Role of MAPK Signaling Pathways in Regulating the Hydrophobin Cryparin in the Chestnut Blight Fungus Cryphonectria parasitica

Abstract

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

Fungal hydrophobins are small, secreted proteins that react to the outermost interface of an object, typically conferring a hydrophobic property on fungal cell walls [1,2]. These proteins have been found to impact several morphogenetic processes, including sporulation, fruit body development, and infection structure formation [2]. Cryparin (Crp) is a small, abundant cell-surface hydrophobic protein in chestnut blight fungus Cryphonectria parasitica; it is a class II hydrophobin [1,3,4]. Antibody staining has indicated that cryparin is exclusively abundant in the fungal fruiting body walls on its natural host, although it has also been found on the surfaces of fungal hyphae grown in artificial culture. Functional analysis of cryparin has indicated that it is required for stromal pustule eruption through the bark of the chestnut tree; this function plays an essential role in the fitness of the fungus [4]. In C. parasitica, Crp mRNA is abundantly expressed, and the cryparin transcript has been shown to account for 25–30% of the total mRNA after 2 days of liquid culture [5]. Promoter analysis of the cryparin gene has indicated that it contains a small core promoter region, which is necessary for high cryparin expression levels [6]. Interestingly, the infection of C. parasitica by Cryphonectria hypovirus 1 (CHV1) results in a drastic change in the accumulation of Crp mRNA, and Crp expression has been shown to be transcriptionally downregulated (between 50% and 70%) in a CHV1-713-infected strain (UEP1), compared with an isogenic virus-free strain (EP155/2) [4,5]. However, no other regulatory mechanisms have been characterized for cryparin regulation.

Signaling cascades play critical roles in modulating diverse regulators, including protein kinases and transcription factors. Among these, the mitogen-activated protein kinase (MAPK) signaling pathway is of particular interest because several genes in the MAPK pathways of C. parasitica and their downstream effectors are closely implicated in symptom development such as reduced sporulation, pigmentation, laccase production, and virulence by the hypovirus infection [7,8,9,10,11,12,13,14]. The MAPK signal transduction pathway consists of three functionally interlinked cascade kinases: MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase (MAPK). The MAPK pathways are highly conserved across a wide range of organisms from yeast to humans [15,16]. In the most studied fungus Saccharomyces cerevisiae, five different MAPK pathways, governing pheromone response, filamentous growth, osmotolerance (HOG1), cell wall integrity (CWI), and ascospore formation, have been identified [17]. As an analogy, three signaling pathways regulating the pheromone response, CWI, and the osmoregulation/stress response have been suggested in filamentous fungi [18]. Fungal hydrophobins, in general, play an important role in the growth and development of most fungi [19]. Thus, both biotic and abiotic environmental conditions have been proven to affect the expression of hydrophobin genes and various signaling pathways such as cAMP/PKA, Ca²⁺/calmodulin, MAPK, and the cell cycle have been identified as regulatory pathways for fungal hydrophobin expression [20,21,22,23,24]. Among these, MAPK signaling pathways are particularly promising. Depending on the fungi, different MAPK pathways have been implicated in the regulation of hydrophobin. Specifically, PMK1, an ortholog of yeast Kss1/Fus3, was shown to regulate the MPG1 hydrophobin gene in Magnaporthe oryzae, and VdHog1, an ortholog of yeast HOG1, was shown to be essential in the expression of VDH1 hydrophobin in Verticillium dahliae [22,25].

In this study, using the advantage of the availability of mutants in three representative MAPKs, we explored the MAPK regulation of cryparin production in C. parasitica.

MATERIALS AND METHODS

Fungal strains and culture conditions

C. parasitica strains used in this study are listed in Table 1. Strains were grown on potato dextrose agar containing L-methiononine (100 mg/L) and biotin (1 mg/L) (PDAmb) plates under constant low light at 25℃ [26]. The mycelia were harvested from 5-day-old cultures, lyophilized, and stored at −70℃ for RNA extraction as previously described [27].

Table 1. List of strains in this study.

Hydrophobicity of mycelial mats

Hydrophobicity of the mycelia of 7-day-old cultures was evaluated by observing the assimilation of 9–10 water droplets (3 µL each) on mycelial surfaces on culture plates.

Cryparin extraction from mycelia

Lyophilized mycelia of 7-day-old cultures were finely ground, well suspended in 60% ethanol (5 µL ethanol per 1 mg mycelia) and centrifuged at 8,000 ×g for 15 min [3]. Extracts from all preparations were further suspended in 6× polyacrylamide gel electrophoresis (PAGE) loading dye and 15% glycerol, followed by separation on a 12% sodium dodecyl sulfate (SDS)-PAGE gel. Following separation, gels were stained with Coomassie blue to visualize the extracted proteins.

