Hypoviral-regulated HSP90 co-chaperone p23 (CpCop23) determines the colony morphology, virulence, and viral response of chestnut blight fungus Cryphonectria parasitica.

We previously identified a protein spot that showed down-regulation in the presence of Cryphonectria hypovirus 1 (CHV1) and tannic acid supplementation as a Hsp90 co-chaperone p23 gene (CpCop23). The CpCop23-null mutant strain showed retarded growth with less aerial mycelia and intense pigmentation. Conidia of the CpCop23-null mutant were significantly decreased and their viability was dramatically diminished. The CpCop23-null mutant showed hypersensitivity to Hsp90 inhibitors. However, no differences in responsiveness were observed after exposure to other stressors such as temperature, reactive oxygen species, and high osmosis, the exception being cell wall-disturbing agents. A severe reduction in virulence was observed in the CpCop23-null mutant. Interestingly, viral transfer to the CpCop23-null mutant from CHV1-infected strain via anastomosis was more inefficient than a comparable transfer with the wild type as a result of decreased hyphal branching of the CpCop23-null mutant around the peripheral region, which resulted in less fusion of the hyphae. The CHV1-infected CpCop23-null mutant exhibited recovered mycelial growth with less pigmentation and sporulation. The CHV1-transfected CpCop23-null mutant demonstrated almost no virulence, that is, even less than that of the CHV1-infected wild type (UEP1), a further indication that reduced virulence of the mutant is not attributable exclusively to the retarded growth but rather is a function of the CpCop23 gene. Thus, this study indicates that CpCop23 plays a role in ensuring appropriate mycelial growth and development, spore viability, responses to antifungal drugs, and fungal virulence. Moreover, the CpCop23 gene acts as a host factor that affects CHV1-infected fungal growth and maintains viral symptom development.

A fungal GPI-anchored protein gene functions as a virulence and antiviral factor.

We show that a gene (CpGap1) encoding a glycosylphosphatidylinositol-anchored protein (GPI-AP) of the chestnut blight fungus Cryphonectria parasitica is differentially expressed by Cryphonectria hypovirus 1 (CHV1) infection. Functional analysis using a CpGap1-null mutant results in no observed changes in cultural morphology other than hypersensitivity to ROS. Analysis of the protein product of the CpGap1 gene (CpGAP1) confirmed motifs with antioxidizing properties. The virulence of the CpGap1-null mutant is significantly decreased, and phytotoxic activity is seen in the peptides of CpGAP1. CHV1 transfer to the CpGap1-null mutant results in severely retarded colonial growth, and virus-titer is significantly increased in the mycelia of CHV1-infected CpGap1-null mutant. These results indicate that CpGAP1 functions as a protective barrier against plant defenses, but also acts as a virulence factor. Moreover, our study demonstrates that the CpGap1 gene is a host-tolerating antiviral factor that helps maintain fungal growth and suppress viral titer after CHV1 infection.

Molecular characteristics of a novel hypovirus from Trichoderma harzianum.

We report a novel mycovirus with a positive-sense single-stranded (+)ss RNA genome, belonging to the family Hypoviridae, infecting Trichoderma harzianum strain M6. The complete genome sequence is 13,813 nucleotides long, excluding the poly(A) tail at the 3' end. Sequence analysis revealed that the genome has a single large open reading frame (ORF) encoding a 4,118-amino-acid polyprotein harboring five conserved motifs of a protease, two conserved domains of a protein of unknown function, an RNA-dependent RNA polymerase, and a helicase. Sequence comparisons revealed that the deduced amino acid sequence of the polyprotein is similar to those of other hypoviruses and is most similar to that of Bipolaris oryzae hypovirus 1 (35.1% identity). Phylogenetic analysis using full-length RdRp and helicase sequences showed that this virus clustered closely with known members of the proposed genus "Alphahypovirus" of the family Hypoviridae. We accordingly designated this novel mycovirus "Trichoderma harzianum hypovirus 2" (ThHV2).

Interaction between hypoviral-regulated fungal virulence factor laccase3 and small heat shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis.

Functional Analysis of an Essential GSP1/Ran Ortholog Gene, CpRan1, from the Chestnut Blight Fungus Cryphonectria parasitica Using a Heterokaryon.

