<C3D6C1BEBAB8B0EDBCAD D E687770>

Transcription

1 제출문 농림부장관귀하 본보고서를 Cryphonectria parasitica 의신호전달관련저병원성화유전자 조절과국내특이 mycovirus 를이용한식물병방제 과제의최종보고서로제출합 니다 년 11 월 14 일 주관연구기관명 : 전북대학교총괄연구책임자 : 김대혁세부연구책임자 : 김대혁협동연구기관명 : 충북대학교협동연구책임자 : 차병진 1

2 요약문 Ⅰ. 제목 Cryphonectria parasitica의신호전달관련저병원성화유전자조절과국내특이 mycovirus를이용한식물병방제 Ⅱ. 연구개발의목적및필요성 Cryphonectria parasitica (Murrill) Barr는밤나무에줄기마름병을유발하여치명적인손상을야기하는강력한식물병원성진균으로, 북미대륙에서는밤나무산림의멸종을야기한바있고, 국내밤나무의경우도 C. parasitica에의해심각한수준으로감염되어있으며, 수령의노화와함께병균에의한피해가심각한수준에있는것으로으로보고되고있다. 그러나이러한특징을지니는 C. parasitica에곰팡이특이적인 mycovirus가전파되어감염을일으키면, 병원균의병원성이감소되고 (Hypovirulence), 여러가지형태학적, 생화학적및분자생물학적변화를유발하게되는데, 본과제에서는 C. parasitica에 mycovirus CHV1의감염에의한 hypovirulence를유도하는 C. parasitica의신호전달체계를규명하며, 국내 CHV의다양성및유전체분석을통하여 mycvovirus에의한진균의저병원성화기작을분자, 개체, 집단수준에서밝히고자하였다. Ⅲ. 연구개발내용및범위 CHV1에의해영향을받는 C. parasitica의유전자발현조절기작으로는, CHV1 감염이 C. parasitica의정상적인 G-protein 관련신호전달과정 (signal-transduction pathway) 을제어한다는가설이제시되었으나, 최근의본연구진의결과는 G-protein 외의세포내신호전달과정의 Ser/Thr protein kinase(cppk1) 가 mycovirus에의한 진균의유전자조절에서중요한 target이된다는것을증명하게되어, mycovirus에 2

3 특이적인신호전달계의주요유전자에대한규명과그들의발현조절기작을연구할필요성이제기되고있다. 따라서본과제에서는첫째, CHV1에의해조절받는 novel protein kinase(cppk1) 의특성분석및 cppk1과 interacting하는단백질및유전자들을분리 특성화하여생물적기능을검색하여 cppk1-관련신호전달체계를규명하고자하였으며, 둘째로는국내의 C. parasitica에존재하는 mycovirus의다양성및 type을결정하고, 이들과국내 C. parasitica 균주의 mycoviral symptom간의상관관계를확립하여각 symptom당특이 mycovirus 유전체를분석함으로국내균주의 hypovirulence유도에가장효과가있는 mycovirus를규명하고자했다. 이러한연구의결과로, CHV1에특이적인 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 의성장과관련된생물적기능을확인하였고, 이유전자산물과반응하는세포내단백질인 Enolase, Bck1 like MEKK, cplc 유전자를각각 cloning하고특성을밝혀 CHV1 관련신호전달과정의일부를규명하였다. 그외에국내의밤나무에서분리된 C. parasitica 균주를대상으로균사융합성의다양성을알아보고자 vegetative compatibility group (VCG) 을결정하였으며, 균주의 VCG type간 hyphal fusion을통한 mycovirus의병징을확인하고 ds-rna를분리특성화한뒤, 이를바탕으로저병원성전이균주제조하였다. 이들저병원성전이균주의제조는 field trial의기본적방제방법으로적용이될것이다. Ⅳ. 연구개발결과및활용에대한건의국제적으로도본과제와관련된 C. parasitica 균주를대상으로 genomics, proteomics, 등의연구가시작되고있으며, Dr. Nuss등에의해주도적으로추진되는세계적인 Genome project group에본연구진의참여가예상되고있는데, 이는본과제를통해발표된연구결과물들이국제적인인정을받았기때문으로생각된다. 또한본과제를통해밝혀진균주의유전적다양성에대한 field study가분자생물학적방법을이용하여분석되었는데, 이러한방법은환경친화적병방제를추구하는유사한진균병의생물적방제에응용모델로활용이가능할것이다. 또한본연구는새로운형태의병방제기술에관련된기본자료를확보하는것외에사상성진균의유전자발현및조절에관련된분야이므로, 산업적으로활용이많은진균을통한유용물질생산을극대화할수있는유전자발현정보를확보하였다고할수있다. 따라서이차대사산물또는재조합단백질생산에필요한 host로써의진균의이용가치를더욱높일수있을것이다. 3

4 SUMMARY ( 영문요약문 ) The chestnut blight fungus Cryphonectria parasitica virtually eliminated the American chestnut tree in the early 20th century. However, strains that contain the double-stranded (ds) RNA virus Cryphonectria hypovirus1(chv1) display characteristic symptoms of reduced virulence, i.e., hypovirulence, as well as diverse hypovirulence-associated traits, such as reduced pigmentation, sporulation, laccase production, and oxalate accumulation. We previously isolated the cppk1 gene encoded the Ser/Thr protein kinase of Cryphonectria parasitica and was transcriptionally up-regulated by the presence of hypovirus CHV1-EP713. To determine the biological function of cppk1, in this study, we constructed a cppk1-null mutant using targeted homologous recombination. The phenotype of pure cppk1-null mutant exhibited dramatic changes in colony morphology showing the characteristics of yeast-like microcolonial growth, no sporulation, and no hyphal differentiation into feeding hyphae. Moreover, hyphae of the cppk1-null mutant were shortened and hyperbranched with globose to bulbose cells. Electron microscopy of the cppk1-null mutant revealed the presence of intrahyphal hyphae, which was the most striking ultrastructural change, indicated that cppk1 is important for coordinating growth with development and maintaining cell wall integrity. To isolate the target proteins of Cppk1, we further analysed the cell-extracts of C. parasitica phosphorylated by Cppk1 using 2-D PAGE. Four distinctive protein spots were randomly selected and the MALDI-TOF MS analysis conducted, followed by a protein data base search was conducted. The homology search, based on the molecular weight of digested protein fragment, indicated that the proteins appeared seemed to be a fungal enolase, Bck1 like MEKK, and phosphatidyl inositol-specific phospholipase C (PLC) homologue. To get clone of the each genes, we conducted the multiple alignments of each proteins, designed several degenerated primers from the conserved region of each proteins, and PCR using the degenerated primers. The screening of the genomic λ library of C. parasitica, using the cloned fragments as a probe, we finally isolated eno1, Bck1 like MEKK, and cplc1 genes. To better characterize fungal gene regulation by hypovirus, we selected the cplc1 gene for further analysis, because hypovirus infection of the chestnut blight fungus C. parasitica is known to downregulate the fungal laccase1 (lac1), the modulation of which is tightly governed by the inositol triphosphate (IP 3 ) and calcium second messenger system in a virus-free strain. Sequence 4

5 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, and the gene organization appeared to be highly similar to that of a δ type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. Accordingly, reduced virulence of the cplc1-null mutant as compared to the wild type was observed, which can be ascribed to the growth defect. However, other PLC-associated characteristics including temperature sensitivity and osmosensitivity did not differ from the wild-type strain, indicating that the cplc1 gene is required for normal mycelial growth rate and colony morphology, and regulates the lac1 expression, which is also modulated by the hypovirus. In this study, we also isolated a total of 87 C. parasitica strains showing hypovirulence from 670 C. parasitica isolates, which were collected from major chestnut plantations all over Korea. To determine the genetic diversity of the fungal strains, we classified all strains into 121 vegetative compatibility group, and among them, 40% of total strains were shown to belong in three major VC groups, indicating there are a few major VC group in Korea and it is possible to control the infection of C. parasitica by transfer dsrna through hyphal fusion in nature. To determine the transfer of ds-rna between fungal strains, we selected 7 strong virulent strains and 5 ds-rna containing strains, tried hyphal fusion between them, and finally got 11 hypovirulent strains which containing same ds-rna isolated from parental strains. The results obtained through this studies we obtained the information of fungal signal transduction, that regulate many important mycovirus-specific genes related in the virulence of C. parasitica. These results will be help to understand the natural biological control of fungal pathogen at the molecular level, which can be applied for the establishment of better strategies to prevent fungal pathogen. 5

6 CONTENTS ( 영문목차 ) Submission form SUMMARY (Korean) SUMMARY (English) CONTENTS (English) CONTENTS (Korean) Chapter 1. Introduction Part 1 Purpose of the R&D Part 2 Necessity of the R&D Chapter 2. Status of the technology Part 1 Status of international research Part 2 Status of domestic research Chapter 3 Contents and Results of the R&D Part 1 Determination of the signal transduction pathway of cppk1, which regulated specifically by mycovirus Part 2 Determination Mycoviral diversity isolated from C. parasitica in Korea Chapter 4. Achievement and contribution of the R&D Chapter 5 Plan to apply the results of R&D Chapter 6 R&D information collected from foreign country Chapter 7 Reference Attachment

7 목 차 제 1 절연구개발의목적 제 2 절연구개발의필요성 제1절국외의기술동향및수준 제 2 절국내의기술동향및수준 제1절 C. parasitica에서 mycovirus에특이적인신호전달 protein kinase, cppk1의 pathway 규명및기능확인 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 의생물적기능확인 Cppk1 단백질의 in vitro 특성확인 Cppk1 target 단백질의확인및분리 Cppk1 target 단백질 coding 유전자의 cloning 및특성화 제 2 절국내 C. parasitica 에감염하는 Mycovirus 의다양성 국내 C.parasitica 집단에존재하는 mycovirus 의 typing 균주의 VCG type 간 hyphal fusion 을통한 mycovirus 의병징확인 국내 mycovirus 의유전자구조를기준으로한 typing 국내주요 mycovirus 의유전체 cloning 및특성화 저병원성전이균주제조 Vegetative compatibility를이용한전이균주선발