Cloning and characterization of MADS-box transcription factors

The C. parasitica genome database (http://genome.jgi-psf.org/Crypa1/Crypa1.home.html) was screened for yeast MCM1 and RLM1 homologues. Polymerase chain reaction (PCR) amplification of CpMcm1 and CpRlm1 was performed with primers CpMcm1-gF1/CpMcm1-gR1 and CpRlm1-gF1/CpRlm1-gR1, respectively (Table 2). The resulting 1.6-kb PCR amplicon of CpMcm1 and 5.1-kb amplicon of CpRlm1 were cloned separately into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced.

Table 2. List of primer sequences.

RT-PCR, reverse transcriptase PCR; qRT-PCR, quantitative real-time PCR.

^aPCR to obtain full gene.

To obtain cDNA clones of CpMcm1 and CpRlm1, PCR was performed using reverse transcriptase PCR (RT-PCR) with primers CpMcm1-mF1/CpMcm1-mR1 and CpRlm1-mF1/CpRlm1-mR1 at nucleotide positions (nt) −1 to 19/917 to 937 and (nt) −15 to 7/1,961 to 1,981 (relative to the start codon). The resulting 0.7-kb PCR amplicon of CpMcm1 and 1.8-kb amplicon of CpRlm1 were cloned separately into the pGEM-T Easy vector and sequenced.

Phylogenetic analysis of MADS-box transcription factors

The phylogenetic relationships of CpMcm1 and CpRlm1 with other fungal MADS-box transcription factor homologs were analyzed using MEGA 7 [28]. An evolutionary matrix was inferred using the Henikoff and Henikoff (1992) method [29], and the branching pattern was generated using the neighbor-joining method [30,31].

Analysis of transcript accumulation using quantitative real-time RT-PCR

RNA from all tested strains was extracted from culture 5 days after inoculation, and quantitative real-time RT-PCR was conducted as previously described [10]. Analyses were conducted at least twice, in triplicate for each transcript, from at least two independent RNA preparations of the same sample. Transcript levels relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (gpd) of C. parasitica were used as an internal control. The primer pairs used for each target gene are listed in Table 2.

Statistical analyses

All quantitative real-time RT-PCR transcripts were analyzed by ANOVA using SPSS software ver. 23.0 (IBM Corp., Armonk, NY, USA). The significance of all effects was determined using the Student-Newman-Keuls method at p = 0.001.

RESULTS AND DISCUSSION

Phenotypic changes of MAPK mutants

To determine the surface hydrophobicity of the aerial mycelia, the easy-wet phenotype was screened by placing a water droplet on the surface of aerial hyphae that had grown for 7 days on PDAmb. Normally, when applying a water droplet to the surface of aerial hyphae, the surface hydrophobicity of the hyphae prevents the water droplet from being absorbed by aerial mycelia and the water remains a discrete drop. However, a cryparin-null mutant quickly absorbs the drop of water. Therefore, we examined the easy-wet phenotype of the mutants representing the three MAPK pathways (Fig. 1). The wild-type EP155/2 showed non-absorbed, intact water droplets and the water drops remained intact even after long periods of incubation (> 24 hr). However, the cryparin-null mutant quickly adsorbed the water droplet, a response characteristic of the easy-wet phenotype [4]. Both the CpSlt2-null mutant and its sectored progeny exhibited the absorption of water droplets, indicating changes in the surface hydrophobicity of the aerial mycelia. The hydrophobicity of the CpBck1-null mutant was also estimated using water droplets. However, the near absence of aerial hyphae in the CpBck1-null mutant made it difficult to determine the surface hydrophobicity. Thus, the easy-wet phenotype was measurable only in the sectored progeny of the CpBck1-null mutant. The sectored progeny of the CpBck1-null mutant exhibited the characteristic easy-wet phenotype, although compact aerial mycelia were produced on the colony of the sectored progeny, and not on the parental CpBck1-null mutant. These results clearly indicate that mutants in the CWI pathways have impaired surface hydrophobicity. We also tested the surface hydrophobicity of the mutant in the mating-responsive MAPK pathway. The mutation in the cpmk2 gene, an ortholog of yeast FUS3 gene, resulted in different growth patterns in response to solid and liquid media [11]. The cpmk2-null mutant showed reduced colonial growth but no growth defect in liquid media. Interestingly, absorption of water droplets on top of the aerial mycelia was not observed (> 24 hr), indicating that the surface hydrophobicity was not changed. Previous studies on the cpmk1 mutant, an ortholog of the yeast HOG1 gene, demonstrated that transcription of the cryparin gene was severely downregulated, such that it was even lower than that of the hypovirulent UEP1 strain, which has been known to downregulate the cryparin gene. It has been reported that the easy-wet phenotype on the aerial mycelia is not obvious [10]. We verified the easy-wet phenotypes of the cpmk1-null mutant and the hypovirulent UEP1 strain and found that in both most water droplets were completely absorbed after long periods of incubation. This difference between the current study and previous studies may be attributed to different colony ages, adaptive changes of the mutants during long periods of storage, or uneven distribution of surface hydrophobicity.