Functional analysis of a GSP1/Ran ortholog, CpRan1, from Cryphonectria parasitica was conducted. Genotype analysis revealed that the putative CpRan1-null mutant was a heterokaryotic transformant harboring two different types of nuclei, one with the wild-type CpRan1 allele and the other with the CpRan1-null mutant allele. The mycelial growth and colony morphology of the heterokaryotic transformant was normal. Microscopic analysis of the resulting conidia (aseptate and monokaryotic asexual spores) demonstrated that although normal germinating spores were observed from conidia harboring a nucleus with the wild-type CpRan1 allele, a number of residual conidia that did not germinate existed. Complementation analysis using protoplasts from the heterokaryon with the wild-type CpRan1 allele confirmed that the CpRan1 gene is essential to C. parasitica. Complementation analysis using the various CpRan1 chimera constructs allowed us to perform a functional analysis of essential amino acids of the CpRan1. Among the four suggested essential amino acids, Lys-97 for ubiquitination was determined to not be an essential residue. Moreover, the CpRan1-null mutant allele was successfully complemented with mouse Ran gene, which suggested that the biological function of Ran gene is evolutionary conserved and that our heterokaryon rescue can be applied for the functional analysis of heterologous genes.

Distinct Roles of Two DNA Methyltransferases from Cryphonectria parasitica in Fungal Virulence, Responses to Hypovirus Infection, and Viral Clearance.

Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1, not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1-null mutant is hypervirulent, while the CpDmt2-null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations.IMPORTANCE Although relatively few in number, studies of DNA methylation have shown that fungal DNA methylation is implicated in development, genome integrity, and genome defense. While fungal DNMTase has been suggested as playing a role in genome defense, studies of the biological function of fungal DNMTase have been very limited. In this study, we have shown distinct biological functions of two DNA methyltransferases from the chestnut blight fungus C. parasitica We have demonstrated that DNMTases are important to fungal development and virulence. In addition, these genes are shown to play an important role in the fungal response to hypoviral CHV1 infection, including severely retarded colonial growth, and in viral clearance, which has never been previously observed in mycovirus infection. These findings provide a better understanding of the biological functions of fungal DNA methyltransferase and a basis for clarifying the epigenetic regulation of fungal virulence, responses to hypovirus infection, and viral clearance.

Transcriptome Analysis of Cryphonectria parasitica Infected With Cryphonectria hypovirus 1 (CHV1) Reveals Distinct Genes Related to Fungal Metabolites, Virulence, Antiviral RNA-Silencing, and Their Regulation.

Comprehensive transcriptome analysis was conducted to elucidate the molecular basis of the interaction between chestnut blight fungus, Cryphonectria parasitica, and single-stranded RNA (ssRNA) mycovirus Cryphonectria hypovirus 1 (CHV1), using RNA-sequencing (RNA-seq). A total of 1,023 differentially expressed genes (DEGs) were affected by CHV1 infection, of which 753 DEGs were upregulated and 270 DEGs were downregulated. Significant correlations in qRT-PCR analysis of 20 randomly selected DEGs and agreement with previously characterized marker genes validated our RNA-seq analysis as representing global transcriptional profiling of virus-free and -infected isogenic strains of C. parasitica. Gene Ontology (GO) analysis of DEGs indicated that "cellular aromatic compound metabolic process" and "transport" were the two most enriched components in the "biological process." In addition, "cytoplasm" was the most enriched term in the "cellular component" and "nucleotide binding" and "cation binding" were the two most enriched terms in the "molecular function" category. These results suggested that altered expression of genes encoding numerous intracellular proteins due to hypoviral infection resulted in changes in specific metabolic processes as well as transport processes. Kyoto Encyclopedia of Genes and Genomes function analysis demonstrated that pathways for "biosynthesis of other secondary metabolites," "amino acid metabolism," "carbohydrate metabolism," and "translation" were enriched among the DEGs in C. parasitica. These results demonstrate that hypoviral infection resulted in massive but specific changes in primary and secondary metabolism, of which antiviral fungal metabolites were highly induced. The results of this study provide further insights into the mechanism of fungal gene regulation by CHV1 at the transcriptome level.

