. In model filamentous fungi, including Neurospora crassa and A. nidulans, these pathways are important for reproducible processes and hyphal morphogenesis as well as carbon metabolism [1,8]. In phytopathogenic fungi such as Magnaporthe oryzae and Ustilago maydis, cAMP signaling is an important trigger for pathogenesis [4,5]. Involvement of the PKA pathway in asexual and sexual development, trichothecene production, and virulence in F. graminearum was also recently reported [13].

Filamentous fungi produce a multitude of secondary metabolites such as mycotoxins, antibiotics, and pigments [32]. Previous studies have revealed that the synthesis of most fungal secondary metabolites is under the control of the G-protein-cAMP-PKA signaling pathway [13,27,28]. In Aspergillus species, the expression of AFLR, the gene that encodes an activator for aflatoxin biosynthesis, is negatively controlled by FadA, the α-subunit of a heterotrimeric G-protein [32]. When the PKA catalytic subunit is active, AFLR expression and the subsequent production of sterigmatocystin are blocked. PKA also influences post-transcriptional control of AFLR via RasA, a member of the family of small GTP-binding proteins [27,28]. In F. graminearum, the production of deoxynivalenol (DON), a virulence factor for the infection of wheat, is regulated by G-protein signaling and the PKA pathway [13,24]. The transcription factor AreA regulates DON production by interacting with Tri10, a transcriptional activator of DON biosynthetic cluster genes, and is known to be a putative target of PKA [12].

In this study, we investigated the regulatory role of the PKA pathway on ZEA production in F. graminearum. Deletion of the PKA regulatory subunit gene (PKR) and the PKA catalytic subunit gene (CPK1) resulted in negative and positive regulation of ZEA production, respectively, demonstrating that activation of the Cpk1-dependent PKA pathway represses ZEA biosynthesis (Fig. 5). Based on previous studies that showed that the cAMP-PKA pathway is downstream of G-protein signaling and that G-protein signaling is involved in ZEA production [24], ZEA production was expected to be regulated by the PKA pathway in F. graminearum.

Fig. 5.Proposed model of the cAMP-PKA signaling pathway for the regulation of ZEA biosynthesis. The PKA pathway, which is activated by a high cAMP (red circles) level, affects the transcriptional and posttranscriptional regulation of ZEB2L. At low cAMP levels, the inactive PKA complex, which contains two PkaRs (R in the yellow circles) and two PkaCs (C in the black circles), induces ZEB2L transcription and produces a high Zeb2L/Zeb2S ratio, leading to the activation of ZEA biosynthesis in F. graminearum. Under ZEA-inducing conditions, two components of the PKA pathway, Cpk1 and Pkr, have active roles in the regulation of ZEA biosynthesis. The green and orange ovals labeled with L and S indicate Zeb2L and Zeb2S homooligomers, respectively. The complexes of green L-labeled ovals and orange S-labeled ovals represent Zeb2L-Zeb2S heterodimers.

The production of both ZEA and DON is under the control of the PKA pathway. However, the regulatory mechanisms for ZEA and DON production do not entirely overlap in F. graminearum. Both α-subunits (GzGpa1 and GzGpa2) participate in the G-protein signaling that is involved in DON production, but only GzGpa1 is closely related to ZEA biosynthesis [24,30]. The only known putative PKA target, AreA, positively regulates DON production but is dispensable for ZEA biosynthesis [21], suggesting that in F. graminearum, the distinct and divergent regulatory mechanisms for the production of each mycotoxin result primarily from pathways downstream of PKA.

The ZEB2 gene produces an activator, Zeb2L, and an inhibitor, Zeb2S, through alternative transcription, and each isoform is regulated through distinct mechanisms [25]. In this study, disruption of the Cpk1 component of the PKA pathway induced high levels of ZEB2L transcription, whereas transcript levels of ZEB2S did not increase accordingly. Activated Cpk1 relays cellular signals to repress ZEB2L transcription, and other pathways may govern ZEB2S transcription. The Cpk1 pathway may also regulate ZEB2L transcripts posttranscriptionally as well as transcriptionally. Both the cpk1 and pkr mutants that expressed ZEB2L-FLAG under the control of a strong promoter (PEF1α) showed similar patterns of ZEA production compared with those of the cpk1 and pkr mutants that expressed the ZEB2 wild-type allele (Fig. 4), suggesting that the Cpk1 pathway negatively regulates ZEB2L posttranscriptionally. Similarly, in A. nidulans, the PKA pathway regulates AFLR both transcriptionally and posttranscriptionally for sterigmatocystin production [28].

Most of the known functional effects of the PKA pathway in eukaryotic cells are mediated via changes in downstream gene transcription. In particular, several PKA-mediated negative regulatory mechanisms have been reported in mammalian cells. PKA phosphorylates histone deacetylase and prevents its nuclear export, leading to the global inhibition of gene transcription [10]. Activation of the PKA pathway leads to upregulation of the transcriptional repressor ICER via alternative promoter usage, a transcriptional modulator CREM isoform [7]. In fungi, PKA substrates have also been shown to mediate negative gene regulation [32]. Likewise, activation of the PKA pathway strongly repressed ZEB2L transcription in F. graminearum. Because ZEB2S transcript levels were not inhibited accordingly, alteration of the Zeb2L/Zeb2S ratio by the PKA pathway could be responsible for the regulation of ZEA. Reprograming of alternative transcription via the PKA pathway has rarely been known in filamentous fungi.

In conclusion, we showed that the PKA signaling pathway negatively regulated ZEA biosynthesis via transcriptional reprogramming of the specific ZEA transcription factor gene ZEB2 and posttranscriptional regulation. Activation of the PKA pathway highly repressed the transcription of ZEB2L but not ZEB2S and therefore modulated the Zeb2L/Zeb2S ratio to fine-tune the regulation of ZEA production. Our findings provide insight into the mechanistic regulation of secondary metabolite biosynthesis in filamentous fungi.