Fig. 1. Easy-wet morphology to determine the hydrophobicity of mycelia. Wild-type EP155/2, hypovirulent UEP1, cryparinnull mutant (ΔCrp), CpBck1-null mutant (TdBCK1), a sectored progeny of TdBCK1 (TdBCK1-S1), CpSlt2-null mutant (TdSLT2-69), a sectored progeny of TdSLT2-69 (TdSLT2-69-S1), cpmk1-null mutant (TdMK1-23), and cpmk2-null mutant (TdMK2-1) strains are indicated on the panels. Colonies are shown after 7 days of culture. After dropping 3 µL of water on the mycelial surfaces, easy-wet morphology was observed using microscopy (EZ4HD, Leica, Germany).

To gain a better understanding of the amount of cryparin produced, we extracted cryparin from the mycelia of mutants and compared cryparin amounts using SDS-PAGE. As shown in Fig. 2A, a protein band was observed at 24 kDa for cryparin in wild-type EP155/2. It is well known that, compared with wild-type EP155/2, cryparin is significantly downregulated in the CHV1-infected isogenic strain UEP1, which was represented by a very-intensity cryparin band on SDS-PAGE (Fig. 2A). In CpBck1-, CpSlt2-, and cpmk1-null mutants exhibiting the easy-wet phenotype, the cryparin band was nearly absent. Interestingly, a more than two-fold increase of cryparin band intensity was observed in the cpmk2-null mutant, suggesting that more cryparin was produced in the cpmk2-null mutant. We also examined the accumulation of cryparin transcript among mutants using real-time RT-PCR (Fig. 2B). The accumulation of cryparin transcripts was significantly downregulated in strains exhibiting the easy-wet phenotype and less amount of cryparin. Cryparin transcript was significantly increased in the cpmk2-null mutant, indicating increased cryparin production. Analyses of cryparin expression indicated that only two MAPK pathway-related mutants, CWI and Hog1, exhibited clear easy-wet phenotypes, due to the dramatic decrease in cryparin production. The Fus3 MAPK pathway mutant also appeared to be implicated in cryparin production, but in a different way from the other two MAPK pathways. The results regarding on the easy-wet phenotype, protein production, and transcript accumulation were all consistent with one another, and the modulation of cryparin production occurred at the level of transcription, according to real-time RT-PCR.

Fig. 2. The cryparin production (A) and cryparin transcript (B) of mitogen-activated protein kinase pathway mutants. Wild-type EP155/2, hypovirulent UEP1, Crp-null mutant (ΔCrp), CpBck1-null mutant (TdBCK1), a sectored progeny of TdBCK1 (TdBCK1-S1), CpSlt2-null mutant (TdSLT2-69), a sectored progeny of TdSLT2-69 (TdSLT2-69-S1), cpmk1-null mutant (TdMK1-23), and cpmk2-null mutant (TdMK2-1) strains are shown.

In silico analysis of regulatory motifs in the cryparin gene promoter

Regulatory sequence motifs were sought and discovered in the 2.0 kb-promoter region of the cryparin gene, which includes the core 188-bp promoter region, using the MEME suites [32]. MEME analysis demonstrated that the promoter region contained three types of binding motifs at a combined p-value of 8.41e-5 (Fig. 3). Motif 1 occurred five times and motifs 2 and 3 each occurred twice in the promoter region (Fig. 3). Using the yeast motif database SCPD, the motif database-scanning algorithm TOMTOM identified regulatory elements corresponding to binding motifs [33]. The regulatory elements corresponding to motif 1 were stress responsive myocyte enhancer factor 2 (MEF2)-like transcription factor regulated by Hog1 (SMP1), high mobility group box containing transcriptional repressor for lowering sterol content in response to high osmolarity Hog1 and hypoxic repressor (ROX1), homeodomain divergent, pheromone-responsive transcriptional activator (STE12), and homeodomain transcriptional activator of phosphate signal transduction (PHO) pathway (PHO2). The regulatory elements of UASPHR (PHR1 upstream activator sequence sites, a hallmark of genes involved in nucleotide excision repair and recombination, UAS for MGT1, RAD16, RAD9, RAD23, RAD51, MAG1, RAD4, RAD6, RNR3, RNR2, RAD26, RAD7, RAD52, RAD50, PHR1, RAD1, MEC2, SPK1, RAD53, and MGTR) and stress responsive MEF2-like transcription factor regulated by Bck1 (RLM1) were identified as regulatory elements, which likely bind motif 2. UASPHR and ROX1 were identified for motif 3.

