Phytopathology. 2006 May;96(5):468-79. doi: 10.1094/PHYTO-96-0468.
ABSTRACT A 2.8-kb double-stranded RNA (dsRNA) element in strain BK18 of Chalara elegans originally isolated from cotton soil in California was characterized by obtaining a full-length cDNA sequence (2,896 nucleotides in length) from a series of overlapping clones. Sequence analysis revealed the presence of one large open reading frame (ORF I) using the mitochondrial genetic code, with 20 to 34% amino acid identity to the ORF I of other previously reported fungal mitochondrial RNA viruses. The ORF I encoded a putative protein of 705 amino acids and contained the conserved motif characteristic of RNA-dependent RNA polymerases. Purification of mitochondria from strain BK18 confirmed the co-localization of this dsRNA, and northern blot hybridization with a strand-specific probe revealed the (+) single-stranded nature. This Chalara elegans mitovirus (CeMV) is designated as a new member of the genus Mitovirus of the family Narnaviridae. Using dsRNA-specific primers, the ORF I region (positions 427 to 2544) was obtained from an additional 2.8-kb dsRNA element in strain HA2 originating from carrot roots in the Netherlands. Both ORFs had 98% homology at the nucleotide and amino acid levels. CeMV was also found to be present in five additional strains of C. elegans from different geographic locations worldwide, and a 97 to 100% nucleotide sequence identity was observed within a 300-bp region of ORF I in these strains. To determine the biological effects of CeMV on C. elegans, attempts to cure strain BK18 of the dsRNA were made. Sequential transfers of mycelium at 35 to 37 degrees C yielded a colony which lacked the 2.8-kb dsRNA when visualized on agarose gels and also in northern blot hybridization analysis. However, reverse transcription-polymerase chain reaction with specific primer sets revealed a band, indicating that dsRNA replication had been significantly repressed (latent). The wild type and latently infected strains were compared for colony morphology, growth rate, melanin production, various enzymatic assays (polyphenoloxidase, laccase, tyrosinase, and esterase), and virulence on carrot roots. Colony morphology on V8 agar was comparable between the two strains, while growth rate, melanin production, and virulence were enhanced in the latently infected strain. There were no detectable differences in enzymatic activity. Transmission electron microscopy of hyphae of the wild type and latently infected strains revealed differences in the number and size of the mitochondria, which were enhanced in the latently infected strain. Our results show that CeMV is a new member of the genus Mitovirus with some disruptive effects on its fungal host and is present in C. elegans strains from different locations worldwide.
摘要 从加利福尼亚棉土中分离出的旋孢腔菌菌株 BK18 的 2.8kb 双链 RNA（dsRNA）元件，通过一系列重叠克隆获得全长 cDNA 序列（长 2896 个核苷酸）进行了特征描述。序列分析表明，存在一个使用线粒体遗传密码的大型开放阅读框（ORF I），与其他先前报道的真菌线粒体 RNA 病毒的 ORF I 具有 20%至 34%的氨基酸同一性。ORF I 编码了一种推定的 705 个氨基酸的蛋白质，并包含 RNA 依赖性 RNA 聚合酶的特征保守基序。从 BK18 菌株中纯化线粒体证实了该 dsRNA 的共定位，并用链特异性探针进行的 northern blot 杂交显示了（+）单链性质。这种旋孢腔菌病毒（CeMV）被指定为 Narnaviridae 科 Mitovirus 属的新成员。使用 dsRNA 特异性引物，从源自荷兰胡萝卜根的菌株 HA2 的另一个 2.8kb dsRNA 元件中获得了 ORF I 区域（位置 427 至 2544）。两个 ORF 在核苷酸和氨基酸水平上均具有 98%的同源性。还在来自世界各地不同地理位置的另外五个旋孢腔菌菌株中发现了 CeMV，并且在这些菌株的 ORF I 的 300bp 区域内观察到 97%至 100%的核苷酸序列同一性。为了确定 CeMV 对旋孢腔菌的生物学影响，尝试消除 BK18 菌株中的 dsRNA。在 35 至 37 摄氏度下连续转移菌丝体产生了一个菌落，当在琼脂糖凝胶上和 northern blot 杂交分析中观察时，该菌落缺乏 2.8kb dsRNA。然而，用特异性引物对进行的逆转录聚合酶链反应显示出一个带，表明 dsRNA 复制受到显著抑制（潜伏）。比较了野生型和潜伏感染菌株的菌落形态、生长速度、黑色素产生、各种酶测定（多酚氧化酶、漆酶、酪氨酸酶和酯酶）以及对胡萝卜根的毒力。两种菌株在 V8 琼脂上的菌落形态相似，而潜伏感染菌株的生长速度、黑色素产生和毒力增强。酶活性没有可检测到的差异。野生型和潜伏感染菌株菌丝体的透射电子显微镜显示线粒体的数量和大小存在差异，潜伏感染菌株中的线粒体数量和大小增强。我们的结果表明，CeMV 是 Mitovirus 属的一个新成员，对其真菌宿主有一些破坏性影响，并且存在于来自世界各地不同位置的旋孢腔菌菌株中。
