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The experimental verification of viroid-host adaptation

类病毒-宿主适应的实验验证

基本信息

批准号：
12660038
负责人：
SANO Teruo
金额：
$ 2.24万
依托单位：
Hirosaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660038/
关键词：
viroid host adaptation DNA shutting random mutation dominant selection DNAシャッフリング宿主適応変異

项目摘要

All pathogen has host specificity. Viroid, the smallest known pathogen, is an excellent model to analyze which part of the RNA molecule is responsible for host specificity. Hop stunt viroid (HSVd) spreads widely among various crops, and the unique sequence variations are found depending on the host species. To verify experimentally how unique variants has adapted to the varieties of hosts, (1) we have constructed random HSVd-variants consisting of various mutations found in the nature, inoculated them to several hosts, and analyzed how they adapt to each hosts, and (2) we have infected hops with natural HSVd isolates, cultivated them for 10 years, and examined the progeny sequences to analyze how each isolates adapted to hop.The first, using random cDNA shuffling, we constructed random HSVd-variants. They were inoculated to cucumber, grapevine, peach, citrus, hop; the host for HSVd, and A. thaliana; non-host for HSVd. HSVd progeny accumulated in cucumber, grapevine, peach, citrus and hop, but no in A. thaliana. The nucleotide sequence analysis of progeny propagated in each species revealed that only HSVd-grapevine type was adaptable for grapevine but several types of chimeras between HSVd-grapevine and -citrus, as well as HSVd-grapevine type, were dominant in the other hosts. The variations characteristic for HSVd-plum were detected in hop and peach in the early stage of infection, but most of them disappeared in the later stage.The second, hops were infected with four natural HSVd-isolates (HSVd-grapevine, -hop, -plum and -citrus) and cultivated for 10 years. All isolates were extracted every autumn for sequencing to analyze the rate of sequence variation. The rate of variation after the 10 years of persistent infection was the highest in HSVd-citrus and -plum, but significantly lower in HSVd-hop and -grapevine. The result indicated that the fitness of the four isolates to hop was different.

所有病原菌都有寄主特异性。类病毒是已知最小的病原体，是分析RNA分子的哪一部分负责宿主特异性的极佳模型。啤酒花矮化类病毒广泛存在于多种作物中，其序列变异因寄主种类不同而异。为了从实验上验证独特的变异体如何适应寄主的品种，(1)我们构建了由自然界中发现的各种突变组成的随机HSVd变种，将它们接种到几个寄主上，并分析它们如何适应每个寄主；(2)我们用HSVd天然分离物感染啤酒花，培养了10年，并检查了后代序列，以分析每个分离物对HOP的适应情况。分别接种于黄瓜、葡萄、桃、柑橘、啤酒花；HSVd的寄主和拟南星；HSVd非寄主。黄瓜、葡萄、桃、柑橘和啤酒花中均有HSVd子代的积累，而拟南星中无此现象。对每种繁殖后代的核苷酸序列分析表明，只有HSVd-葡萄型适合于葡萄，而在其他寄主上，HSVd-葡萄型和HSVd-葡萄型之间的嵌合体以及HSVd-葡萄型占优势。啤酒花和桃的HSVd-plum在侵染初期即可检测到变异特征，但大部分在侵染后期消失。其次，用4个HSVd天然分离物(HSVd-葡萄、-HOP、-李和-柑橘)侵染啤酒花，并培养10年。每年秋季提取所有分离物进行测序，分析序列变异率。持续侵染10年后，柑橘和李树的变异率最高，葡萄和葡萄的变异率显著低于柑橘和李树。结果表明，4个菌株对啤酒花的适合度不同。