Transcription Factors BbPacC and Bbmsn2 Jointly Regulate Oosporein Production in Beauveria bassiana

ABSTRACT

The entomopathogenic fungus Beauveria bassiana can produce the secondary metabolite oosporein under alkaline conditions or in fungus-killed cadavers. However, the regulatory mechanism of oosporein synthesis is not fully understood. In thisstudy, we found that the pH signaling transcription factor BbPacC is involved in the regulation of oosporein production. Overexpression of BbPacC promotes oosporein production in B. bassiana at pH 6.0 or under alkaline conditions (pH 8.0), but deletion of this gene abolished oosporein production. Under acidic conditions (pH 4.0), no oosporein production was observed in the wild-type and BbPacC overexpression strains. Yeast one-hybrid assays and electrophoretic mobility shift assay (EMSA) confirmed the binding ability of BbPacC with 4 putative PacC-binding sites in the promoter region of BbOpS3, a transcription factor located in the oosporein synthetic gene cluster regulating the expression of oosporein synthetic genes. Overexpression of Bbmsn2, a previously reported negative regulator of oosporein synthesis, in OEPacC or wild-type strains abolished oosporein production in all tested conditions. However, deletion of Bbmsn2 in the BbPacC overexpression strain significantly improved oosporein production even at pH 4.0. These results indicated that BbPacC is a positive regulator of oosporein production and functions jointly with Bbmsn2 to regulate oosporein production in different environments and particularly under alkaline conditions.

IMPORTANCE B. bassiana produces the red dibenzoquinone pigment oosporein under certain specific conditions, such as alkaline conditions and fungus-killed cadavers. Ooporein possesses antibiotic and insect immune inhibition activities and plays multiple roles during the infection process of B. bassiana against insect hosts. Several negative regulators involved in oosporein synthesis have been reported; however, we know little about the positive regulators outside the biosynthetic gene cluster. Here, we found that the pH signaling transcription factor BbPacC positively regulates oosporein production by binding to several PacC-binding sites. In addition, our results also indicate that BbPacC jointly acts with the negative regulator Bbmsn2 to regulate oosporein synthesis. Our results provide insight into understanding the regulatory mechanism of oosporein production as well as targets to engineer B. bassiana strains producing high levels of oosporein.

INTRODUCTION

Entomopathogenic fungi play essential roles in regulating the insect population in nature and have been exploited as fungal biopesticides. After attachment of conidia or other infectious cells on the insect cuticle, entomopathogenic fungi initiate the infection process following germination, penetration of the host cuticle, and proliferation in the host hemolymph. After host death, fungi grow outward (penetrating the cuticle again) and use the insect cadaver as a nutrient source to form mycelium and produce a large number of conidia on the cuticle, which may begin another round of infection under suitable conditions. During this process, varied interactions occur between entomopathogenic fungi and their insect hosts and between these fungi and other saprophytic microorganisms present in the insects, which might be able to feed on the dead insects (1–4). Increasing evidence has suggested that secondary metabolites have important roles in these interactions. Destruxins produced by Metarhizium anisopliae could inhibit host immune reactions, and the loss of synthetic genes of destruxins caused a significant reduction in fungal virulence (5). Similarly, deletion of the beauvericin synthetic gene in Beauveria bassiana also resulted in an approximately 60% reduction in virulence against Galleria mellonella, Helicoverpa zea and Spodoptera exigua (6).

Bacteria in insect cuticles, hemolymph, or gut are also an important factor affecting fungal infection (7–9). In B. bassiana-infected mosquitoes, it was found the opportunistic pathogenic bacterium Serratia marcescens overgrows in the midgut and translocates to the hemocoel, which facilitates fungal killing speed (10). After host death, cadavers are rich in nutrients, not only for fungal but also for bacterial growth. To compete with the bacteria in the cadavers, B. bassiana produces a red dibenzoquinone pigment oosporein after host death to inhibit bacterial growth. Strains with deletion of the oosporein synthetase gene BbOpS1 cannot efficiently compete for nutrients with bacteria in cadavers and cause less fungal growth and conidiation (11). The transcription factor BbOpS3 in the oosporein cluster (Opn) positively regulated the expression of oosporein synthesis genes. Studies have shown that 2 transcription factors, BbSmr1 and Bbmsn2, negatively regulate oosporein production in B. bassiana (11, 12). BbSmr1 is a zinc finger protein involved in the regulation of fungal development and secondary metabolite production. Deletion of Bbsmr1 resulted in upregulation of the oosporein synthetic gene cluster and then improved oosporein production (11). In addition, the transcription factor Bbmsn2, which regulates the stress responses in B. bassiana, is also a negative regulator of oosporein production. Currently, however, we do not know the regulatory details of oosporein production and are unsure whether there are other proteins involved in the regulatory network.

