• The Plant Pathology Journal
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• v.19 no.1
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• pp.30-33
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• 2003

The chestnut blight fungus, Cryphonectria parasitica, and its hypovirus aye a useful model system in the study of the mechanisms of hypoviral infection and its consequences, such as a biological control of fungal pathogens. Strains containing the double-stranded (ds) RNA viruses Cryphonectria hypovirus 1 show characteristic symptoms of hypovirulence and display hypovirulence-associated changes, such as reduced pigmentation, sporulation, laccase production, and oxalate accumulation. Interestingly, symptoms caused by hypoviral infection appear to be the result of aberrant expression of a number of specific genes in the hypovirulent strain. Several viral regulated fungal genes are identified as cutinase gene, Lac1, which encodes an extracellular laccase, Crp, which encodes an abundant tissue-specific cell-surface hydrophobin that mediates physical strength, and Mf2/1 and Mf2/2, which encode pheromone genes involved in poor sporulation in the presence of hypo-virus. Since the phenotypic changes in the fungal host are pleiotropic, although coordinated and specific, it has been suggested that the hypovirus disturbs one or several regulatory pathways (Nuss,1996). Accordingly, several studies have shown the implementation of a signal transduction pathway during viral symptom development. Although further studies are required, hypovirulence and its associated symptom development due to the hypoviral regulation of a fungal hetero-trimeric G-protein have been suggested. In addition, recent studies have shown the presence of a novel protein kinase gene cppk1 and its transcriptional upregulation by hypovirus. In this review, the presence of important components in signal transduction pathway, their putative biological function, and viral-specific regulation will be addressed.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.159-161
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• 2005

Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is a useful model system to study the hypoviral regulation of fungal gene expression. The hypovirus is known to downregulate the fungal laccase1 (lac 1), the modulation of which is tightly governed by the inositol triphosphate ($IP_3$) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), in order to better characterize the fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd ${\beta}$-strand and the ${\alpha}$-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a ${\delta}$ type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. In addition, down regulation of lac1 expression was observed. However, temperature sensitivity, osmosensitivity, virulence, and other hypovirulence-associated characteristics did not differ from the wild-type strain. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for appropriate mycelial growth, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.117.3-118
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• 2003

We tested for the presence of double-stranded RNA (dsRNA) mycovirus in 827 Fusarium graminearum isolated from diseased barley and maize. dsRNA mycoviruses with various sizes were isolated. Of them, it was previously reported that dsRNA from DK2l isolate had pronounced morphological changes, including reduction in mycelial growth, increased to red pigmentation, reduced virulence and sporulation. (Chu et al., Appl. Environ. Microbiol. 2002). For better understanding of this hypovirulence associated with DK2l dsRNA virus, we determined the complete nucleotide sequence of dsRNA genome and named Fusarium hypovirus DK2l strain (Fhv-DK2l ). Genomic RNA of Fhv-DK2l was determined to be 6625 nucleotides in length excluding the poly (A) tail and contained three putative open reading frame. RNA-dependent RNA polymerase (RdRp) and helicase domain were expected in ORF A, 54 to 4709 nucleotide position. ORE B, 4752 to 5216 nucleotide position, and ORF C, 5475 to 6578 nucleotide position, were predicted to encode 16.7kDa and 41.3kDa protein respectively each. We could not detect any conserved domains from these two proteins. Phylogenetic analysis showed Fhv-DK2l was related to Cryphonectria hypovirus 3. Ten additional isolates were found that were infected with dsRNA mycoviruses. These mycoviruses contain 2 to 4 different segments of dsRNAs with the size range of approximately 1.7 to 10-kbp in length. The presence of dsRNAs isolates did not affect colony morphology and were transmissible through conidia and ascospore with incidence of 30-100％. These results indicate that there is genomic diversity of dsRNA mycoviruses that infect F. graminearum isolates and that impact of virus infection on host's morphology and virulence is determined by the interaction between dsRNAs and the fungal host, not by the mere presence of the dsRNAs

• The Plant Pathology Journal
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• v.17 no.6
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• pp.386.2-386
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• 2001

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.450-457
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• 1998

