• The Plant Pathology Journal
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• 제19권1호
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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.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2002년도 총회 및 추계학술발표회
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• pp.53.1-53
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• 2002

• 한국균학회지
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• 제22권4호
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• pp.343-349
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• 1994

밤나무 동고병(胴枯病)에 심하게 감염(感染)된 미국밤나무의 이병(罹病)줄기로부터 병원균(病原菌)인 Cryphonectria parasitica를 102균주(菌株) 분리(分離)하여 배지상에서 배양(培養)하면서 체세포(體細胞) 화합성(和合性)을 조사한 결과, 102 균주는 54 개의 화합성군(和合性群)으로 나누어 졌으며 그중 38개의 화합성군에 단지 한 균주씩, 6개의 화합성군에 각각 2 균주씩 포함되었으며 나머지 52개 균주는 10개의 화합성군에 포함되어 다양한 화합성군으로 분화(分化)되었음을 보여주었다. 체세포 화합성에 있어서 이러한 다양(多樣)한 분화는 병원균이 분리된 지역에서 80년 이상 존재(存在)해 오면서 시간이 경과(經過)함에 따라 유전적(遺傳的)으로 많은 분화가 일어나고 이로 인하여 화합성군의 숫자가 증가(增加)한 것에 기인(起因)하는 것으로 추측된다. 가장 빈도(頻度)가 높은 10개의 화합성군으로부터 각각 한 균주씩을 대표적인 균주로 선발(選拔)하고 이들을 여러 지역에서 분리된 저병원성 균주와 균사융합(菌絲融合)을 시키면서 저병원성(低病原性) 균주로의 변환(變換)을 시도(試圖)하였다. 10개의 대표적인 균주는 모두 최소(最少)한 1개 이상의 저병원성 균주에 의해서 저병원성 균주로 변환되었다.

• Molecules and Cells
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• 제23권3호
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• pp.304-315
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• 2007

Fusarium graminearum causes a serious scab disease of small grains in Korea. The nucleotide sequence of the genomic RNA of a double-stranded RNA (dsRNA) virus, Fusarium graminearum virus-DK21 (FgV-DK21), from F. graminearum strain DK21, which is associated with hypovirulence in F. graminearum, was determined and compared to the genome sequences of other mycoviruses, including Cryponectria hypoviruses. The FgV-DK21 dsRNA consists of 6,624 nucleotides, excluding the 3'-terminal poly(A) tail. The viral genome has 53- and 46-nucleotide 5' and 3' untranslated regions (UTRs), respectively, and five putative open reading frames. A phylogenetic analysis of the deduced amino acid sequence of ORF1, which encodes a putative RNA-dependent RNA polymerase, and those of other mycoviruses revealed that this organism forms a distinct virus clade with other hypoviruses, and is more distantly related to other mycoviruses (3.8 to 24.0% identity). However, pairwise sequence comparisons of the nucleotide and deduced amino acid sequences of ORFs 2 through 5 revealed no close relationships to other protein sequences currently available in GenBank. Analyses of RNA accumulation by Northern blot and primer extension indicated that these putative gene products are expressed from at least two different subgenomic RNAs (sgRNAs), in contrast to the cases in other hypoviruses. This study suggests the existence of a new, as yet unassigned, genus of mycoviruses that exhibits a potex-like genome organization and sgRNA accumulation.

• Journal of Microbiology
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• 제44권5호
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• pp.556-561
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• 2006

A differential display for the expression profiles of wild-type Cryphonectria parasitica and its virally-infected isogenic hypovirulent strain revealed several transcripts of interest, which evidenced significant matches with fungal genes of known function. Among which, we have further analyzed an amplified PCR product with significant sequence similarity to the known fungal stress-responsive thioredoxin gene from Neurospora crassa. The product of the cloned thioredoxin gene, CpTrx1, consists of 117 amino acids, with a predicted molecular mass of 13.0 kDa and a pI of 5.4. Sequence comparisons demonstrated that the deduced protein sequence of the CpTrx1 gene evidenced a high degree of homology to all known thioredoxins, with the highest degree of homology with trx1, a thioredoxin gene from Saccharomyces cerevisiae, and evidenced a preservation of the conserved hall markresidues (Trp-Cys-Gly-Pro-Cys) at the active site of thioredoxin. The E. coli-generated CpTRX1 manifested thioredoxin activity, according to the insulin reduction assay, which indicates that the cloned gene does indeed encode for the C. parasitica thioredoxin.

• Molecules and Cells
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• 제26권5호
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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.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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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

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
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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.

• The Plant Pathology Journal
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• 제16권3호
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• pp.142-146
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• 2000

Ordered differential display using RT-PCR (ODD-PCR) was conducted to have a profile of the differently expressed genes between a hypovirulent strain of Cryphonectria parasitica (UEP1) and its isogenic wild type strain (EP155/2). ODD-PCR has advantages of high sensitivity, reproducibility, proportional representation, and limited number of primer combinations comparing with other differential display methods. RNAs were prepared from 1 and 5 day liquid culture of both hypovirulent and wild type strains, and were further evaluated with the marker genes of C. parasitica such as cryparin and mating factor MF2-1, which were already proven to be specifically down-regulated by the presence of mycovirus CHV1-713. ODD-PCR was conducted using those RNAs and expressed genes were categorized to five groups according to their temporal and quantitative expression patterns. Those fives groups are CPC, CPE, CPL, CPD, and CPU which represent constitutively-expressed, early-expressed, late-expressed, down-regulated, and up-regulated, respectively. Ninety two primer combinations out of a total of 192 have been tested so far. Among the twenty to fifty distinct bands per each reaction, an average of four to ten genes was identified as viral-regulated fungal genes. Those viral-specifc genes were further analyzed by DNA sequencing followed by homology search. Characterization of 30 clones including all five groups were conducted as a preliminary data and more are under investigation.

• The Plant Pathology Journal
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• 제32권4호
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• pp.281-289
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• 2016

The transcription cofactor Swi6 plays important roles in regulating vegetative growth and meiosis in Saccharomyces cerevisiae. Functions of Swi6 ortholog were also characterized in Fusarium graminearum which is one of the devastating plant pathogenic fungi. Here, we report possible role of FgSwi6 in the interaction between F. graminearum and Fusarium graminearum virus 1 (FgV1) strain DK21. FgV1 perturbs biological characteristics of host fungi such as vegetative growth, sporulation, pigmentation, and reduction of the virulence (hypovirulence) of its fungal host. To characterize function(s) of FgSWI6 gene during FgV1 infection, targeted deletion, over-expression, and complementation mutants were generated and further infected successfully with FgV1. Deletion of FgSwi6 led to severe reduction of vegetative growth even aerial mycelia while over-expression did not affect any remarkable alteration of phenotype in virus-free isolates. Virus-infected (VI) FgSWI6 deletion isolate exhibited completely delayed vegetative growth. However, VI FgSWI6 over-expression mutant grew faster than any other VI isolates. To verify whether these different growth patterns in VI isolates, viral RNA quantification was carried out using qRT-PCR. Surprisingly, viral RNA accumulations in VI isolates were similar regardless of introduced mutations. These results provide evidence that FgSWI6 might play important role(s) in FgV1 induced phenotype alteration such as delayed vegetative growth.