Fig. 3. Regulatory motifs of the cryparin promoter in Cryphonectria parasitica. Predictions were obtained from MEME-suite and TOMTOM analysis. Pink, green, and black wedge-shapes indicate motifs 1, 2, and 3, respectively.

Transcription of in silico-suggested transcription factors for cryparin expression

Among the diverse regulatory motifs, the SMP1, STE12, RLM1 binding motifs were of interest because SMP1, STE12, and RLM1 are well-known downstream transcriptional regulators of MAPK pathways in response to high osmolarity, pheromone, and CWI, respectively [34,35]. The SMP1 binding motif is the MADS-box binding motif, which is involved in interactions of the transcription factor containing the MADS-box domain, a conserved amino acid region for DNA binding and dimerization [36]. Based on the sequence similarity, MADS-box proteins are classified as a serum response factor (SRF)-like/Type I and MEF2-like/Type II subfamily. Although S. cerevisiae harbors four MADS-box proteins, a survey of the C. parasitica genome revealed the presence of two MADS-box transcription factors, CpRlm1 and CpMcm1, which were similar to those of other known filamentous ascomycetes [37]. The genomic and near full-length cDNA of CpRlm1 and CpMcm1 were cloned and sequenced using gene-specific primers and RT-PCR as previously described (Table 2) [8,9,10]. Sequence analysis of CpRlm1 indicated that it consisted of three exons, with two intervening sequences of 106 and 82 bp. The deduced protein product consisted of 594 amino acids, with an estimated molecular mass of 64.3 kDa and a pI of 9.28 (GenBank accession No. MF969091). CpMcm1 was predicted to consist of three exons, with two intervening sequences of 202 and 79 bp. The deduced protein product consisted of 217 amino acids, with an estimated molecular mass of 23.5 kDa and a pI of 5.97 (GenBank accession No. MF969092). A homology search of CpRlm1 using the Clustal Omega software revealed that the protein product showed high amino acid identities with other known MADS-box transcription factors of Aspergillus nidulans RlmA (42% amino acid [aa] identity), A. fumigatus RlmA (47%), Neurospora crassa Rlm1 (52%), Fusarium graminearum Rlm1 (57%), M. oryzae MIG1 (64%), and S. cerevisiae Rlm1 (26%). In addition, a homology search of CpMcm1 demonstrated high amino acid identities with other known MADS-box transcription factors of A. nidulans McmA (63% aa identity), A. fumigatus Mcm1 (64%), F. graminearum FgMcm1 (74%), N. crassa Mcm1 (75%), and S. cerevisiae Mcm1 (49%). However, very limited similarity (33.1%) was observed between the protein products of CpRlm1 and CpMcm1. Phylogenetic analysis of CpRlm1 and CpMcm1 indicated that CpMcm1 belonged to the clade containing an SRF-like subfamily, and CpRlm1 belonged to the clade containing a MEF2-like subfamily (Fig. 4).

Fig. 4. Phylogenetic analysis of the CpMcm1 and CpRlm1 genes of Cryphonectria parasitica. Phylogenetic tree was generated using the neighbor-joining method with selected ascomycetes: Mcm1 (CAA88409.1) and Rlm1 (AAB68210.1) from Saccharomyces cerevisiae, MIG1 (ABX79379.1) from Magnaporthe oryzae, FgMcm1 (XP_388872.1) and Rlm1 (XP_389515.1) from Fusarium graminearum, Mcm1 (EAA35381.1) and Rlm1 (EAA36453.3) from Neurospora crassa, Mcm1 (EAL85828.1) and RlmA (EAL92725.1) from Aspergillus fumigatus, McmA (EAA60098.1) and RlmA (EAA63555.1) from A. nidulans. Numbers at the nodes indicate bootstrap values (> 90). Bootstrap analysis was performed with 1,000 replications. Bar represents 10% sequence divergence. MEF2, myocyte enhancer factor 2; SRF, serum response factor.