PacC is a vital component of the Pal/Rim-pH pathway that is responsive to ambient pH changes and regulates the expression of a series of genes, including acid- or alkaline-expressing genes in fungi (13–16). PacC also regulates secondary metabolite production in filamentous fungi. For example, PacC was found to negatively regulate sterigmatocystin (ST) production, and constitutive activation of PacC resulted in 10-fold less production of ST in an Aspergillus nidulans strain (17). In addition, the production of Patulin (PAT) in Penicillium expansum, a mycotoxin that can cause serious health concerns, was also affected by environmental pH (18), and was regulated by PacC. Deletion of PacC in P. expansum resulted in the loss of PAT production in conditions with pH values above 6.0, which was caused by the downregulation of 15 genes in the patulin cluster (19). One or more PacC-binding sites were found in the promoters of 9 PAT clusters, indicating that PePacC may directly regulate the expression of PAT synthetic genes. In B. bassiana, one PacC gene was found and functional characterized (20). Deletion of BbPacC resulted in no production of oxalic acid and the insecticidal compound dipicolinic acid but promoted the production of a yellow-colored compound bassianolone B (20). Luo et al. reported that wild-type B. bassiana produced more oosporein under alkaline conditions than under acidic conditions (12). We also found 4 PacC-binding sites (T/CGCCAAG) in the promoter of the key transcription factor BbOpS3 in the oosporein cluster. Therefore, we hypothesized that BbPacC might be involved in the regulation of oosporein production. In this study, we constructed BbPacC deletion and overexpression strains, and analyzed oosporein production in these strains to clarify this question. Our results indicated that BbPacC of B. bassiana is a positive regulator of oosporein synthesis and functions jointly with the reported negative regulator Bbmsn2 to control oosporein production under different pH conditions.

RESULTS

BbPacC positively regulates oosporein production.

BbPacC is an important pH-responsive transcription factor in B. bassiana and is involved in the fungal stress response and the regulation of several secondary metabolites, such as oxalic acid, dipicolinic acid, and bassianolone B (20). BbOpS3 is a GAL4 transcription factor located in the oosporein synthetic gene cluster and regulates the expression of oosporein synthesis genes. To explore the possible regulatory proteins in oosporein production, we analyzed the promoter region of BbOpS3 and found four PacC-binding sites, 3 sites of TGCCAAG (-941 to -937 bp, -930 to -924 bp, -506 to -500 bp) and 1 site of CGCCAAG (-1251 to -1245 bp) (Fig. 1A). To clarify whether BbPacC is involved in oosporein synthesis, we constructed BbPacC knockout mutants by replacing the encoding region with the Sur resistance gene (Fig. S1A and B). BbPacC overexpression strains were also constructed by placing the coding region of BbPacC under the control of the constitutive promoter PgpdA from the glyceraldehyde-3-phosphate dehydrogenase gene of B. bassiana (Fig. S1C). The expression levels of BbPacC in overexpression strains were detected by real-time PCR (RT-PCR), and 1 strain exhibiting the highest expression level of BbPacC (31-fold higher than wild-type) was selected for further study (Fig. S1D). BbPacC deletion and overexpression strains were inoculated into SDB with different pH values (4.0, 6.0, and 8.0). After being cultured for 3 to 4 days, the BbPacC overexpression strain produced oosporein in media with pH = 6.0 or 8.0 and made the culture medium turn red. However, deletion of BbPacC abolished oosporein production in all tested media at pH 4 to 8. The wild-type strain only produced oosporein under alkaline conditions (pH 8.0) (Fig. 1B). High-performance liquid chromatography (HPLC) analysis results confirmed the oosporein-producing conditions, showing concentrations ranging from 0.039 ± 0.001 to 0.166 ± 0.016 mg/mL (Fig. 1C). To further evaluate oosporein production in fungus-killed cadavers, BbPacC deletion and overexpression strains were inoculated onto G. menollena larvae. After host death, larvae infected by the wild-type and BbPacC overexpression strains appeared red–brown, and HPLC verified oosporein production in the 2 strains. BbPacC overexpression strains produced more oosporein than the wild-type strain. After deletion of BbPacC, no or less oosporein was produced in fungus-killed larvae (Fig. 2). The expression of BbOpS3 in fungus-killed cadavers exhibited an almost similar trend to that of BbPacC, with the highest expression level achieved in the cadavers where the BbPacC overexpression mutant was growing (48 h post-death) (Fig. 2B).