8 본과제에서는밤나무줄기마름병균인 C. parasitica에 mycovirus CHV1의감염에의한 hypovirulence를유도하는 C. parasitica의신호전달체계를규명하며, 국내 CHV의다양성및유전체분석을통하여 mycvovirus에의한진균의저병원성화기작을분자, 개체, 집단수준에서밝히고자하였다. 이를위해 ; 1 CHV1에의해조절받는 novel protein kinase(cppk1) 의특성분석및 cppk1과 interacting하는단백질및유전자들을분리 특성화하여생물적기능을검색하여 cppk1-관련신호전달체계를규명하고자하였으며 2 국내의 C. parasitica에존재하는 mycovirus의다양성및 type을결정하고, 이들과국내 C. parasitica 균주의 mycoviral symptom간의상관관계를확립하여각 symptom당특이 mycovirus 유전체를분석함으로국내균주의 hypovirulence유도에가장효과가있는 mycovirus를규명하고자했다. 1. 과학기술적측면 Cryphonectria parasitica (Murrill) Barr는밤나무에줄기마름병을유발하여치명적인손상을야기하는강력한식물병원성진균으로, 약 100여년전에밤나무육종을위하여아시아에서수입된모본에의해감염되어전파되기시작한북미대륙에서는밤나무산림의멸종을야기한바있다. 또한비교적저항성이있는것으로알려진국내밤나무의경우도 C. parasitica에의해심각한수준으로감염되어있으며 (>30% among all canker area), 수령의노화와함께병균에의한피해가심각한수준에있는것으로으로보고되고있다 (Ju et al., 2002). 8

9 이들이이렇게강력한 pathogen으로작용하는이유는넓은지역과시간에걸쳐쉽게퍼질수있는다량의 spore를갖고있고, 다양한환경속에서도숙주에대한특이성과감염성이매우높아, 쉽게감염하고적응하여숙주의저항성을피해생존하는능력이뛰어나기때문이다. 그러나이러한특징을지니는 C. parasitica에곰팡이특이적인 mycovirus가전파되어감염을일으키면, 병원균내에다양하고새로운특징을나타나게된다. 식물병원성진균에서많이보고되는 dsrna로구성된 virus 혹은 virus-like particle의존재는많은경우에 mycovirus로서일반적현상으로받아들여지고있는데, 특히 C. parasitica의경우세포질에존재하는 dsrna로구성된 mycovirus의감염이 C. parasitica의많은형태적차이및그와관련된병징을나타내는직접적인원인임이분자생물학적수준에서증명된바있다. 즉 mycovirus 감염경로는이병균주 (virus-containing C. parasitica) 와건전균주간의균사간세포융합 (anastomosis; hyphal fusion) 에의해서이병균주에서건전균주로 virus가옮겨지는것으로알려져있는데, 현재까지 mycovirus를순수분리하여기주인진균에직접접종하는방법이성공된바없기때문에 mycovirus를포함하는 C. parasitica의여러변화가 mycovirus의감염에의한것이아니라혹시세포융합중에일어나는기타물질의교환에의한것이아니가하는의문이있어왔으나, mycovirus의병원성재조합체 (infectious cdna copy of dsrna) 로만형질전환된재조합 C. parasitica가 mycovirus를포함하는균주와동일한병징이나타나는것을확인함으로써 mycovirus의감염이균주의다양한병징을나타나게하는직접적원인임을증명하였다 (Choi and Nuss, 1992). 따라서 C. parasitica에서밝혀진 mycovirus와의상호관계는지금까지보고된진균과 mycovirus간의상호작용의예중에서가장많이앞서서연구되고있고, 식물병원성진균의유전자발현기작연구와함께 Neurospora, Aspergillus spp. 등에서알려지지않은 mycovirus에의한진균의유전자발현및조절부분과이로인한식물에대한저병원성현상 (hypovirulence) 이가장잘정립된 model system이기도하다. 현재까지알려진바로는, C. parasitica에 mycovirus가감염되었을때나타나는특징은기주특이적이기보다는 mycovirus strain에따르는것으로밝혀졌다. 즉 mycovirus의어떤 strain에감염되었는지에따라 C. parasitica의병징이결정되는것이다 (Chen and Nuss, 1999). 지금까지가장많은연구가이루어진 mycovirus strain인 Cryphonectria hypovirus 1 (CHV1) 에감염된 C. parasitica 균주의병징은 9

10 아래와같다. 1 저병원성화 (Hypovirulence): CHV1 감염으로인하여밤나무에대한 C. parasitica의병원성이감소된다 (Anagnostakis, 1982; Van Alfen, 1982; Van Alfen et al., 1975). 2 형태적생화학적변화 (Biraghi et al., 1953, Day et al., 1977; Hillman et al., 1990): - 색소체형성감소 - 유성및무성포자형성감소 - laccase (polyphenol oxidase) 생성감소 - Oxalate 생성감소 3 분자생물학적변화 : CHV1감염이 C. parasitica의특정유전자들의발현에영향을미치며, 그기작으로는이들특정유전자들의전사과정을억제 (transcriptional down-regulation) 하는것이확인되었다 (Kim et al., 1995; Rigling and Van Alfen, 1991; Zhang et al., 1993; Kazmierczak et al., 1996). CHV1의존재에의해특이적으로 down-regulation되는 C. parasitica의유전자들은아래와같다 (Kim et al., 1995; Zhang et al., 1993; Zhang et al., 1994; Varley et al., 1992). - Lac1; extracellular laccase gene - Crp1; cell surface associated-fungal hydrophobin gene - Mf2/1 & 2/2; mating pheromone gene - Cut1; cutinase gene 이상의 virus 감염에의해나타나는 C. parasitica 병징의특징을요약하면, 다른 virus의감염의경우와는달리기주인 C. parasitica의특정유전자들의전사과정을억제하며, 그결과균사의생장등영양생식에는영향을주지않으나유 무성포자생성등과균사의분화및발달과정 (differentiation, development) 에영향을끼쳐미성장상태 (juvenile stage) 에머물도록하는특징을나타내는것을알수있으며, 이러한병징과관련된특징들은 mycovirus에의한진균의유전자발현조절을관찰 연구하는데좋은 marker로서활용이가능하다. CHV1 에의해영향을받는 C. parasitica 의유전자발현조절기작으로는, CHV1 10

11 감염이 C. parasitica의정상적인신호전달과정 (signal-transduction pathway) 을 "disturb" 한다는가설이 G-protein(G αi 와 G β) 의 cloning과이들유전자를 knock-out 시킨 null mutant에서보여지는부분적인 viral symptom-like phenotype들을통해제시되었으나 (Gao and Nuss, 1996), 이들 G protein의발현에대한 mycovirus CHV1의직접적인인과관계는좀더연구가필요한현실이다 (Zhang et al., 1998). 그러나최근본연구진의결과는 G-protein외의세포내신호전달과정의 Ser/Thr protein kinase(cppk1) 가 mycovirus에의한진균의유전자조절에서중요한 target 이된다는것을증명하게되어 (Kim et al., 2002), mycovirus에특이적인신호전달계의주요유전자에대한규명과그들의발현조절기작을연구할필요성이제기되고있다. 이러한유전자조절의결과로얻어지는병징간의 1:1 대응관계를정립하는연구를시행하여, virus에의한기주의유전자발현조절기작이라는기초학문분야와생물적방제의분자수준의이해및활용이라는응용면에서도많은기여를하리라기대되어계속적인관심과연구가필요하다고하겠다. 2. 경제 산업적측면국내의경우밤은표1에서와같이단일임산물로는가장경제성이높을뿐아니라, 단일농산물로는가장수출이많은품목이다. 하지만최근의연구에의하면비교적저항성으로알려진국내밤나무의경우도 C. parasitica에의해심각한수준으로감염되어있으며 (Ju et al., 2002), 그로인한생산성저하는, 저가의가격으로수입되는중국산밤과의경쟁력또한심각한수준으로떨어져이에대한대책이필요한실정이다. C. parasitica의병방제를위한외국의연구진에의한시도를살펴보면, mycovirus의 DNA copy(infectious cdna copy of dsrna) 를이용하여병원성균주를형질전환시켜병원성균주를저병원성균주로전환하는데성공하였다 (Choi and Nuss, 1992). 따라서이렇게얻어진형질전환체를 C. parasitica의집단내에살포함으로병방제를시도하고기대하였으나, 이들형질전환체의 genome에삽입된병원성재조합체 (infectious cdna copy of dsrna) 의전사과정결과생기는 dsrna는 CHV1과마찬가지로 C. parasitica의유성생식을억제하기때문에 (Zhang et al., 1993), 염색체내에병원성재조합체 (infectious cdna copy of dsrna) 를포함하면서다양한체세포융합군 (Vegetative Compatibility Group) 에속하는 progeny를기대하기어렵게되어전체적인집단내의 mycovirus의전파를이용한식물병방제에한계를드러냈다. 11

12 현재밤나무줄기마름병균에대한방제를위해다각적인노력을기울인결과, CHV1 이감염된저병원성균주로부터병원성균주로의 CHV1의전달이가장효과적인것으로나타났다. 그러나이와같은 CHV1의매개방법은순수분리한 CHV1 particle을병원성균주로직접적으로감염시킬수없기때문에현재까지그유일한매개방법으로는저병원성균주와근접한병원성균주간의균사융합 (hyphal fusion or anastomosis) 을통한것이유일한것으로밝혀졌다. 따라서효과적인생물적방제를위해서는전체 C. parasitica집단에서균주간균사융합을가능하게하는 vegetative compatibility group(vcg) 의조성이매우중요한것으로보고되고있다. 실제유럽의경우에서는유럽전체의 C. parasitica가 2 또는 3개의 VC group에속함으로 VCG 조성이매우단순하여집단내어느한균주에 mycovirus가감염되게되면집단내 VCG 조성이단순한관계로주위의다른균주와균사융합이용이하여쉽사리 CHV1 의전파가이루어져전체집단이저병원성화됨으로식물병방제의효과가매우높으나, 북미대룩의경우는전체 C. parasitica의 VC group이매우다양하여집단내에어느균주에 mycovirus가감염되어도저병원성균주와주위의다른균주와 VC group 이상이한관계로균사융합을이루지못함으로결과적으로이들 mycovirus의전파가이루어지지않게되어높은방제효과를거둘수가없음이증명되어이를이용한방제에서한계를나타내었다. 하지만본연구진은그간의연구결과 (Ju et al., 2002) 국내균주의 VCG 조성이비록복잡하나어느한 VCG가대부분을차지하는우점현상이존재하며, 따라서이들균주에 dsrna를도입하여적극활용한다면국내전체 population을대상으로한효과적인생물적방제가가능하리라기대하고있으며, 예비실험결과국내에도 dsrna 로구성된 mycovirus가존재함으로국내균주에대한효과적인국산 mycovirus의탐색과특성규명이시급하다고하겠다. 3. 사회 문화적측면사상성진균의유전자발현및조절에관한본연구는유용물질의생산및활용에서두각을나타내는고부가가치진균의생명공학적가치를제고할뿐만아니라, hypovirulence에대한정확한기작규명은식물병방제에서환경및생태계에많은부정적인영향을미치는화학적약제처리를대체하는생물학적방제에대한기초지식을제공하여이미자연계에존재하는기주-기생체간의상호관계를응용함으로써환경 12