The accumulation of CpRlm1, CpMcm1, and cpst12 transcripts was analyzed using real-time RT-PCR. In order to understand the downregulation of cryparin and the easy-wet phenotype, we examined the expression of putative regulatory elements using real-time RT-PCR. The accumulation of CpRlm1 transcript was significantly reduced in the CpBck1 and CpSlt2 mutant strain in the CWI MAPK pathway, suggesting that the cloned CpRlm1 appeared to be the downstream transcription factor of the CWI pathway (Fig. 5A). Compared with the wild type, transcription of the CpRlm1 gene was significantly downregulated in Crp- and cpmk1-null mutant strains, which exhibited the easy-wet phenotype and reduced amounts of cryparin. However, no significant changes in the CpRlm1 transcript were observed in the hypovirulent UEP1 strain, which showed reduced amounts of cryparin and the corresponding transcript. These results suggest that CpRlm1 is important for the expression of cryparin, but that the hypoviral regulation of cryparin may not be directly attributed to CpRlm1. When we examined the accumulation of CpMcm1 transcripts, the CpMcm1 transcript was not significantly changed in UEP1 and the sectored progeny of the CpBck1-null mutant, which showed reduced amounts of cryparin and their transcription. More interestingly, the accumulation of the CpMcm1 transcript was significantly increased in the cryparin-null mutant (Fig. 5B). These results indicate that CpMcm1 had very little effect on cryparin expression. We also analyzed the transcription of cpst12, an ortholog of yeast STE12 [9,10]. The accumulation of cpst12 was significantly reduced in mutant strains of cpmk2, an ortholog of yeast FUS3, which suggested that the cpst12 was a downstream transcription factor of cpmk2. In tested strains showing the easy-wet phenotype and reduced amounts of cryparin, the accumulation of cpst12 transcript was significantly decreased compared with that of the wild-type EP155/2 (Fig. 5C). Thus, cpst12 appeared to be essential for cryparin expression. cpst12 was transcriptionally downregulated in the cpmk2-null mutant showing the increased cryparin production. Considering that cpst12 is a downstream factor of cpmk2, its downregulation in the cpmk2 mutant is not uncommon. However, the cpmk2-null mutant demonstrated increased cryparin in terms of transcript accumulation and protein levels. Additionally, the accumulation of CpRlm1 transcript was also downregulated in the cpmk2-null mutant. Thus, the modulation of cryparin expression appears to be more complex in that, although transcriptional regulation by both CpRlm1 and cpst12 occurred, there might be another tier of regulation mediated by a pheromone-responsive pathway. Thus, it will be interesting to examine cryparin production in the CpRlm1- and cpst12-null mutant strains.

Fig. 5. Quantitative real-time reverse transcription polymerase chain reaction analysis of cpst12, CpMcm1, and CpRlm1. Changes in expression of CpRlm1 (A), CpMcm1 (B), and cpst12 (C) with the wild-type (EP155/2), hypovirulent (UEP1), Crp-, CpBck1-, CpSlt2-, cpmk1-, and cpmk2-null mutant strains relative to levels of glyceraldehyde-3-phosphate dehydrogenase (gpd). Y-axis values were normalized to the transcript levels of the corresponding gene in the wild-type strain. Error bars indicate standard deviation based on three independent measurements.

In conclusion, the promoter region of the class II fungal hydrophobin cryparin contained a MADS-box binding element and cpst12-binding domain. Among the two putative MADS-box transcription factors of C. parasitica, CpRlm1, not CpMcm1, was the putative transcription factor that showed dramatic transcriptional downregulation in mutant strains exhibiting the easy-wet phenotype. Additionally, cpst12 is a putative transcription factor, showing a transcript downregulation pattern in mutant strains with the easy-wet phenotype and reduced amounts of cryparin. Thus, among three characterized MAPK pathways, two pathways, cpmk1 and CpSlt2, orthologs of yeast HOG1 and SLT2, respectively, showed downregulation of CpRlm1 and transcriptional downregulation of cryparin (Fig. 5). These results suggest that CpRlm and cpst12 might be putative transcription factors for the motif in the promoter region of cryparin. However, further studies on the binding affinity of CpRlm1 and cpst12 to the motif in the cryparin promoter region should be conducted to examine specific downstream effects of cryparin expression.

In response to extreme changes in the environment, precise responses must be coordinated by the fungi through MAPK signaling pathways. Our studies provide a framework for examining comparative studies of MAPK signaling and indicate that all three MAPK pathways play a role in the regulation of the fungal hydrophobin cryparin in C. parasitica.