Functional analysis of an essential Ran-binding protein gene, CpRbp1, from the chestnut blight fungus Cryphonectria parasitica using heterokaryon rescue.

A Ran binding protein (RanBP) homolog, CpRbp1, from Cryphonectria parasitica, has been identified as a protein that is affected by hypovirus infection or tannic acid supplementation. In this study, functional analyses of CpRbp1 were performed by constructing a knockout mutant and analyzing the resulting heterokaryon. Transformation-mediated gene replacement resulted in two putative CpRbp1-null mutants and genotype analyses identified these two mutants as heterokaryotic transformants consisting of two types of nuclei, one with the wild-type CpRbp1 allele and another with the CpRbp1-null mutant allele. Although stable mycelial growth of the heterokaryotic transformant was observed on selective medium containing hygromycin B, neither germination nor growth of the resulting conidia, which were single-cell monokaryotic progeny, was observed on the medium. In trans complementation of heterokaryons using a full-length wild-type allele of the CpRbp1 gene resulted in complemented transformants. These transformants sporulated single-cell monokaryotic conidia that were able to grow on media selective for replacing and/or complementing markers. These results clearly indicate that CpRbp1 is an essential gene, and heterokaryons allowed the fungus to maintain lethal CpRbp1-null mutant nuclei. Moreover, in trans complementation of heterokaryons using chimeric structures of the CpRbp1 gene allowed for analysis of its functional domains, which was previously hampered due to the lethality of the gene. In addition, in trans complementation using heterologous RanBP genes from Aspergillus nidulans was successful, suggesting that the function of RanBP is conserved during evolution. Furthermore, in trans complementation allowed for functional analyses of lethal orthologs. This study demonstrates that our fungal heterokaryon system can be applied effectively to determine whether a gene of interest is essential, perform functional analyses of a lethal gene, and analyze corresponding heterologous genes.

Comparative Transcriptomic Analysis of MAPK-Mediated Regulation of Sectorization in Cryphonectria parasitica.

Fungal sectorization is a complex trait that is still not fully understood. The unique phenotypic changes in sporadic sectorization in mutants of CpBck1, a mitogen-activated protein kinase kinase kinase (MAPKKK) gene, and CpSlt2, a mitogen-activated protein kinase (MAPK) gene, in the cell wall integrity pathway of the chestnut blight fungus Cryphonectria parasitica have been previously studied. Although several environmental and physiological factors cause this sectoring phenotype, genetic variants can also impact this complex morphogenesis. Therefore, RNA sequencing analysis was employed to identify candidate genes associated with sectorization traits and understand the genetic mechanism of this phenotype. Transcriptomic analysis of CpBck1 and CpSlt2 mutants and their sectored progeny strains revealed a number of differentially expressed genes (DEGs) related to various cellular processes. Approximately 70% of DEGs were common between the wild-type and each of CpBck1 and CpSlt2 mutants, indicating that CpBck1 and CpSlt2 are components of the same MAPK pathway, but each component governs specific sets of genes. Functional description of the DEGs between the parental mutants and their sectored progenies revealed several key pathways, including the biosynthesis of secondary metabolites, translation, amino acid metabolism, and carbohydrate metabolism; among these, pathways for secondary metabolism and translation appeared to be the most common pathway. The results of this comparative study provide a better understanding of the genetic regulation of sector formation and suggest that complex several regulatory pathways result in interplays between secondary metabolites and morphogenesis.

Characterization of a Hypovirus-Regulated Septin Cdc11 Ortholog, CpSep1, from the Chestnut Blight Fungus Cryphonectria parasitica.