13 친화적이며, 따라서사회적인식측면에서도긍정적인효과를나타내리라기대한다. 구분 13

14 제 1 절국외의기술동향및수준 현재국외의경우 C. parasitica의 hypovirulence 연구분야에서많은업적을남기는 group으로는미국 U. of California at Davis 대학의 Dr. N. K. Van Alfen교수와 U. of Maryland의 Dr. D. L. Nuss 그리고 Cornell 대학의 Dr. M. G. Milgroom 등이있다. 먼저 Dr. Van Alfen 교수의경우는 C. parasitica의존재하는 ds-rna와 hypovirulence와의관계를제시하였고, ds-rna의감염에의해 C. parasitica의주요유전자가 down-regulation됨을처음으로증명하였으며 (Kazmierczak et al., 1996), 이들특정유전자들의생물학적인기능을유전자치환의방법에의해연구함으로써병원성에밀접한유전자의확보에관한연구를계속수행하고있다 (Kim et al., 1995). Dr. N. L. Nuss의경우는 hypovirulence의연구에서특히 mycovirus인 CHV1의유전자지도와이들의기능 (Choi et al., 1991) 그리고앞서기술한바처럼병원성재조합체 (infectious cdna copy of dsrna) 를이용하여병원성진균을형질전환함으로이들을저병원성균주로유도할수있음을증명하였으며 (Choi and Nuss, 1991) 또한이와같은병원성재조합체 (infectious cdna copy of dsrna) 를이용하여 C. parasitica와유연관계에있는다른진균에서도병원성재조합체 (infectious cdna copy of dsrna) 를도입하면진균의세포질내에 ds-rna의 mycovirus가생성됨을제시함으로 hypovirulence 현상이다른여러진균에서도이용될수있음을제시하였다 (Choi and Nuss, 1992). 또한이렇게얻어진형질전환체를 C. parasitica의집단내에살포함으로병방제를시도하고기대하였으나, 이들형질전환체의 genome에삽입된병원성재조합체 (infectious cdna copy of dsrna) 의전사과정결과생기는 dsrna는 CHV1과마찬가지로 C. parasitica의유성생식을억제하기때문에 (Zhang et al., 1993), 염색체내에병원성재조합체 (infectious cdna copy of dsrna) 를포함하면서다양한체세포융합군 (Vegetative Compatibility Group) 에속하는 progeny를기대하기어렵게되어전체적인집단내의 mycovirus의전파를이용한식 14

15 물병방제에한계를드러냈다. 또한최근에는 CHV1 감염에의한 C. parasitica의 signal transduction pathway에관련된 G-protein유전자를 cloning 하여이들의생물학적기능에관한연구에많은관심을기울이고있다 (Chen et al., 1996). Dr. M. G. Milgroom은 C. parasitica의 population biology에많은연구를하고있으며, 북미지역의균주는많은종류의 VC group으로구성되어있음을밝혔으며이로인해균사간의세포융합이용이하지못하고따라서 mycovirus의전파가억제되므로북미지역에서는 CHV1에의한효과적인방제가이루어질수없었음을제시하고있다 (Milgroom and Lipari, 1995). 하지만현재유럽의경우에서는유럽전체의 C. parasitica가 2 또는 3개의 VC group에속함으로 VCG 조성이매우단순하여집단내어느한균주에 mycovirus가감염되게되면집단내 VCG 조성이단순한관계로주위의다른균주와균사융합이용이하여쉽사리 CHV1의전파가이루어져전체집단이저병원성화됨으로식물병방제의효과가매우높게나타나고있다. 제 2 절국내의기술동향및수준 국내의경우 C. parasitica의존재와피해에관한부분적인역학조사가오래전에보고된이후이에대한연구가미미하였으나최근산림환경연구원을비롯하여 C. parasitica에관한오랜연구를수행하고귀국한강원대학교의이종규박사님그리고충북대학의차병진교수님등에의해 etiology에관한연구가진행되고있다. 또한 mycovirus에의한버섯의병해등에관하여는경상대학교의이현숙박사님에의해 mycovirus의동정및특성화에관한연구가활발하며, 서울대학교의이용환박사님등에의해분자생물학적인방법으로 Fusarium spp. 에서 dsrna와병원성과의상관관계에관한연구및 Magnaporthe grisea에서 camp 와 PKC등이관련된신호전달에대한연구가활발히진행되고있다. 그밖에경상대학교의이창원교수님에의해사상성진균에서 PLC의 cloning 및기능에대한연구가진행되고있으며, 진균의 proteomics에대한연구도진행중이다. 15

16 제1절 C. parasitica에서 mycovirus에특이적인신호전달 protein kinase, cppk1, 의 pathway 규명및기능확인 1. CHV1에특이적인 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 의생물적기능확인 1) cppk1 유전자의특징및발현양상 cppk1 full-length 유전자를 cloning 하여 sequencing 하여분석한결과모든 kinase의 conserved한 domain을가지는 Ser/Thr protein kinase임을확인하고, homology search를실시하여진균에서다른 homologue를검색하였다. 또한 cppk1 유전자의발현을 Northern blot analysis를이용하여분석한결과이유전자는 mycovirus에의해특이적으로발현이증가하는 up-regulation되는유전자임을확인하였다 (Fig. 1). 2) cppk1 mutant 제조 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 의생물적기능확인을위하여이유전자의기능이결손된 null-mutant를제조하였다. 이를위해항생물질저항성유전자인 hygromycin 유전자를 cppk1 유전자내에치환시켜재조합벡터를제조한후진균에형질전환하여재조합벡터상의 DNA와진균의 chromosome에존재하는 DNA간의 2차에걸친 homologous recombination (double crossover) 을유도하였다. Target 유전자가치환된 null-mutant의선발은치환된유전자의염기서열에서유도된 PCR primer를이용하여각각의형질전환체의 DNA로부터 PCR을실시한후예상되는 DNA의증폭유무를가지고 1차검증하고, 나아가 restriction enzyme을사용하는 Southern blot analysis를실시하여최종확인하였다 (Fig. 2). Fig. 2의 A는 cppk1의 genomic DNA주위의여러제한효소 site를이용하여 cppk1-null mutant의유전자지도이다. Fig.2의 B는 hygromycin에의해치환된 cppk1의유전자결손을 cppk1 및 hygromycin 유전자를 probe로하여확인한그림이다. 본실험을통해확보된 prototype의형질전환체는 cppk1-null type의핵과 16

17 cppk1-wild type의핵이하나의세포에동시에존재하는 heterokaryon임을확인하였으며, cppk1-null type의핵으로만된 clone을확보하기위하여서는 heterokaryon 을배양하여무성포자를획득한다음형질전환에사용된선택표지인 hygromycin이있는배지에서배양함으로 cppk1-null mutant type의핵을가진균사를선별적으로획득할수있었으며, 순수분리된 cppk1-null mutant는포자형성을하지못하는것으로확인되었다. 2) cppk1-null mutant의형태적세포생물학적특징유전자치환방법으로유도된 cppk1-null mutant 는 PDAmb 배지에서형태적으로효모의 colony (yeast-like micro-colonial growth) 와같은형태로성장을했다. 이를광학현미경및전자현미경으로관찰했을때 hyphae의성장이크게제한되는것으로나타났으며제한된 hyphase의모양이실모양이아니고구형의모습으로나타났다. 또한전자현미경을통해관찰했을때 hyphae 내에또다른 hyphae가존재하는 intrahyphal hyphae(hypahe within hyphae) 형태로성장하고있음을확인했다. 이는세포벽의합성에문제가있는것으로생각되어 mutant의 cell-wall을염색해본결과비정상적인 chitin의축적으로인한것으로나타났다 (Fig. 3). 3) cppk1-null mutant와 wild type 간의 cppk1유전자의발현확인 cppk1 null-mutant와 wild type 간의 hypovirulent symptom관련특징을비교하기위해서는 methionine과 biotin을포함하는 potato dextrose agar (PDAmb) plate에서의 hypovirulent symptom과상관관계에있는 pigmentation, sporulation, laccase production의감소현상등을관찰하고, 수피접종을통한 virulence test를수행하고자하였으나 growth 자체가 wild-type 균주와차이가컸으므로이를직접비교하는것은의미가없는것으로사료되어 mutant에서의 cppk1의발현을 cppk1-specific antibody를사용해서확인하였다 (Fig 4). 2. Cppk1 단백질의 in vitro 특성확인 1) 활성있는재조합 Cppk1 단백질의발현, 분리및정제. 단백질의순수분리를위해 cppk1 cdna 클론을대장균전용의 expression vector 에삽입시켜대장균에형질전환시킨뒤발현시켜세포를초음파파쇄하여 17

18 SDS-PAGE를통해유전자산물을확인하였다. 이렇게발현된단백질산물은 6개의 His-아미노산을포함하는 fusion protein으로발현됨으로, 발현된단배질의말단에붙은 6xHis-tag에대한특이성을지닌 Ni-affinity chromatography (Novagen Inc.) 를이용하여순수분리하였다 (Fig. 5). 본연구에서는전체 cppk1 유전자를발현시켰고 kinase domain 이포함된 partial 유전자도발현하여확인하였다. 2) Cppk1의 in vitro phosphorylation assay 확립순수분리된 Cppk1의 kinase assay는 Myelin basic protein (MBP) 를 substrate로하고방사성동위원소로표식된 [gamma- 32 P]ATP를이용하여실시한뒤 SDS-PAGE를수행하여 kinase activity를확인하였다. Fig. 6에나타냈듯이대장균에서분리정제된 Cppk1 단백질은 MBP에대하여 kinase activity가있음이확인됐다. 따라서 cppk1 유전자를 E. coli를이용하여다음단계인 cppk1-interacting protein을검색할수있는 assay 방법을확보하였다. 3. Cppk1 target 단백질의확인및분리 1) cell-free extract를 substrate로이용하여 Cppk1 assay C. parasitica 내에서 Cppk1의 target protein의수및크기를확인하고자 cell-free extract를 substrate로하여 in vitro phosphorylation assay를진행했다. 그결과 50 kda 및 44 kda의크기를갖는단백질 band를확인했다 (Fig. 7). 2) Cppk1 단백질의 target 단백질을 2-D analysis를통해분리및동정 Cppk1 단백질의 target 단백질을분리확인하기위하여, In-vitro kinase assay를통해동위원소로표식된 target protein (50kDa and 44kDa) 들을 2-DE(dimensional electrophoresis) 과정을통해확인분리하고자시도하였다. 그결과두개의 spot을 2-D gel 상에서확인하였다 (Fig. 8). 4) MALDI, Q-TOF를통한 target 단백질 sequence 결정 E. coli에서발현시켜순수 분리정제된재조합 CpPK1 단백질에의해 kinasing되는 C. parasitica의단백질을 2-D PAGE를실시하여각각의단백질을 PAGE gel에 display 하였으며이때확인된 spot 들은 Tandem Mass를이용하여부분적이단백질 18