FIG 1.

BbPacC positively regulates oosporein production. (A) Analysis of the BbOpS3 promoter. The putative PacC-binding sites were indicated as PacC-1, PacC-2 (contained 2 sites) and PacC-3 sequentially. The potential binding sites of BbPacC and Bbmsn2 are highlighted by the different colors green and gray, respectively. (B) Oosporein production of BbPacC deletion and overexpression strains was detected in SDB buffered for different pH values (4, 6 and 8). (C) Oosporein concentration detected by HPLC. **, P < 0.01; ns, not significant.

FIG 2.

Deletion of BbPacC affects oosporein production in G. mellonella cadavers. The expression of BbOpS3. (A) and BbPacC (B) was analyzed by RT-PCR after host death. The Actin gene was used as the reference gene for normalization. The inset image in A shows the color change of cadavers due to oosporein production. (C) HPLC analysis of oosporein extracted from cadavers killed by BbPacC deletion or overexpression strains. *, P < 0.05; **, P < 0.01; ns, not significant.

Interactions between BbPacC and the BbOpS3 promoter.

Interactions of BbPacC and predicted PacC-binding sites in the BbOpS3 promoter were analyzed by yeast one-hybrid and electrophoretic mobility shift assay (EMSA). For yeast one-hybrid assays, 4 predicted BbPacC-binding sites were cloned into 3 vectors. As there is only a 6 bp spacer between the second site (-941 to -937 bp) and the third site (-930 to -924 bp) (Fig. 1A), we designed these 2 sites in 1 fragment, named PacC-2. The results revealed that BbPacC interacts with all 3 tested fragments containing PacC-binding site(s), with fragment 2 (PacC-2), which contains 2 sites exhibiting the strongest binding ability (Fig. 3A). The interaction of BbPacC with the BbOpS3 promoter was further analyzed by EMSA. Biotin-labeled DNA probes containing the predicted binding site of the BbOpS3 promoter (-946 bp to -929 bp) were synthesized and incubated with extracted total proteins of fungal strains. A protein-DNA interaction band was observed in the presence of BbPacC protein from BbPacC overexpression and wild-type strains. However, for the total proteins from the BbPacC deletion strain, no interaction was observed (Fig. 3B).

FIG 3.

Yeast one-hybrid and EMSA of BbPacC-binding to the BbOpS3 promoter. (A) Yeast one-hybrid of BbPacC to 4 potential PacC-binding sites in the BbOpS3 promoter. PacC-binding sited were indicated in Fig. 1A. (B) EMSA of BbPacC bound to a putative PacC site from the BbPacC promoter. Proteins were extracted from B. bassiana wild-type, BbPacC deletion and overexpression strains and used for EMSA. An unlabeled probe was added in Lanes 2, 4 and 6.

BbPacC and Bbmsn2 regulate jointly oosporein production.

