Kum-Kang So

Rapid screening of an ordered fosmid library to clone multiple polyketide synthase genes of the phytopathogenic fungus Cladosporium phlei

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.

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.

Changes in the mycovirus (LeV) titer and viral effect on the vegetative growth of the edible mushroom Lentinula edodes.

This study attempted to cure the edible mushroom Lentinula edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygen-depleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. These results indicate that LeV infection has a deleterious effect on mycelial growth.

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.

Characterization of NpgA, a 4'-phosphopantetheinyl transferase of Aspergillus nidulans, and evidence of its involvement in fungal growth and formation of conidia and cleistothecia for development.

The null pigmentation mutant (npgA1) in Aspergillus nidulans results in a phenotype with colorless organs, decreased branching growth, delayed of asexual spore development, and aberrant cell wall structure. The npgA gene was isolated from A. nidulans to investigate these pleiomorphic phenomena of npgA1 mutant. Sequencing analysis of the complementing gene indicated that it contained a 4′-phosphopantetheinyl transferase (PPTase) superfamily domain. Enzymatic assay of the PPTase, encoded by the npgA gene, was implemented in vivo and itvitro. Loss-of-function of LYS5, which encoded a PPTase in Saccharomyces cerevisiae, was functionally complemented by NpgA, and Escherichia coli-derived NpgA revealed phosphopantetheinylation activity with the elaboration of 3′5′-ADP. Deletion of the npgA gene caused perfectly a lethal phenotype and the absence of asexual/sexual sporulation and secondary metabolites such as pigments in A. nidulans. However, a cross feeding effect with A. nidulans wild type allowed recovery from deletion defects, and phased-culture filtrate from the wild type were used to verify that the npgA gene was essential for formation of metabolites needed for development as well as growth. In addition, forced expression of npgA promoted the formation of conidia and cleistothecia as well as growth. These results indicate that the npgA gene is involved in the phosphopantetheinylation required for primary biological processes such as growth, asexual/sexual development, and the synthesis of secondary metabolites in A. nidulans.

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.

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.

Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome

Two different diketopiperazines, cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were isolated from the culture filtrate of Epichloe typhina and found to be inducers of phleichrome production, were chemically synthesized and evaluated for use in the improved production of phleichrome from wild-type and UV-mutagenized strains (M0035) of Cladosporium phlei. When supplemented with PDA and V8 juice agar media, both inducers showed significant increases in the production of phleichrome. Phleichrome production was increased in a dose-dependent manner up to a concentration of maximum yield for both inducers. No further significant induction was observed by supplementing inducers over the concentration of maximum yield. Among the two inducers, cyclo-(L-Pro-L-Phe) showed better inducing capability than cyclo-(L-Pro-L-Leu). The maximum yield was observed from the M0035 strain grown on V8 juice media supplemented with 150 μM cyclo-(L-Pro-L-Phe), which was estimated to be 232.6 mg of phleichrome per gram of mycelia and 10.2 mg of secreted phleichrome per 20 agar-plugs. Interestingly, growth inhibition was observed on V8 juice agar media with 100, 150, and 200 μM cyclo-(L-Pro-L-Phe) but not on PDA with the same amount of inducer, which suggests that the inhibitory effect might be through the overproduction of phleichrome rather than the toxic effect of the inducer itself. Superoxide production by purified phleichrome was dramatically stimulated upon illumination, thus demonstrating photodynamic production of superoxide in vitro by phleichrome.

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.

Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.