19 sequence를확인한결과 4개의 interacting candidate를선발할수있었으며 (Fig. 9), 그 4개는다음과같다. - 2개의 signal transduction 관련유전자 a) BCK1 homologue b) PLC homologue - 2개의 metabolic 유전자 c) enolase d) glyceraldehyde-3-phosphate dehydrogenase. 4. Cppk1 target 단백질 coding 유전자의 cloning 및특성화 1) Cppk1 target 단백질 coding 유전자의 cloning (1) Enolase 유전자의 cloning GenBank Database를검색하여진균유래의 enolase 아미노산서열을분석하여, 공통으로나타나는부위를대상으로 degenerated primer (forward: 5'-CTTGAATCTTCCCCAGTTA-3', reverse: 5'-TTCACTAACTACCGGCCAA-3') 를합성한후 C. parasitica의 genomic DNA를대상으로 PCR을수행하였다. 이렇게확보된 PCR replicon은 DNA 염기서열을통해유전자진위를확인하고이를방사선동위원소로 labeling하여 C.parasitica genomic library에서 screening하여확인하였다. 이때확인된 clone은 subcloning 과정을거쳐유전자염기서열을결정하여 GenBank에등록하고유전자분석을실시하였다 (Fig. 10, Fig 11). (2) Bck1 homologue 유전자의 cloning GenBank Database를검색하여진균유래의 Bck1 like MEKK 아미노산서열을분석하여 (Fig. 12), 공통으로나타나는부위를대상으로 degenerated primer를상기와같은방법으로제작하여 C. parasitica의 genomic DNA를대상으로 PCR을수행하였다 (Fig 13). 이렇게확보된 PCR replicon은 DNA 염기서열을통해 C. parasitica 의 Bck1 like MEKK유전자로확인됐고현재 subclonig 중에있다. 이는향후 subcloning 과정을거쳐유전자염기서열을결정하여 GenBank에등록하고유전자분석및세포내에서의기능이밝혀질것이다. 19

20 (3) PLC homologue 유전자의 cloning GenBank Database를검색하여진균유래의 PLC 단백질의아미노산서열을분석하여공통으로나타나는 X와 Y부위인 SSHNTY 및 GYVLKP를대상으로 degenerated primer를제작하였다 (Forward: 5'-CAGYTCNCAYAAYACNTAYCT-3', Reverse: 5'-CGGGATCCGGYTTNAGNACRTANCC-3'). 이를이용하여 C.parasitica 의염색체 DNA를대상으로 PCR fragment를확보했고이렇게확보된 PCR replicon은 DNA 염기서열을통해유전자진위를확인하고이를방사선동위원소로 labeling하여 C. parasitica genomic library에서 screening한후 cloning 하였다. 이때확인된 clone은 subcloning 과정을거쳐유전자염기서열을결정하여 GenBank에등록하고유전자분석을실시하였다. 본연구에서확보한 C.parasitica 유래의 PLC 유전자는 736 bp 의 PLC-delta type으로분석되었다 (Fig. 14 및 Fig. 15). 1 PLC 유전자가결손된 mutant 의분리 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 의 target 유전자로확인된 PLC 유전자의생물적기능을확인하기위하여이유전자의기능이결손된 null-mutant를제조하였다. 이를위해항생물질저항성유전자인 hygromycin 유전자를 cplc 유전자내에치환시켜재조합벡터를제조한후진균에형질전환하여재조합벡터상의 cplc와진균의 chromosome에존재하는 cplc간의 2차에걸친 homologous recombination (double crossover) 을유도하였다. Target 유전자가치환된 null-mutant의선발은치환된유전자의염기서열에서유도된 PCR primer를이용하여각각의형질전환체의 DNA로부터 PCR을실시한후예상되는 DNA의증폭유무를가지고 1차검증하고, 나아가 restriction enzyme을사용하는 Southern blot analysis를실시하여최종확인하였다 (Fig. 16). Fig. 16의 A는 cplc의 genomic DNA주위의여러제한효소 site를이용하여 cplc-null mutant의유전자지도이다. Fig.16의 B는 hygromycin에의해치환된 cplc의유전자결손을 cplc 및 hygromycin 유전자를 probe로하여 Southern blot analysis를확인한그림이다. 2 cplc 유전자가결손된 mutant 의생리적특징본실험을통해확보된 cplc-null 형질전환체의생리적특징을살펴보고자 temperature sensitivity, osmosensitivity 등을 wildtype과비교하였다 (Fig. 17 및 Fig. 18). 또한 cplc 유전자의기능을확인하기위하여 yeast Saccharomyces 20

21 cerevisiae 유래의 PLC를이용하여 complementation을실시하였다 (Fig. 19). 또한 cplc에의해조절되는유전자의종류를알아보기위하여 laccase 유전자를비롯한 marker 유전자를대상으로 Norhern blot analysis를시도하였다 (Fig. 20). 그결과 cplc 유전자가결손된 mutant는정상균주보다성장이느리고 aerial mycelia가거의없는어두운오렌지색을띠는 mycelial growth 특징을나타내었다 (Fig. 21). 따라서병원성테스트에서정상균주보다병원성정도가약하게나왔으나이는 mutant 균주의 growth-defect에의한것으로사료된다. 그외에 temperature sensitivity, osmosensitivity 등은정상균주와큰차이가없었고 lac1 유전자의발현이 cplc 유전자에의해조절받는것으로나타났다. lac1 유전자는효모의 PLC complementation에의해발현이회복되는것으로나타났다. 따라서 cplc 유전자는정상적인 growth 및 colony morphology를위해반드시필요하며 hypovirus에의해조절되기도하는 lac1 유전자의발현에반드시필요한유전자인것으로판명되었다. 21

22 제 2 절국내 C. parasitica 에감염하는 Mycovirus 의다양성 1. 국내 C.parasitica 집단에존재하는 mycovirus 의 typing 1) 균주확보아래그림에나타나있는각지역으로부터총 670여균주의 C. parasitica를분리해내었다. 이들의균총모양은균총이전체적으로노란색을띠면서포자를많이만드는것, 균총의가운데만노랗고가장자리는하얀균사로둘러싸인것, 그리고균총이전체적으로흰균사들로만이루어져있으며포자형성도아주불량한것등 3가지정도로크게나눌수있었다. 또한, 균총의가장자리가균일한것과불규칙한것으로구분할수있었다. 병원성이센균주들은대개균총의색은노랗고가장자리의모양은불규칙한것들이었으며, 분리한균주들은대개이런범위에속하는것들이었다. Jun-kok 8(S) Euyjongpu 4(T) Yang-pyung 18(U) Kwang-ju 30(V) Hwa-sung 4(X) Seo-san Ye-san Pu-yeo Kong-ju 3(O) 16(P) 25(Q) 25(R) Chun-cheon 48(h) Hong-cheon 31(j) Hoeng-sung 7(i) Won-ju 15(g) Choong-ju 49(e) Jin-cheon 7(f) Chung-ju 18(m) Young-dong 39(l) Ik-san 15(H) Jung-eup 9(G) Im-sil 7(E) Soon-chang 3(F) Yung-cheon 14(I) Kyung-ju 21(J) Pung-san 4(K,M) Yung-yang 5(L) Koo-rye 13(D) Kok-sung 18(C) Soon-cheon 29(B) Kwang-yang 30(A) Ha-dong 41(Y) Jin-ju 31(Z) San-chung 54(a,b) Ham-an 27(d) 22

23 2) Bavendamm test C. parasitica isolate들의 phenol oxidase 활성을조사한결과 (Fig. 21) 분리균주의대부분인 576균주가병원성인것으로보였으며, 87균주가저병원성인것으로보여저병원성균주로보이는것의비율이약 13% 에달했다. 저병원성으로보이는이들균주는 mycovirus, 즉 ds-rna에감염되어있을가능성이높은것들이라고할수있다. 3) C. parasitica isolate들의 vegetative compatibility C. parasitica는유전적변이가심하여균주간에균사융합성이다양한것으로알려져있는진균이다. 미국에서는동등한비중의 64개의 VC group이밝혀져있으며, 유럽에서는지역에따라큰차이가있지만미국에서보다는다양성이적은것으로알려져있다. 그러나우리나라의균주들을대상으로한이번연구의결과 (Fig. 22) 를보면우리나라에는 121개의 VC group이존재하는것으로밝혀져 C. parasitica의유전적다양성이다른나라보다우리나라에서훨씬더심함을알수있었다. 이러한현상은 mycovirus에감염된저병원성균주를이용한생물적방제의걸림돌이될수도있다. 하지만, 다행스러운것은다양한 VC group 중에서도지역적으로우점을하고있는 group들이존재한다는사실이다. 실제로 VC group 중 KR-VC104에는총 670여균주중 164균주가속해있었으며, KR-VC105에는 62균주, KR-VC43에는 42균주가속해있어이세 group에속하는균주들이전체의 40% 에이르고있었다. KR-VC104 는전북을제외한어느지역에서나가장높은빈도를보이고있었다. 따라서 KR-VC104를우리나라의우점 C. parasitica VCG로판단해도무리가없을것으로보인다. 4) 수피접종시험저병원성표준균주인 UEP-1보다작은병반을만드는균주는총 670균주중 164 균주였으며, 병원성표준균주인 EP 155-2보다큰병반을만드는균주는모두 378 균주였다. 나머지 128 균주는 UEP-1보다는크고 EP155-2보다는작은병반을만들었다. 5) ds-rna의검출현재총 670균주중 650균주에서 ds-rna의존재를검정한결과 77균주에서 23

24 ds-rna 특이적밴드를검출할수있었다. 우리나라의 C. parasitica가 mycovirus에감염되어있는양상은모두 4 종류로서 (Fig. 23), ds-rna의크기별로 12 kb에만감염되어있는것, 12 kb와 2.7 kb에감염되어있는것, 3 kb에만감염된것, 그리고 3 kb와 2 kb, 1.8 kb에동시에감염되어있는것이었다. 이중 12 kb에단독감염된것이 46 균주로가장많았으며, 다음이 12와 2.7 kb로 25 균주, 그리고나머지두종류는각각 3 균주씩이었다. 우리나라에존재하는 121개의 VCG 중 29개에서 ds-rna를검출할수있었으며, 다행스럽게도 KR-VC104를비롯하여빈도수가높은 VCG들은모두감염되어있었다. 지역적으로는경기와강원을제외한나머지도에서는모두 ds-rna 감염균주를찾을수있었으나, 충북에서는단 1 균주, 충남과전북에서는 5 균주, 경북에서 7 균주등중부지방에서는매우적었다. 반면에밤나무의주재배지역인경남에서 43 균주, 전남에서 16 균주가 ds-rna에감염되어있음을확인하였다. 또한, ds-rna 검출결과와 Bavendamm test 결과를비교하여보면, Bavendamm test에서저병원성으로보이던 87 균주중 45 균주에서만 ds-rna가검출되었고, 병원성으로보이던균주중 32 균주에서역시 ds-rna를분리할수있었다. 밤나무가지에접종하였을때의병원성과비교하면, UEP1 보다병원성이낮은균주중 20 균주만이 ds-rna에감염되어있었으며, EP-155-2보다병원성이큰균주중 44 균주가감염된것이었다. ds-rna의존재와잘부합하는특성은균총의형태였다. 일반적으로병원성인것으로이해되고있는노란균총을만드는 450개의균주중단 8 균주만이 ds-rna에감염되어있었고, 중간형태또는흰균총을만드는균에서 69 균주가감염되어있었다. 2. 국내균주의 VCG type 간 hyphal fusion 을통한 mycovirus 의병징확인 10개 VCG에서선발된균주로부터약 300개의유도균주가선발되었다 (Fig. 24). 이들균주는현재 dsrna 분리와병원성검정, phenol oxidase 활성검정실험중에있다. 3. 국내 mycovirus 의유전자구조를기준으로한 typing 1) ITS-RFLP를이용한유연관계비교 189개균주의 DNA를 ITS-PCR로증폭하여제한효소를처리한결과, 178개균주 24