Bbmsn2 has been shown to negatively regulate oosporein production in B. bassiana (12). To test whether Bbmsn2 and BbPacC regulate jointly oosporein production, BbPacC was overexpressed or deleted in Bbmsn2 strains, including ΔBbmsn2 and OEBbmsn2. Firstly, we constructed ΔBbmsn2 and OEBbmsn2 strains by replacing the encoding region with the Sur resistance gene or placing the encoding region of Bbmsn2 under the control of a constitutive promoter Pb3 from the glyceraldehyde-3-phosphate dehydrogenase gene of B. bassiana (Fig. S2A and B). Then, we overexpressed or deleted BbPacC in ΔBbmsn2 and OEBbmsn2 strains, obtaining the strains OEBbPacCOEBbmsn2, OEBbPacCΔBbmsn2, and ΔBbPacCΔBbmsn2. The expression levels of BbPacC and Bbmsn2 in different overexpression and knockout strains were detected by RT-PCR, and some strains exhibiting the highest expression level of BbPacC was selected for further study, as well as verification of the knockout gene (Fig. S2G). These strains were cultured in SDB buffer with various pH values for 4 days, and oosporein production was detected. Although the expression of BbPacC promoted oosporein production at pH 6.0 and 8.0, overexpression of Bbmsn2 completely inhibited oosporein production under these tested conditions regardless of whether BbPacC is overexpressed (Fig. 4). Conversely, deletion of Bbmsn2 in the BbPacC overexpression strain further improved oosporein synthesis even at pH 4.0, in which wild-type and OEBbPacC strains do not synthesize oosporein (Fig. 4). These results were similar with that of Luo et al., in which Bbmsn2 mutant produced oopsorein under pH 5.1 to 7.4 in SDB (12). In double deletion mutants of BbPacC and Bbmsn2, no oosporein production was observed in media with various pH values. The oosporein production of the OEBbOps3 strain is also presented. Oosporein was produced under 3 pH conditions, and the content increased from pH 4.0 to pH 8.0. The expression levels of the polyketone synthetase genes BbOpS1 and BbOps3 exhibited a positive relationship with the oosporein production in these tested strains (Fig. 5). These results indicated that BbPacC and Bbmsn2 play positive and negative regulatory roles, respectively, in oosporein production and jointly regulate oosporein synthesis in various environments.

FIG 4.

Bbmsn2 and BbPacC jointly regulate oosporein production in B. bassiana. Fungal strains were cultured in SDB buffered for different pH values (4, 6, and 8) for 3 days. The BbOpS3 overexpression strain (BbOpS3^OE) was used as a positive control.

FIG 5.

Expression analysis of the oosporein synthetic gene (BbOpS1) and the regulatory gene (BbOpS3) in various strains. Fungal strains were cultured in SDB (pH 4, 6, and 8) for 3 days, and total RNA was extracted for RT-PCR analysis. The Actin gene was used as the reference gene for normalization.

BbPacC and Bbmsn2 exhibited different expression models under various pH values.

It has been reported that alkaline conditions can promote oosporein production, but acidic conditions inhibit oosporein synthesis (12). To understand the possible mechanism regulating oosporein production under various pH conditions, we analyzed the expression levels of BbPacC and Bbmsn2 in the wild-type strain in SDB buffered for different pH values. BbPacC was expressed at the highest level at pH 8.0 but the lowest at pH 4.0; however, a converse expression pattern of Bbmsn2 was observed, with the highest expression at pH 4.0. High-level expression of Bbmsn2 at pH 4.0 explains why the BbPacC overexpression strain cannot synthesize oosporein under this condition (Fig. 6).

FIG 6.

Expression of BbPacC and Bbmsn2 in B. bassiana under different pH values. Wild-type B. bassiana was cultured in SDB buffered for various pH values (4, 6, and 8) for 3 days. Total RNA was extracted, and RT-PCR was performed as described in Materials and Methods. The Actin gene was used as the reference gene for normalization. **, P < 0.01.

DISCUSSION

Oosporein is reported in Oospora colorans, Beauveria brongniartii and some soil/endophytic fungi (21–23). Based on the genomic DNA sequence, the oosporein synthetic gene cluster was also identified in the B. bassiana genome, and the biosynthetic pathway and biological functions were verified experimentally (24–26). Two negative regulators of oosporein production, BbSmr1 and Bbmsn2, have been reported (8, 9), and our results showed that the transcription factor BbPacC positively regulates oosporein production. Overexpression of BbPacC significantly improved oosporein production in SDB under pH 6 or 8; however, deletion of BbPacC severely impaired oosporein synthesis. In addition, our results showed that BbPacC functions jointly with the negative regulator Bbmsn2 to affect oosporein synthesis under various pH conditions. This provides insight into the regulatory mechanism of oosporein synthesis.

The production of fungal secondary metabolites is impaired by different environmental signals, such as nutrients, temperature, light, pH, and stressors (27). Therefore, transcription factors involved in nutrient source, light intensity, redox status, and ambient pH values, such as LaeA, AreA, HapX, and PacC, are responsible for the regulation of the production of some secondary metabolites (28). In entomopathogenic fungi, PacC is involved in acid/alkaline responses, many physiological processes, and mycelial growth, stress response, virulence, and secondary metabolite production (20, 29). Deleting PacC (MrPacC) from Metarhizium robertsii reduced fungal tolerance to salt/metal challenges and full virulence and downregulated the expression of chitinase and glucosyltransferase genes (29). In B. bassiana, BbPacC regulates not only fungal responses to various stressors, including osmotic stress and SDS, but also the production of oxalic acid, dipcolinic acid, and bassianoline B (20). In this study, BbPacC was also found to regulate oosporein production. It is worth noting that orsellinic acid is the intermediate product of the oosporein synthesis pathway produced by the polyketide synthetase BbOpS1 in the oosporein cluster. These results indicated BbPacC plays important roles in the production of acid substances in B. bassiana and we may explore more secondary metabolites with BbPacC deletion or overexpression strains.