The cryparin of Cryphonectria parasitica belongs to a cell wall associated fungal hydrophobin. The cryparin, though it is encoded by a single copy gene, is known for the high expression during the liquid culture of C. parasitica, and it turns out that 22% of total mRNA was transcribed for cryparin at 48hr after the liquid culture. In addition, it is also known as one of down-regulated fungal proteins by the presence of double stranded RNA virus, Cryphonectria hypovirus 1. In previous studies (Kim et al., 1999), we have constructed a cryparin-null mutant by replacing the cryparin gene with hygromycin B resistance gene due to site directed homologous recombination. In order for the promoter analysis of cryparin which seems to be very strong as well as mycoviral specific, it is preferable to have a strain with only a target promoter replaced and a discernable target site for incoming vectors. However, the cryparin-null mutant revealed the presence of an additional copy of transforming vector except the one which replaced the cryparin gene. In addition, the cryparin-null mutant did not contain any markers for targeted integration of incoming vectors. This prompts us to design an experiment to obtain a strain for promoter analysis of cryparin gene. A different mating type strain EP6(Mata, $met^-$) was mated with the cryparin-null mutant ${\triangle}$Crp194-7(MatA, Crp${\triangle}$::hph) to make the progenies with only a single replacement vector and $met^-$ characteristic remained. Nutritional assay as well as Southern blot analysis revealed that the progeny, ${\triangle}$Crp194-a6, was the methionine auxotroph with a single replacing vector in genome. Northern blot analysis and PAGE showed that there was no cryparin produced in this bred strain either.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2000.10a
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• pp.65.1-65
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• 2000

• Molecules and Cells
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• v.26 no.5
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• pp.496-502
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• 2008

Cryparin, encoded as a single copy gene (Crp) of the chestnut blight fungus Cryphonectria parasitica, is the most abundant protein produced by this fungus. However, its accumulation is decreased remarkably in C. parastica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus 1. To characterize the transcriptional regulatory element(s) for strong expression and viral regulation, promoter analysis was conducted. Serial deletion of the Crp promoter region resulted in a step-wise decrease in promoter activity, indicating a localized distribution of genetic elements in the cryparin promoter. Promoter analysis indicated two positive and a repressive cis-acting elements. Among them, the promoter region between nt -1,282 and -907 appeared to be necessary for hypoviral-mediated down-regulation. An electrophoretic mobility shift assay (EMSA) on the corresponding promoter region (-1,282/-907) indicated two regions at (-1,257/-1,158) and (-1,107/-1,008) with the characteristic AGGAGGA-N42-GAGAGGA and its inverted repeat TCCTCTC-N54-TCCTCCT, respectively, appeared to be specific binding sites for cellular factors.

• 한국균학회소식:학술대회논문집
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• 2018.05a
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• pp.11-11
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• 2018

Cryphonectria parasitica, chestnut blight fungus, has a characteristic of decreasing pathogenicity when infected with Cryphonectria hypovirus 1. C. parasitica is known to be one of the most representative model systems used to observe the interaction between viruses, plants and fungi. The mitogen-activated protein kinase (MAPK) pathway, which is well conserved in various organisms ranging from yeast to humans, functions in relaying phosphorylation-dependent signals within MAPK cascades to diverse cellular functions involved in the regulation of pheromone, cell wall integrity, and osmotolerance in filamentous fungi. Several genes in the MAPK pathway were revealed to be regulated by hypovirus, or to be involved in pathogenicity in C. parasitica. Among these pathways, the CWI pathway has aroused interest because CpBck1, an ortholog of yeast Bck1 (a CWI MAPKKK), was previously reported to be involved in cell wall integrity and sectorization. Interestingly, sporadic sectorization was observed in the CpBck1 mutant and sectored phenotypes were stably inherited in the progeny that were successively transferred from sectored mycelia. In this study, we analyzed the biological function of CpSlt2, downstream gene of CpBck1, to confirm whether the sectorization phenomenon occurred in the specific single gene or cell wall integrity (CWI) pathway. As results, the CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, abnormal pigmentation, CWI-related phenotypic defects, and dramatically impaired virulence. 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. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.

• The Korean Journal of Mycology
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• v.25 no.3 s.82
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• pp.191-201
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• 1997

A mutant (HSMl) of Cryphonectria parasitica created during transformation reproduced the hypovirulent symptoms in virus-free wild type. Its phenomena have been proved with morphological marker such as reduced sporulation, pigmentation, and laccase production. In addition to the changes in phenotypic characteristics, down-regulations of Lac1, Crp1, Vir1 and Vir2 were also observed. The integration of transforming vector was confirmed and located within genome by marker rescuing. Vector integration occurred between two genes, Cpg2 and Cpg3, which resulted in the disruption of neither Cpg2 nor Cpg3. Both Cpg2 and Cpg3 genes, sized at 1.8 kb and 1.9 kb respectively, were rarely transcribed genes in Cryphonectria parasitica. Cpg2 expression was significantly overexpressed from 4 to 5 day old culture of both UEP1 and HSM1 while no differences were observed in Cpg3 expression. It appears that an aberration from the normal expression of Cpg2, not Cpg3, results in the hypovirulent symptoms in virus-free wild type.