25 가대조구로사용한유럽의 CHV1 균주와동일한밴드형태를보였으며, 11개의균주만이다른밴드형태를보였다. AluⅠ을처리했을때 CHV1은 430 bp와 220 bp의 2가지밴드를보인반면이들 11개균주는절단되지않았다. 또한 CfoⅠ을처리했을때, CHV1 균주가 290 bp와 50 bp 밴드를보인것과는달리, 이들은 300, 270, 그리고 80 bp의밴드를보였다 (Fig. 25). 밴드형태가다른 11개의균주중 9균주가전남에서, 2균주는경남에서분리된균주였으며, 6균주에서는 dsrna가검출되었다 kb dsrna를가진것은 2균주였으며, 각각전남과경남에서분리된균주들이었다. 4균주는 3.0 kb 또는 kb를가진균주로 3균주는전남에서, 1균주는경남에서분리되었다. 11개균주중전남에서분리된 4균주는 VCG-14에속해있었으며, 나머지 7개균주는각각다른 7개의 VCG에속해있었다. 2) RT-PCR을이용한유연관계비교 C. parasitica에서검출되는 dsrna의다양성을알아보기위해 cdna를이용해증폭한결과 5 non-coding region에서약 600 bp, ORF B에서 1200 bp 크기의 PCR 산물을얻었으며 (Fig. 26), dsrna의염기서열은유럽의 CHV1-EP713과비교하였다. 1 5 non-coding region 총 14개의균주는 5개의그룹으로나뉘었으며 (Fig. 27) 유럽의 EP713과는약 90% 이상의유사성을보였다. 우리나라균주간의유사성을보면전남에서분리된 AS112 와경남의 ZS312 등두균주의염기서열은 100% 로동일하였으며, 같은장소에서분리된 an422와 as522는 0.2% 의차이를보였다. 경남의 as522와경북의 IE412는 92.7% 로가장낮은유사성을보였다. BS222은 ITS-RFLP에서특이한밴드형태를보였던균주였으나 Y121과 98% 의높은유사성을보였다. 2 ORF B 5 non-coding region과비슷하게 5개의그룹으로나뉘었고 (Fig. 28) EP713과는약 81% 이상의유사성을보였다. IE412와 RN421, an422와 as522가각각 99.8% 와 99.5% 로유사성이가장높았으며, 5 non-coding region에서염기서열이동일했던 AS112와 ZS312는 92.3% 의유사성을보였다. 특히전북에서분리된 HE522는유럽의 EP713과 98.1% 의높은유사성을보였다. 25

26 4. 국내주요 mycovirus 의유전체 cloning 및특성화 분리된 dsrna 중 L-dsRNA인 12.7 kb를제외한 3.0 kb, 2.7 kb, 2.0kb, 1.8 kb dsrna를정제하였다 (Fig. 29). 이들 dsrna는외국에서보고되어있는 s-dsrna보다크기가더작으며 L-dsRNA가존재하지않는균주에서도존재했다는점에서우리나라균주의특징일가능성이매우높다. 이 dsrna들이 L-dsRNA의일부인지를확인하기위하여분리된 ds-rna들을정제하였으며, 다시 RT과정을통해 cdna 합성을하였고현재 cloning 과정에있다. Cloning 결과가나오면우리나라 C. parasitica 의특이성을확인할수있을것이다. 5. 저병원성전이균주제조위의 Fig. 21에서도볼수있듯이같은 VCG에속하는균주들끼리는대치배양으로유도한균사융합에의하여 mycovirus에감염됨, 즉저병원성이유도되는것을확인할수있었다. 그러나, VCG가다른균주들끼리는이러한대치배양에의하여저병원성전이균주를만들어낼수없었다. VCG가다른균주들간에는균사융합이일어나지않고대치한자리에서균사가더이상자라지않는것을확인하였다. 이러한현상은 PDA에서뿐만아니라수피에접종하였을경우에도마찬가지였다. 따라서, VCG가다른균주간에대치배양법에의하여새로운저병원성균주를만들어내는것은가능성이매우낮다고결론지었다. 그러나, 식물의경우바이러스가침입하려면반드시상처가있어야하지만, 식물세포의원형질체를사용하면상처없이도바이러스가침입하여감염이되는현상을이용하면저병원성균주가없는 VCG의병원성균주들로부터전이균주를유도해낼수있는가능성을찾을수도있을것이다. 따라서, C. parasitica의원형질체를만들고이에저병원성균주에서분리한 dsrna를접종하는방법도제안이가능하다. 6. Vegetative compatibility 를이용한전이균주선발 1) 사용균주 1999~2000년전국의밤나무재배단지에서분리한균주중 VC group 104에서병원성 6 균주, 저병원성5 균주, 그리고 VCG 85의병원성 1균주 (a1) 를사용하였다. VCG85의균주, a1은다른 VCG 균주사이의전이가능성을알아보기위해사용하였다. - 병원성균주 26

27 Colony type Phenol oxidase pathogenicity ds-rna a 1 Irregular yellow B411 Irregular yellow Q1 Irregular yellow QE412 Irregular yellow R112 Regular center yellow RW412 Regular center yellow Ya Irregular yellow 저병원성균주 Colony type Phenol oxidase pathogenicity ds-rna AS112 Irregular center yellow IE311 Regular white RN421 Regular white Y112 Irregular white ZW411 Regular white ) 균주의선발선발된 12 균주는 PDA에서 25, 4일간배양후, 1/4 PDA 배지에병원성균주와저병원성균주한쌍을나란히치상하였다. 또는 25, 3일간배양후두균주의균사가맞닿는부분을해부현미경 (40X) 으로관찰하여균사가융합한것으로보이는균사를잘라 PDA에치상하였다. 분리된균사는 25, 4일간배양하였고섹터가생기는부분은균일한균총으로자랄때까지계속해서계대배양하면서분리하였다. 이렇게분리된균주는 colony type, phenol oxidase activity, VC test, ds-rnadetection을조사하였다 (phenol oxidase activity test: bavendamm 배지, VC test: PDAvc 배지, ds-rna detection: cellulose chromatography 방법이용 ). 그결과 1/4 PDA에서균사가융합된것으로보이는 314개의균주를분리했다 (Table 3). 3) 선발된균주의 vegetative compatibility test 27

28 VCG 104 분리균주들은모균주와모두균사융합성을보였고, VCG 85균주는배지상에약한경계를형성하였다. 4) Colony type 분리된이들균주들의균총형태는대부분병원성균주의균총과유사했으며 (Fig. 30), 이들중 10 균주만이저병원성균주와비슷하게나타났다. 즉균총색이하얀색은아니지만균사가일정한형태로균일하게자라는형태를보였다 (Fig. 31). 5) phenol oxidase activity 분리된 314개의균주의 phenol oxidase activity를조사한결과, 총 75개의균주가병원성모균주보다활성이낮았고저병원성모균주와는비슷하거나낮았다 (Table 4)). 특히괄호로표시되어있는 9 균주는병원성모균주와저병원성모균주보다 phenol oxidase activity가낮게나타났다. 또한균총의형태는저병원성균주와비슷했지만하얀색을띠지는않았고병원성균주보다포자형성과색소침착이줄어든것을볼수있다 (Fig. 32). 6) ds-rna의검출 314개균주의 ds-rna를확인한결과, 11개의균주에서 ds-rna가검출되었다. 다른 VCG인 VCG 85에서분리한 a1-전이균주와, 동일한 VCG인 B411, Q1-전이균주에서는전혀검출되지않았다. 이렇게 dsrna가검출된 11개균주는상기의실험에의해분리된 9 균주와 Ya-RN421 1균주, R112-IE311 1균주이다 (Table 5). 이들균주에서는저병원성모균주에서관찰되었던 ds-rna가존재함을알수있었다 (Fig. 33). 7) 생물적방제용 Field trial 현재까지체세포융합성을통해분리한전이균주들에대해 CHV1의 full genome이삽입되었는지정확하게확인하지는못하였지만, 전기영동에서보여준크기를보면정상적으로전이가된것으로여겨지며, 전이균주와저병원성모균주의 ds-rna는차후 ds-rna의염기서열비교로가능할것으로생각된다. 하지만, Chen &Nuss (1999) 은 CHV1 cdna clone을삽입했을때모균주와동일한 phenotype을보였다고보고했었으나, 현재본연구진에의해분리된전이균주들의병원성은, 저병원성모균주와동일한형태를보이고있지는않다. 따라서계대배양을통해균주를안정화시키고이에따른변화는계속적인실험을통해확인하여야할것이라사료된다. 끝 28

29 으로현재 ds-rna가확인된 11개균주를이용하여 Field trial을시도하여야하나계절상현재시점에서는늦가을이기때문에실험조건이좋지않아접종에관련된 data는포함시키지않았다. 29

30 구분연구목표평가의착안점 1 차년도 (2002) 2 차년도 (2003) CHV1 에특이적인 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 의생물적기능확인 Cppk1 단백질의 in vitro 특성확인 국내 C.parasitica 집단에존재하는 mycovirus 의 typing Cppk1 target 단백질의확인및분리 Cppk1 target 단백질 coding 유전자의 cloning 및특성화 국내분리의 mycovirus 접종실험및특성화 국산 mycovirus 를이용한 hypovirulence 유도 cppk1 의생물적기능및 kinase assay 확립 국내 Mycovirus 의순수분리및다양성조사 cppk1 의 target 단백질분리및특성화 국내특이 Mycovirus 의유전체결정 목표달성도및 / 기술발전기여도 CHV1 에특이적인 C. parasitica 신호전달 protein kinase 유전자 (cppk1) 가결손된 mutant 를제작하여그생물적기능을확인하여목표치 100% 달성함 Cppk1 단백질의 in vitro phosphorylation 특성확인하여목표치 100% 달성함 국내 C.parasitica 집단의 VCG typing 하여목표치 100% 달성함 2-D PAGE 및 phosphory- lation 방법을적용하여 4 종류의 Cppk1 target 단백질의확인및분리로목표치 100% 달성 VCG 에따른 mycovirus 의분리및 typing 으로목표치 100% 성공함 3 차년도 (2004) cppk1 관련 pathway 의확립 저병원성유도균주의유성생식에의한새로운 VC group 의출현검색 생물적방제 cppk1 pathway 규명 국내 mycovrius 및재조합균주를이용한생물적방제효과검정 cppk1 는 enolase, Bck1 like MEKK 및 PLC 관련신호전달과정에관련된것으로결론하여 100 목표달성 국내 mycovirus 를이용하여저병원성유도균주제조및생물방제기초확립 30