BbOpS3 is a Gal4-like transcription factor in the oosporein synthesis gene cluster. Overexpression of BbOpS3 can constitutively activate oosporein, but the BbOpS3 mutant caused no oosporein production, indicating that BbOpS3 directly regulates oosporein production (26). Therefore, the regulatory mechanism of BbOpS3 expression is the key to understanding oosporein synthesis. Our results showed that BbPacC can bind the putative PacC sites in the promoter of BbOpS3, indicating that BbPacC may directly regulate the expression of BbOpS3. Deletion of BbPacC abolished oosporein production, further verifying that BbPacC is a positive regulator in oosporein production. However, it is unclear whether other positive regulatory factors in B. bassiana control oosporein production.

Studies have shown that the production of oosporein in artificial media is not consistent and greatly depends on nutrient compositions and pH values. A study found that only a small fraction (5 out of 16) of B. bassiana strains freshly isolated from cadavers could produce oosporein under the tested conditions, and serial passage on artificial media caused the oosporein-ng strains to lose oosporein-producing ability (30). Therefore, there may be a complex regulatory cascade in oosporein synthesis in B. bassiana. Although BbPacC is a positive regulator of oosporein production, overexpression of BbPacC did not lead to oosporein production at pH 4.0. Deleting another transcription factor, Bbmsn2, from the OEBbPacC strain resulted in oosporein synthesis at pH 4.0, indicating that, at low pH Bbmsn2 blocked oosporein synthesis even with high levels of BbPacC. RT–PCR analysis showed that Bbmsn2 has a higher expression level under acidic conditions in the B. bassiana wild-type strain, but the expression of BbPacC exhibits the opposite trend: alkaline > neutral > acidic conditions, which also explains why B. bassiana produces oosporein in alkaline environments. In the BbOpS3 promoter, there is also a putative msn2-binding site AGGGG (-436 to -500 bp). We have tried to analyze the interaction between Bbmsn2 and the putative binding site. However, no evident interaction was observed (data not shown). Currently, we are not sure whether the regulatory mechanism of Bbmsn2 on BbOpS3 expression is direct or indirect. In a previous study, we found that the transcription factor BbSmr1 also negatively regulates oosporein production. However, the relationship among these transcriptional factors needs further study. Combining these results, we hypothesize that there is a complex regulatory cascade for oosporein production in which more regulatory proteins, except those examined here, may be involved. Further studies will clarify the detailed regulatory mechanism of oosporein synthesis, which will aid in understanding the biological roles of oosporein in B. bassiana.

MATERIALS AND METHODS

Strains and culture conditions.

B. bassiana strain Bb0062 (CGMCC 7.34) was used in this study and typically cultured in Czapek-Dox Broth and Czapek-Dox Agar (CZB)/CZA, Potato Dextrose Broth (PDB)/PDA, or Sabouraud Dextrose Broth (SDB)/SDA. Escherichia coli DH5α and Agrobacterium tumefaciens AGL-1 were used for routine DNA manipulation and fungal transformation, respectively.

Construction of deletion and overexpression strains.

The primers used for DNA manipulation are listed in Table S1. To construct the BbPacC deletion mutant, a 651 bp sequence in the BbPacC ORF was replaced with the Sur gene cassette (1694 bp). The upstream (952 bp) and downstream (1510 bp) regions were amplified by PCR using genomic DNA as a template and cloned into the EcoRI/SpeI and XbaI/HindIII sites of the pK2-bar vector. The resultant vector was verified by sequencing and transformed into A. tumefaciens. B. bassiana transformation mediated by A. tumefaciens was performed as described (31). Correct mutants were screened by PCR using the primers PacC-t1/t2 and subsequently verified by RT–PCR. Using the same method for the BbPacC mutant, the Bbmsn2 mutant was constructed by replacing a 464 bp fragment with this gene with the Sur gene cassette (2560 bp). The upstream (1532 bp) and downstream (1230 bp) regions of the Bbmsn2 deletion region were amplified and cloned into pK2-Sur. The obtained Bbmsn2 deletion construct was transformed into B. bassiana wild-type and ΔBbPacC strains, and the Bbmsn2 mutant (ΔBbmsn2) and double deletion mutant (ΔBbmsn2ΔBbPacC) were obtained.