We identified a protein spot showing downregulation in the presence of Cryphonectria hypovirus 1 and tannic acid supplementation as a septin subunit with the highest homology to the Aspergillus nidulans aspA gene, an ortholog of the Saccharomyces cerevisiae Cdc11 gene. To analyze the functional role of this septin component (CpSep1), we constructed its null mutant and obtained a total of eight CpSep1-null mutants from 137 transformants. All CpSep1-null mutants showed retarded growth, with fewer aerial mycelia and intense pigmentation on plates of potato dextrose agar supplemented with L-methionine and biotin. When the marginal hyphae were examined, hyperbranching was observed in contrast to the wild type. The inhibition of colonial growth was partially recovered when the CpSep1-null mutants were cultured in the presence of the osmostabilizing sorbitol. Conidia production of the CpSep1-null mutants was significantly increased by at least 10-fold more. Interestingly, the conidial morphology of the CpSep1-null mutants changed to circular in contrast to the typical rod-shaped spores of the wild type, indicating a role of septin in the spore morphology of Cryphonectria parasitica. However, no differences in the germination process were observed. Virulence assays using excised chestnut bark, stromal pustule formation on chestnut stems, and apple inoculation indicated that the CpSep1 gene is important in pathogenicity.

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization.

Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK) of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase), demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Mutation of the Slt2 ortholog from Cryphonectria parasitica results in abnormal cell wall integrity and sectorization with impaired pathogenicity.

We assessed the biological function of CpSlt2, an ortholog of the cell wall integrity (CWI) MAPK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica. The CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, and abnormal pigmentation. In addition, the CpSlt2-null mutant exhibited CWI-related phenotypic defects including hypersensitivity to cell wall-disturbing agents and other stresses. Electron microscopy revealed the presence of abnormal hyphae such as intrahyphal hyphae. In addition, virulence assays indicated that the CpSlt2 gene plays an important role in fungal pathogenesis. As cultivation of the mutant strains progressed, the majority of the colonies showed sporadic sectorization and mycelia from the sectored area stably maintained the sectored phenotype. Although mycelial growth was partially recovered, the sectored progeny had dramatically impaired virulence, confirming the CpSlt2 gene has a role in pathogenicity. Compared to a previous mutant of the CpBck1 gene, a MAPKKK gene in CWI pathway, the CpSlt2-null mutant showed similar, although not identical, phenotypic changes and most phenotypic changes were less severe than those of the CpBck1-null mutant. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.

Heterokaryon analysis of a Cdc48-like gene, CpCdc48, from the chestnut blight fungus Cryphonectria parasitica demonstrates it is essential for cell division and growth.

Functional analysis of a cell division cycle 48 (CDC48) ortholog, CpCdc48, from Cryphonectria parasitica was performed via construction of a CpCdc48-null mutant. Genotype analysis revealed that the putative CpCdc48-null mutant was a heterokaryotic transformant containing two different types of nuclei (i.e., one with the wild-type CpCdc48 allele and the other with the CpCdc48-null mutant allele). Although stable mycelial growth of the heterokaryotic transformant was observed on media containing hygromycin B, neither germination nor growth of the resulting spores was observed on selection media, suggesting that the CpCdc48 gene is essential. Microscopic analysis of germinated conidia from the heterokaryon demonstrated that although there were normal germinating spores due to the wild-type conidia, there were many residual conidia that did not germinate. However, with prolonged incubation, non-germinating conidia began to swell into gigantic globose spores. DAPI staining and FACS analysis of the gigantic spores revealed the presence of multiple nuclei. These gigantic conidia did not show any signs of polarized growth and underwent autolysis with further incubation. These findings indicate that the CpCdc48 gene is responsible for delayed cell cycle during spore germination, resulting in some karyokinesis, but not following spore cytokinesis. Thus, CpCdc48 is essential for cell division and polarized growth.

A Mutant of the Bck1 Homolog from Cryphonectria parasitica Resulted in Sectorization with an Impaired Pathogenicity.

CpBck1, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of Saccharomyces cerevisiae, was cloned and characterized from the chestnut blight fungus Cryphonectria parasitica. The CpBck1-null mutant displayed cell wall integrity-related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Compared with the wild type, both the parental CpBck1-null mutant and the sectored progeny exhibited marked impaired virulence. The present study revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization.

Mycoflora and enzymatic characterization of fungal isolates in commercial meju, starter for a korean traditional fermented soybean product.

Mycoflora was assessed in the commercial meju from four well-separated geographic origins. A total of 112 fungal isolates were identified by phenotypic characteristics and molecular taxonomy using sequencing the internal transcribed spacer of the rDNA and revealed 19 species from 13 genera. Enzymatic characteristics of protease and amylase, and mycotoxin production were analyzed.