31 본연구의수행을통해, 식물병의중요한부분을차지하며나아가최근유용단백질을생산하는생명공학분야에서간단한진핵세포생물로관심을가지게되는진균의유전자발현에대한기작을신호전달체계와함께일부규명하였고, 비교적많은기초연구가선행된일부진균에서는존재하지않지만, 그외의다양한많은종류의진균에서그존재가보고되는 mycovirus 혹은 dsrna에의한조절작용을연구하므로써본연구결과는기초분자생물학적분야에서비교적새롭고독창적인유전자조절 model system 을제공하였다. 또한기주 (C. parasitica)-기생체 (mycovirus) 간의상호작용결과에대한유전자조절은식물병 ( 밤나무줄기마름병 ) 에대한안전하고좋은생물적방제의기초자료로사용이가능하므로환경친화적인생물적방제의학문적근거와응용의예를제시할것으로사료된다. 또한본연구는밤나무 origin에속하는북동아시아의한축인국내밤나무에감염된 C. parasitica에서존재하는 mycovirus의유전적, 생태적다양성에대한연구를진행하였으므로 mycovirus의전파및오랜역사를지닌우리지역에서 mycovirus의진화에대한기본적인 data를제공할수있게되었다. 이는세계적인분포를지닌 mycovirus의 genotype과국내특이적인 genotype의확인을통해기주-기생체간의 coevolution을설명할수있으며, 이는독립된지역에서가장잘적응한 genotype을유추하여가장효과적인생물적방제를실시하는데기초자료로또한기여할수있게된다. 이상의결과들은국내연구진으로하여금진균의유전자발현및 mycovirus에의한진균의유전자조절그리고 mycovirus의다양성및진화에관한과학기술적측면에서국내뿐아니라세계적으로선도적역할을가능하게하리라기대되어본연구에대한계속적인지원이기대된다. 31

32 본연구과제와관련된균주인 C. parasitica를대상으로 High throughput 연구를통한 genomics, proteomics, 등의연구가본과제와유사한방법으로시작되고있으며, 일부보고되고있다. 또한 Dr. Nuss등에의해주도적으로추진되어오는본균주에대한 Genome project의 funding이가시화되고있다는소식과, Genome project group에본연구진의참여를제안받았는데, 이는본과제를통해발표된연구결과물들이국제적인인정을받았기때문으로생각된다. 본연구를통한결과및향후의연구결과들은추후본연구진이세계적인 Genome project group에참여하여연구하는데결실을맺어크게기여하는부분이생기리라기대된다. 32

33 1. Biraghi,A Rep. Congr. Int. Union Forest Res. Org. 11th Rome, 1953 pp Chen,B., Gao,S., Choi,G.H. and Nuss,D.L Proc. Natl. Acad. Sci. USA. 93: Choi,G.H., Pawlyk,D.M. and Nuss,D.L Virology 183: Choi,G.H., and Nuss,D.L Science 257: Choi.G.H., Chen,B., and Nuss,D.L Proc. Natl. Acad. Sci. USA 92: Day,P.R., Dodds,J.A., Elliston,J.E., Jaynes,R.A., and Anagnostakis,S.L Phytopathology 67: Hillman,B.I., Shapira,R., and Nuss,D.L Phytopatology 80: Ju, Y.J., Kim, D.H., and Cha, B.J Plant Pathol. J. 18(1): Kang, H.S., Choi, J.W., Park, S.M., Cha, B.J., Yang, M.S., and Kim, D.H Plant Pathol. J. 16: Kazmierczak,P., Pfeiffer,P., Zhang,L., and Van Alfen,N.K J. Virol. 70: Kim,D.H., Rigling,D., Zhang,L., and Van Alfen,N.K Mol. Plant-Microbe Interact. 8: Kim, M.J., Choi, J.W., Park, S.M., Cha, B.J., Yang, M.S. and Kim, D.H Mol. Microbiol. 45: Rigling,D. Van Alfen,N.K J. Bacteriol. 173: Van Alfen,N.K., Jaynes,R.A., Anagnostakis,S.L., and Day,P.R Science. 189: Van Alfen,N.K Ann. Rev. Phytopathol. 20: Varley,D.A., Podila,G.k., and Hiremath,S.T Mol. Cell. Biol. 12: Zhang,L., Churchill,A.C.L., Kazmierczak,P., Kim,D.H, and Van Alfen,N.K Mol. Cell. Biol. 13:

34 34

35 Table 2. Characteristics of 11 Korean isolates of Cryphonectri parasitica which had different band patterns in ITS-RFLP Isolates Province VCG dsrna type Characteristics (kb) Pathogenicit Colony Phenol oxidase y type activity AE322 Jeonnam VCG high white moderate AN122 Jeonnam VCG-14 none low white moderate B211 Jeonnam VCG-13 none low white moderate B422 Jeonnam VCG-58 none moderate white moderate BW112 Jeonnam VCG-14 none low white moderate BW412 Jeonnam VCG-14 none high white moderate BS222 Jeonnam VCG high yellow moderate BS322 Jeonnam VCG high white moderate BN122 Jeonnam VCG low center yellow moderate aw411 Gyeongnam VCG low white moderate bs131 Gyeongnam VCG high white moderate 35

36 Table 3. Isolation of hypovirulent by hyphal fusion. hypovirulent AS112 IE311 RN421 Y112 ZW411 a B Q virulent QE R RW Ya

37 Table 4. Numbers of the hypovirulent strains shown low phenol oxidase activity. hypovirulent AS112 IE311 RN421 Y112 ZW411 a B Q virulent QE412 4 (2) 5 (3) R (1) 1 1 RW412 5 (3) Ya

38 Table 5. Numbers of the hypovirulent strains containing dsrna molecule. hypovirulent AS112 IE311 RN421 Y112 ZW411 QE virulent R RW Ya

39 Fig. 1. Northern blot (A-D) and RT-PCR (F and G) analysis of marker gene expression in virus-infected UEP1 and virus-free wild type EP155/2 strains. A; cppk1, B; cryparin, C; Mf2/1, D; glyceraldehyde-3-phosphate dehydrogenase (gpd), F; gpd, G; cppk1, E; EtBr-stained RNA gel for equal loading of RNA samples 39

40 Fig. 2. Restriction map and Southern blot analysis of heterokaryotic parental strain and pure cppk1-null mutant. Note that heterokaryotic parental strain contains both wild type nuclei and cppk1-null type nuclei in the same mycelia due to the coenocytic(mutiple nuclei in a single cell) characterics of filamentous fungi. 40

41 Fig 3. Phenotypic characteristics of cppk1-null mutant. (A) (B) (C) Morphology of colony forming units of a heterokaryotic spore suspension (TdPK1) grown on non-selective (I) and selective (II) PDAmb plates. The characteristic colony types are shown in enlargements of the areas marked by boxes. Both cultures are 10 days old. Microscopic observations of the wild type EP155/2 (I and III) and cppk1-null mutant TdPK1-1 (II and IV). Bars, 25 mm (I, II) and 10 mm (III, IV). I. Typical extended filamentous hyphae (H) are seen in the wild type EP155/2. II. Bead-like catenulate hyphae (BH) consisting of bulbous and globose cells (arrows) are seen in the cppk1-null mutant TdPK1-1. III. Transverse and longitudinal sections of the wild-type EP155/2 strain showing normal fungal hyphae (H). IV. Transverse and longitudinal sections of the cppk1-null mutant TdPK1-1 showing parts of bead-like hyphae (BH). Note that all the fungal cells are markedly hypertrophied to varying degrees. Fluorescent microscopy to detect chitin accumulation. Young hyphae of wild type EP155/2 (I) and cppk1-null mutant TdPK1-1 (II) were stained with 10 mg/ml Calcofluor White for 20 min and photographed through a fluorescence microscope. Septa are indicated by arrowheads. Photographs were taken at a magnification of x 400. (D) Transmission electron microscopic images of mycelia of the wild type EP155/2 (I), and hypertrophied globose cells (II) and intrahyphal hyphae (III) of the cppk1-null mutant TdPK1-1. H, hyphae; S, septae; HH, intrahyphal hyphae; W, cell wall; Bars, 2 mm. 41

42 Fig. 4. Western blot analysis (A) and colony morphology (B) of representative C. parasitica strains transformed by anti-sense copies of cppk1. (A) Western blot analysis of representative anti-sense transformants. Total cell-free extracts prepared from wild type EP155/2 (wt), cppk1-null mutant TdPK1-1 (cppk1), and representative anti-sense transformant types I, II, and III were probed with CpPK1-specific antisera. The numbers at left are the protein size in kda. (B) Colony morphology of representative anti-sense transformants. Three examples of representative types of colony morphology of the anti-sense transformants are shown in comparison with the nontransformed wild-type EP155/2 strain and the cppk1-null mutant TdPK

43 Fig. 5. SDS-PAGE of purified E. coli-expressed full-length and truncated CpPK1. Lanes 2 and 3 contain E. coli-expressed full-length and truncated CpPK1, respectively. Lane 1 contains the size markers and numbers at left refer to the protein sizes in kda. 43

44 Fig. 6. Kinase assay of CpPK1 using cell-free extracts. (A) Lane 1 shows the phosphorylation pattern of endogenous proteins without E. coli-expressed CpPK1. Lanes 2, 3, 4, and 5 are phosphorylation patterns of endogenous proteins with 2 ng, 4ng, 8ng, and 16ng of E. coli-expressed CpPK1, respectively. (B) Kinase assay using bovine serum albumin as a control. Each lane contains the same amount of BSA as described above. Arrows indicate the potential intracellular substrates of CpPK1. Numbers at left refer to the protein sizes in kda. 44

45 Fig. 7. Kinase assay of cell-free extracts. Kinase assay of endogenous proteins was conducted using cell-free extracts from 1-day and 5-day old culture of both Ep155/2 and UEP1. Lanes 1 and 2 contain endogenous proteins from 1-day and 5-day old culture of Ep155/2, respectively, and lanes 3 and 4 are from 1-day and 5-day old culture of UEP1, respectively. Arrows indicate the phosphorylated proteins, and numbers at left and right refer to the protein sizes in kda. 45

46 Fig. 8. 2D-PAGE of phosphorylated proteins of C. parasitica. A panel indicated the Coomassie blue stained gel and B panel shows the autoradiogram of the same PAGE gel. 46