To construct the BbPacC overexpression vector, the ORF was amplified with the primers Pacc-O1/O2 using genomic DNA as a template. The BbPacC fragment was cloned downstream of the constitutive promoter PgpdA in the pK2-bar-PgpdA vector using a seamless cloning kit (ClonExpress, Vazyme). BbPacC overexpression strains were verified by PCR using the primers pgpdA-up, located in the promoter, and pgpdA and PacCcDNA2.

Oosporein extraction and detection.

Fungal strains were cultured in 0.5 × SDB buffered with 0.2 M Na₂PO₄-0.1 M citric acid to pH = 4 and 6, and 0.05 M glycine-0.05 M NaOH to pH = 8, respectively. Fungal cultures were grown at 26°C for 3 days, and oosporein was extracted and detected by HPLC as described (11). HPLC was performed with an Agilent ZORBAX SB-C18 column (25 cm × 4.6 mm, 5 μm) with a formic acid (0.1‰)/acetonitrile gradient consisting of 5% acetonitrile (0 to 3 min), 5 to 100% acetonitrile (3 to 18 min), and 100% acetonitrile (18 to 21 min). Purified oosporein was used to prepare a standard curve.

RT-PCR analysis.

Total RNA was extracted from various strains after being cultured in 0.5 × SDB for 3 days or from fungus-killed G. mellonella larvae. Real-time PCR was performed following the previously described method (11), with the indicated primers in Table S1. The relative transcript levels of target genes were normalized to actin (GenBank accession no. HQ232398).

Fungal infection.

Fungal strains were grown on PDA plates for 2 weeks, and conidial suspensions were prepared with 0.05% Tween 80 and adjusted to 10⁷ conidia/mL. Fourth instar G. mellonella larvae were immersed in conidial suspension for 15 s and were kept in Petri dishes (ϕ = 150 mm). After the fungi killed the larvae, cadavers were transferred into a new Petri dish, and oosporein production was observed by a color change and HPLC analysis.

Yeast one-hybrid assay.

To perform a yeast one-hybrid assay, the BbPacC ORF (2004 bp) was amplified with the primer pair BbPacC-AD42-F/R using B. bassiana cDNA as a template and cloned into pB42AD to obtain pB42AD-BbPacC (effector). PacC-binding sites containing 3-bp of its upstream and downstream sequences respectively were repeated two or three times, and designed into a primer pair (Table S1). As there is only 6-bp space between second and third sites, they were designed into one fragment and repeated two times (Fig. 1A). Primers (10 μm) were treated by polynucleotide kinase and used to form double stranded DNA with EcoRI and XhoI sites at both ends, respectively. The obtained fragments were cloned into the pLacZi vectors to construct reporter vectors. The effector and reporters were co-transferred into the EGY48 yeast strain to confirm interactions in yeast cells. Transformants were grown on SD/-Ura/-Trp medium at 30°C for 3 to 5 days, and positive colonies were transferred onto SD/Gal/Raf medium supplemented with X-gal (80 mg/L) for color development, with empty pB42AD and pLacZi serving as negative controls.

Fungal protein extraction and EMSA analysis.

B. bassiana wild-type, BbPacC^OE, and ΔBbPacC strains were cultured in 0.5 × SDB (pH = 8) for 4 days. Fungal proteins were extracted from mycelium using a filamentous fungal protein extraction kit (BestBio, China). EMSA was performed using the LightShift Chemiluminescent EMSA kit (Thermo Fischer Scientific) following the manufacturer’s instructions. The biotin-labeled DNA fragment containing the PacC-binding site in the BbOpS3 promoter (-946 bp to -929 bp) was synthesized (Huada Biological Technology). In competition assays, a 200-fold unlabeled probe was added.

Data analyses.

Experiments were performed in triplicate and repeated three times. Gene expression analyses oosporein concentration were graphed using GraphPad prism 7.0 software (GraphPad) and significance was assessed with one-way ANOVA. Asterisks were used to denote significance (*, P < 0.05; **, P < 0.01).