• 한국균학회지
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• 제25권3호통권82호
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• pp.191-201
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• 1997

유전자의 삽입에 의해 발생하는 C. parasitica의 돌연변이체중 mycovirus에 감염된 것과 같이 색소와 포자를 적게 형성하는 균주(HSM1)를 선발하였다. 선발된 균주는 형태학적 병징외에도 laccase효소의 역가와 같은 생화학적 그리고 표지 유전자들을 통해 분자 생물학적인 특징도 virus에 감염된 균주와 동일한 특징을 나타냈다. HSM1에서 돌연변이가 일어날 부위를 cloning하여 조사한 결과, 유전자 삽입 부위는 C. parasitica의 두 유전자(Cpg2와Cpg3)의 사이(intergenic space)이며 유전자의 삽입 결과, HSM1에서 Cpg2의 발현이 오히려 증가됨이 관찰되었고, 나아가 이와 같은 현상은 mycovirus 감염 균주(UEP1)에서도 일어나고 있음을 확인하였다.

• 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.

• Journal of Applied Biological Chemistry
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• 제48권4호
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• pp.222-225
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• 2005

Strains showing antifungal activity against Cryphonectria parasitica were isolated from coast soil of Taean , Korea. Of 152 strains isolated, 6 strains showed antifungal activity in vitro against C. parasitica. Ta24 strain showed highest activity with 1.6 cm clean inhibition zone. For strain identification, the morphological characteristic and 16S rDNA sequences were determined. Ta24 strain showed 99% homology with Streptomyces sampsonii and was identified as Streptomyces sp.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.

• Journal of Applied Biological Chemistry
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• 제50권3호
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• pp.173-174
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• 2007

• 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.

• 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.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2018년도 춘계학술대회 및 임시총회
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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.

• 한국균학회지
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• 제26권4호통권87호
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• pp.450-457
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• 1998

Cryparin은 Cryphonectria parasitica의 세포벽에 풍부한 소수성 단백질에 속한다. cryparin은 비록 하나의 유전자에 의해 발현되지만 액체배양 후 48시간이 지나면 발현된 전체 유전자중에서 22%를 차지할 정도의 높은 발현 양상을 나타낸다. 또한 cryparin은 RNA mycovirus인 Cryphonectria hypovirus 1의 감염에 의해 발현이 현저히 억제되는 유전자로 알려졌다. 이미 지난 실험(Kim et al., 1999)에서 상동염색체간의 재조합을 이용하여 cryparin 유전자를 항생제 hygromycin B 저항성 유전자로 치환한 치환체를 제조하였다. 발현율이 매우 높으면서도 virus에 의해 밀접하게 영향받는 cryparin 유전자의 promoter 분석을 위하여서는 대상이 되는 유전자 치환을 위한 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 어느 한곳에만 삽입되도록 하는 성질을 가진 균주의 개발이 필요하다. 그러나 지난번 실험의 결과 얻어진 cryparin 치환체는 치환용 vector외에도 무작위로 삽입된 vector가 존재하고 나아가 새로운 vector들이 어느 한곳에만 삽입되도록 하는 성질을 갖지 못하였다. 따라서 본 실험에서는 cryparin 유전자 치환체와 영양요구성 돌연변이체인 균주간의 교잡을 이용하여 분석 대상이 되는 유전자의 치환에 이용된 vector만을 포함하며, 분석에 이용될 여러 유전자 운반체들이 genome내의 어느 한곳에만 삽입되도록 하는 성질을 가진 균주를 제조하였다.