Structural and Functional Analysis of Linear Plasmid DNAs of Plant Pathogenic Fungus

1.在114株立枯丝核菌田间分离物中，有48株检测到类质粒DNA(PlDNA)。这48个分离物在日本立枯丝核菌的9个融合群(AG)和种内群(ISG)中的分布如下：AG-1中0个(Sasaki型)，AG-1中1个(网枯型)，AG-2-1中0个，AG-2-2中11个(冲突型)，AG-2-2中10个(根腐型)，AG-3中10个，AG-4中8个，AG-5中4个，AG-6中4个。每个分离物携带一个、两个或三个经凝胶电泳鉴定的plDNA。电子显微镜分析表明，这些plDNA均为线形分子。以nick翻译的plDNA为探针，通过Southern印迹分析比较了代表性菌株中plDNA之间的序列同源性。从相同的AG和ISG分离株中获得的plDNA之间有相当大的序列同源性。根据序列同源性将AG-2-2分离株中的plDNA分为两组。在立枯丝核菌(Rhizoctonia Solani)融合群4(AG-4)的RI-分离物中发现了3个线形DNA质粒。这些质粒命名为pRS64-1、-2和-3，大小相同，为2.7kb。限制性内切酶图谱和Southern杂交分析表明，pRS64-1、-2和-3在两个末端都有同源区域。即使用蛋白酶K或碱处理后，质粒DNA对3‘-核酸外切酶和5’-核酸外切酶都具有抗性。在变性条件下，限制性内切酶消化产生的两个末端片段的长度都增加了一倍，表明线形质粒DNA的两端都有发夹环。对总DNA的Southern blotting分析表明，pRS64 DNA存在两种二聚体形式。一种是头对头二聚体，另一种是尾对尾二聚体。这些独特的DNA结构在复制中的作用

1. Plasmid-like DNA (plDNA) was found in 48 out of 114 field isolates of Rhizoctonia solani. These 48 isolates were distributed as follows among the nine anastomosis groups (AG) and intraspecific groups (ISG) recognized among Japanese isolates of R. solani : 0 in AG-1 (sasakii type), 1 in AG-1 (web-blight type), 0 in AG-2-1, 11 in AG-2-2 (rush type), 10 in AG-2-2 (root rot type), 10 in AG-3, 8 in AG-4, 4 in AG-5 and 4 in AG-6. Each isolate carried one, two or three plDNAs identified by gel electrophoresis. Electron microscopic analysis revealed that all these plDNAs were linear molecules. The sequence homology among plDNAs found in representative isolates was examined by Southern blot analysis, using nick-translated plDANs as probes. Considerable sequence homology was observed among plDNAs obtained from isolates within the same AG and ISG. The plDNAs occurring in the isolates of AG-2-2 were classified into two groups on the basis of the sequence homology.2. Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization anlysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3'-exonuclease and 5'-exonuclease even after treatment with protease K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of th