47 Fig. 9. Results of MALDI-TOF analysis of one of CpPK1-dependent phosphorylated protein spot followed by the homology search using Mascot. 47

48 Fig. 10. Amino acid sequence alignment of predicted gene product with other homologous genes from filamentous fungi. Identical amino acids are highlighted in white on the black background. The homology (percent identity) of the C. parasitica gene in relation to other genes is given in parenthesis at the end of each sequence. The numbering of the residues from the N-terminus of the whole protein is shown on the right. Cp_Eno, Af_Eno, Ao_Eno, Nc_Eno, and Pc_Eno are the enolases from C. parasitica, A. fumigatus, A. oryzae, N. crassa, and P. citrinum, respectively. 48

49 Fig. 11. Nucleotide and deduced amino acid sequence of interacting gene from C. parasitica. The intron and 5'- and 3'-flanking regions are shown in small letters. The 7-bp conserved element is underlined and the transcription initiation site shown by a bold letter. The putative poly (A) signal is indicated in the box. 49

50 Fig.12. Matched the peptide sequence with Bck1 like MEKK of P. anserina were indicated by red color. Using these peptide sequence, degenerate primers were generated. 50

51 Fig.13. As a result of PCR amplification with two degenerate primers, 400bp DNA fragment was cloned. DNA sequence of this PCR amplicon was identified as a Bck1 like MEKK of P. anserina 51

52 Fig.14. The schematic diagram of the predicted cplc1 gene product. The conserved X, Y, and C2 domains are indicated in the boxes. The unique amino acid extension between the 2nd b-strand and the a-helix of the TIM-barrel is indicated by a bold line. Numbering of the residues from the N-terminus of the whole protein is shown at the top. 52

53 -1108 gtcgactgcttgcccaattggccgaggctttcttccgctccgcgacgccgggttcagctccc gatgcatctgaagatgctgttgtgctggctgaggatcttcttatagtcccacagagatgcgacggaaggagg -972 gacagctgcacccagagcccaaatacaaatgtcaaattggaaactgtgctataacgctgacctgagccttca -899 cctaaagtagactgaataggcgaacaaaacaccactctagcacgtgtaagccgcgggtcgtcgatttgttgt -827 tcttctttcctgaaccatcggtttgcttccatctcgttcagggtccaaccccccattatccgtccagaccct -755 ttaattcttgcggaggcagctaaaacgaaggcccctaattgagggctacgtgggcagccatattattaagat -683 gggccacccgttctagccgggtccagcccctgaaacggcccgcaaatctggcccgagagagagagagaaagg -648 gaaggaaggcctcgaagctggcgacctgctaggtgacgtcttagggtcctcggttgctgcttctccctctct -576 attttgggtcgggcgacggcaagtgaggatgaggtcgaaaccaaggaagccagagcgattatcaagtcttca -504 ggatgatgtgtacatgagccatcctgccttgattttcttctttactgcataaaacacatgtcattcccttac -432 tcgcccagtcacacggtgacacgcgagcggtgtctcgcgtatccatttgtcctctccaaagctcgagctcgt -360 ggtgggggaaaaaggcccttgtccgttacgacacaagtctggccgggaagaatacccactttcagggctcga -288 cttccactcttgtatcaaggcgaagctgggcctccaaacctattttcctggagatcgccaggcggtgaggtg -216 gacgtggtgtcaaaattgatggactttggttgagccgagtatgtggcgcgattgcacacgtactcactcact -144 tactcgttgtctagcatagagtttcttcttcccaccgccgtcgaagccctccttttatttggaataaacaga - 71 ttcccacatacatcgtaacatctcctcccccgtccaggacagagtggtgcaaacactcctacaacagtcgtc 1 ATGGCGGCGGCTGCACCACAAGAGCAGCAGCAGCAGCAGCAGAAGGAGGAGGATGGAGAACTCCCTCCGCCC 1 MMMAAAAAAAAAAAAPPPQQQEEEQQQQQQQQQQQQQQQQQQKKKEEEEEEDDDGGGEEELLLPPPPPPPPP 0073 AGTGAGGCCATCACCCAGGCCGGTGGTGGCGTCTCCGGCGCGGGTCCCGCCCGCAGCATCACCACTGTCTCC 0025 SSSEEEAAAIIITTTQQQAAAGGGGGGGGGVVVSSSGGGAAAGGGPPPAAARRRSSSIIITTTTTTVVVSSS 0145 GCTGCTGTGCTCCCATACCTCGAGAAAATCTTCAACTGTCATGCCGACACCAGCCAGGCCTGGCACCGTGGC 0049 AAAAAAVVVLLLPPPYYYLLLEEEKKKIIIFFFNNNCCCHHHAAADDDTTTSSSQQQAAAWWWHHHRRRGGG 0217 CAGGCAGAGACCTTCATCCGATGCACTCAGGCTGGCGACCCGAATGGCAGCGCCGAGGGCCTCCCTGCCGAC 0073 QQQAAAEEETTTFFFIIIRRRCCCTTTQQQAAAGGGDDDPPPNNNGGGSSSAAAEEEGGGLLLPPPAAADDD 0289 CTGGCTACCAAGGACGAGCTGGACTTCAATGATTTTCTGCGCTACATGACCAGTGATGCTACCAGCGCCGTC 0 97 LLLAAATTTKKKDDDEEELLLDDDFFFNNNDDDFFFLLLRRRYYYMMMTTTSSSDDDAAATTTSSSAAAVVV 0361 ACACCCCTGAATGCACAAACGGGTGGGGACCTGTCCTATCCGCTGAGCAGCTACTTCATCTCCAGCAGCCAC 0121 TTTPPPLLLNNNAAAQQQTTTGGGGGGDDDLLLSSSYYYPPPLLLSSSSSSYYYFFFIIISSSSSSSSSHHH 0433 AACACGTACCTCACGGGAAACCAGCTCAGCAGTGATGCCAGCACGGACGCGTACAAGAACGTCCTGCTACGC 0145 NNNTTTYYYLLLTTTGGGNNNQQQLLLSSSSSSDDDAAASSSTTTDDDAAAYYYKKKNNNVVVLLLLLLRRR 0505 GGGTGTAGATGCATCGAGATTGATGTCTGGGATGGGGACGAGTCAGACTCAGAGGCCAGCGGTTACTCGTCG 0169 GGGCCCRRRCCCIIIEEEIIIDDDVVVWWWDDDGGGDDDEEESSSDDDSSSEEEAAASSSGGGYYYSSSSSS 0577 AGTGACCTCGAGGATCATGACCCCAAGAAGGCGGCTGCAGCTCGCGCAAAGAGGAAGGCAAAGGTCGACAAG 0193 SSSDDDLLLEEEDDDHHHDDDPPPKKKKKKAAAAAAAAAAAARRRAAAKKKRRRKKKAAAKKKVVVDDDKKK 0649 GCCAAGGCCAAGATTCCCAAGTCCGTGCTGCAGAAGCTTGAGCAGACATCGCTCGGCAAGAAGCTCGAGAAG 0217 AAAKKKAAAKKKIIIPPPKKKSSSVVVLLLQQQKKKLLLEEEQQQTTTSSSLLLGGGKKKKKKLLLEEEKKK 0721 TATGTCGAGAAGAAGACGGAGCCCAAGGCGCCCGCCTCGCCGTCTACCTCGTCAGCAGCAGCAGCAGCAGCA 0241 YYYVVVEEEKKKKKKTTTEEEPPPKKKAAAPPPAAASSSPPPSSSTTTSSSSSSAAAAAAAAAAAAAAAAAA 0793 GCGCCAGCGCCAGCACCCGCAGCGAAGGATGATTCGAAAGTGAAAGTCGCCAGCCAGGCCTCCTCCTCTCCT 0265 AAAPPPAAAPPPAAAPPPAAAAAAKKKDDDDDDSSSKKKVVVKKKVVVAAASSSQQQAAASSSSSSSSSPPP 0865 GCGCCGCCGTTGGTGGAGAAGCTCCCGGCCCTCGCGGCGGCTGTTATCGAACCCCGTGTACTACATGGTTAC 0289 AAAPPPPPPLLLVVVEEEKKKLLLPPPAAALLLAAAAAAAAAVVVIIIEEEPPPRRRVVVLLLHHHGGGYYY 1937 ACGCTAACCAAGGAGGTCTCCTTCCGCGAGGTGTGTTTTGCTATCAAGGAGTACGCCTTTTCCGTGACGGAT 0313 TTTLLLTTTKKKEEEVVVSSSFFFRRREEEVVVCCCLLLAAAIIIKKKEEEYYYAAAFFFAAAVVVTTTDDD 1009 CTTCCGCTCATCGTGAGTCTCGAGGTGCATGCAGGGCCTGAACAGCAGGAGATCATGGTCAAGATCATGAAC 0337 LLLPPPLLLIIIVVVSSSLLLEEEVVVHHHAAAGGGPPPEEEQQQQQQEEEIIIMMMVVVKKKIIIMMMNNN 1081 GAGACCTGGGCCGGCCTCCTACTCGATCCGCCTGAGAAGGAGGCCGACGTACTCCCGTCGCCTAGCGACCTG 0361 EEETTTWWWAAAGGGLLLLLLLLLDDDPPPPPPEEEKKKEEEAAADDDVVVLLLPPPSSSPPPSSSDDDLLL 1153 CGTCGCAAAATTCTCGTCAAGGTCAAGTACGCGCCCCCTGGCCAGGAGGTCTCTGCGGCCGCCGCATCCGAC 0385 RRRRRRKKKIIILLLVVVKKKVVVKKKYYYAAAPPPPPPGGGQQQEEEVVVSSSAAAAAAAAAAAASSSDDD 1225 GAGGATGTCTCTACGCCGGGCCAAGTCGCTGCCCCTGGGTCTCCAGAGGCCAAGAAGAAGAAGAAGCCCTCC 0409 EEEDDDVVVSSSTTTPPPGGGQQQVVVAAAAAAPPPGGGSSSPPPEEEAAAKKKKKKKKKKKKKKKPPPSSS 1297 AAGATCATTCATGCTCTGAGTGCCCTGGGCATCTACACCAAGGCCGTTTCGTTCAAGTCGCTTCACCAACCG 0433 KKKIIIIIIHHHAAALLLSSSAAALLLGGGIIIYYYTTTKKKAAAVVVSSSFFFKKKSSSLLLHHHQQQPPP 1369 GAAGCCACTATGCCGTCACACGTCTTTTCCCTGGGCGAGAAGAGCGTCAGAGAGGTGCACGAGAAGCAGGGT 0457 EEEAAATTTMMMPPPSSSHHHVVVFFFSSSLLLGGGEEEKKKSSSVVVRRREEEVVVHHHEEEKKKQQQGGG 1441 CAGGACCTGTTTGAGCACAACCGAAAATATCTCATGCGGGCGTACCCATCTGGCTTCAGAGTTGGCTCGTCC 0481 QQQDDDLLLFFFEEEHHHNNNRRRKKKYYYLLLMMMRRRAAAYYYPPPSSSGGGFFFRRRVVVGGGSSSSSS 1513 AATCTGGACCCTACGCCGTTCTGGAGGAAGGGGATCCAGATTGCCGCGTTGAACTGGCAGCACTGGGATGAG 0505 NNNLLLDDDPPPTTTPPPFFFWWWRRRKKKGGGIIIQQQIIIAAAAAALLLNNNWWWQQQHHHWWWDDDEEE 1585 GGCATGATgtgagtgggaagcaaaagcatccagaagcgctggcctgattataattgctgacccattgtccct 529 GGGMMMMM 1657 acccatacccaccatacaggctcaacgagggcatgtttgccggctcaggcggttacgttctcaagcctcaag 0532 LLLLLLLLLLLLLLLLLLLLLLLNNNEEEGGGMMMFFFAAAGGGSSSGGGGGGYYYVVVLLLKKKPPPPPPG 1729 GCTACCGACACGACAAGCCTAACGCGCCCGCCGAGACCGCCCACGTGCAGCACCGCACGCTGGACCTCGAGA 0549 GGYYYRRRHHHDDDKKKPPPNNNAAAPPPAAAEEETTTAAAHHHVVVQQQHHHRRRTTTLLLDDDLLLEEEI 1801 TCAAGGTTCTCGCCGCTCAGAACCTGCCGCTCCCGCCCGAGACCTCGGTCAAGTCGTTTAACCCTTACGTCA 0573 LLKKKVVVLLLAAAAAAQQQNNNLLLPPPLLLPPPPPPEEETTTSSSVVVKKKSSSFFFNNNPPPYYYVVVK 1873 AGGTCGAGCTTCACGTCGAAGCCGGTGGGGAGCGCCACGGCCACTTCCCCGCCGACGGTCACGAGTGTGACC 0597 KKVVVEEELLLHHHVVVEEEAAAGGGGGGEEERRRHHHGGGHHHFFFPPPAAADDDGGGHHHEEECCCDDDL 1945 TGGCCACCGCGGCCGCTGCCAAGGCCGCAGCCGCCAGGCGCAGCGAGGACGATGAGGAGGTCGAGGGCGAGT 0621 LLAAATTTAAAAAAAAAAAAKKKAAAAAAAAAAAARRRRRRSSSEEEDDDDDDEEEEEEVVVEEEGGGEEEY 53

54 2017 ACAAGGCGCGCACCAAGACCGCCAAGGCCACGTGCGATCCGGACTATAAGGGCGAAATACTCGAGTTCAAGG 0645 YYKKKAAARRRTTTKKKTTTAAAKKKAAATTTCCCDDDPPPDDDYYYKKKGGGEEEIIILLLEEEFFFKKKA 2089 CCATCCCAGGCGTCGTCGAGGAACTGAGCTTCGTGCGCTTCATCGTGCGCGACGACGTCAAGTTTAGGCGGG 0669 AAIIIPPPGGGVVVVVVEEEEEELLLSSSFFFVVVRRRFFFIIIVVVRRRDDDDDDVVVKKKFFFRRRRRRD 2161 ATGATCTCGCTGCATGGGCGTGTGTACGCCTGGACCGGCTGCGGATGGGGTACCGCTTTGTGAGACTTATGG 0693 DDDDDLLLAAAAAAWWWAAACCCVVVRRRLLLDDDRRRLLLRRRMMMGGGYYYRRRFFFVVVRRRLLLMMMD 2233 ATGCCAAGGGGAAGGAGAGTGAGGGGGCTATTCTTGTCAAGGTCATGAAGAAGGTCTATTAGttcgggttga 717 DDAAAKKKGGGKKKEEESSSEEEGGGAAAIIILLLVVVKKKVVVMMMKKKKKKVVVYYY*** 2305 ttgggttgcggaagttttgtgtaaggggagacacacagagagagaatggagggaggggtggggatatttagc 2377 actttcgtggctgcgtggcttggtacagagccagtcagtagtatcctttccaacaggtttggggtaacataa 2451 aaatcaaagagagacagaaaggttcttatgattctatactctgtttttatggcccattgcatgctagtgctc 2523 tgtacagtcttgaacgcaacaagtttgaatttcattctccatgtactcccctgggccatgatctgtctaatc 2595 gctgaccccttaaaaaaagcatgtcaaagttccataaccatctcgcactataaatcgctgagagctcttccg 2667 gctgtttgtatgtgggagcccttaaaaatcccatttgcctggcatatcaacgccaaactccattcatccctt 2739 ctgaaaatgacaat Fig. 15. Nucleotide and deduced amino acid sequence of the cpcl1 of C. parasitica. The intron and 5'- and 3'-flanking regions are shown in small letters. The regions used to design degenerate PCR primers are indicated in the boxes. The amino acid residues of the X domain, Y domain, and C2 domain are underlined, double-underlined, and double-wavylined, respectively. The unique 133 amino acid extension (from the conserved Asp 176 to His 310) is indicated by a dotted lined. The transcription initiation site is shown by a bold letter, and the conserved hallmark residues for the binding of InsP 3 and calcium in the catalytic domain are highlighted. The putative poly (A) signal is underlined. The GenBank accession number for the cpcl1 sequence is AY

55 Fig 16. Restriction and Southern blot analyses of the cplc1-null mutant (TdPLC7) and the wild-type EP155/2. (A) Restriction map of the cplc1 genomic region and the gene-replacement vector pdplc1, which contains 616-bp and 716-bp fragments from the 5'- and 3'-flanking regions, respectively. The arrows show the direction of transcription. The box and the line indicate regions in which the sequences have or have not been determined, respectively. B, BamHI E, EcoRI N, NotI P, PstI S, SalI ScI, SacI ScII, SacII. (B) Southern blot analysis of the wild-type EP155/2 strain (lane 1) and the cplc1-replaced transformant TdPLC7 (lane 2). All of the DNA samples were digested with BamHI. The blots were probed with the 0.6-kb SacII/SacI fragment (probe1) and the 0.8-kb EcoRI/BamHI-hph fragment (probe2). The TdPLC7 transformant has undergone the desired replacement at cplc1, as evidenced by the changes in size of the fragments that hybridize with probe 1, and the lack of hybridization with probe 2. The probes are indicated in the restriction map of the cplc1 gene in the upper panel (A). 55

56 Fig.17. Colony morphologies on PDAmb. The colony morphology after 14 days of cultivation is shown. The strains used, indicated above the panel, were the virus-free wild-type (EP155/2), its isogenic virus-containing hypovirulent strain (UEP1), the cplc1-null mutant (TdPLC7), and the cplc1-complemented strain (TcPLC1).Colony morphology on PDAmb. 56

57 Fig.18. Colony morphology under the hyperosmotic conditions. The numbers on the top refer to molar concentrations of sorbitol. The numbers I, II, and III at left indicate the wild-type (EP155/2), the osmosensitive cpmk1-null mutant (TdMK1-23), and the cpcl1-null mutant (TdPLC7). 57

58 Fig. 19. Northern blot analysis of lac1 using RNA samples from strains that were complemented with the yeast PLC1 gene. The numbers 1, 2, 3, and 4 indicate the EP155/2, UEP1, cplc1-null mutant (TdPLC7), and yeast PLC1-complemented strains, respectively. RNA was prepared from mycelia that were grown on PDAmb plates, and equal loading of RNA samples is shown in the bottom panel with a parallel blot that was hybridized with the Gpd probe as an internal control. 58

59 Fig. 20. Molecular characteristics of the cplc1-null mutant. Northern blot analyses of lac1 using RNA samples from liquid cultures (A) and CHX-induced plates (B). The identity of each strain is given above the line, and the numbers indicate days after inoculation (A) and hours after transfer to the CHX-containing medium (B). Equal loading of RNA samples is shown in the bottom panel, along with a parallel blot that was hybridized with the Gpd probe as an internal control 59

60 10 Radial growth (diameter, cm Days after inoculation Fig. 4. Radial growth rate on PDAmb at 25 o C. The square, triangle, and diamond indicate the EP155/2, UEP1, and cplc1-null mutant (TdPLC7) strains, respectively. Open and closed marks indicated the radial growth with and without Ca2+ supplementation, respectively. Error bars represent the standard deviation of three replicates in three independent experiments. Of the three different Ca2+ concentrations tested, representative experiments using 2.0 mm Ca2+ supplementation are shown. 60

61 Fig. 21. Bavendamm test. Colonies were grown on tannic acid-containing medium, as previously described (Rigling et al., 1989). The level of brown coloration correlated with the laccase activity of each strain. 61

62 Fig. 22. Vegetative compatibility test 62

63 A B C Fig. 23. Four types of ds-rna infection of Cryphonectria parasitica. A: 12 kb and 12 kb kb, B: 3.0 kb, C: 3.0 kb kb kb. 63

64 Fig. 24. Construction of hypovirulent strain by hyphal fusion. A: virulent, B: hypovirulent, C: hypovirulent isolate (arrow) converted from virulent 64

65 Fig. 25. Agarose gel electrophoresis of PCR products of 4 Korean Cryphonectria parasitica isolates digested with Alu Ⅰ(A) and CfoⅠ (B). Lane 1: 1 kb DNA ladder, Lane 2: positive control (CHV1) isolates, Lanes 3-6: Korean isolates. 65

66 Fig. 26. Agarose gel electrophoresis of PCR products of 5' non-coding region (A) and ORF B (B) of Korean isolates of Cryphonectria parasitica. Lane 1: 1 kb DNA ladder, Lane 2: positive control (CHV1) isolates, Lanes 3-4: Korean isolates 66

67 Fig. 27. Relationships among Korean isolates and European isolate (EP713) of Cryphonectria parasitica in sequences of 5 non-coding region. Horizontal distance indicates the degree of relatedness. 67

68 Fig. 28. Relationships among Korean isolates and European isolate (EP713) of Cryphonectria parasitica in sequences of ORF B. Horizontal distance indicates the degree of relatedness. 68

69 Fig. 29. dsrna forms isolated from Korean type Cryphonectria parasitica 69

70 A B C Fig. 31. Construction of hypovirulence strain by hyphal fusion. A, Virulent strain; B, Hyphal fused strain; C, Non virulent strain 70

71 Fig. 32. Phenol oxidase activity of hypovirulent strains that constructed from the virulent strain and hypovirulent strain by hyphal fusion. Upper, Virulent strain, Lower, Hypovirulent strain, Left and Right, Hyphal fused strain 71

72 Fig. 33. dsrna forms isolated from hyphal fused strains. M, DNA ladder; C:EP43 (CHV1); V, virulent strain; T, dsrns transferred strain; HV: Hypovirulental parent strain 72