The experimental verification of viroid-host adaptation

所有病原体都具有宿主特异性。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好模型。啤酒花矮化类病毒（HSVd）广泛存在于各种作物中，不同的寄主种类其基因序列存在差异。为了通过实验验证独特的变异体如何适应各种宿主，（1）我们构建了由自然界中发现的各种突变组成的随机HSV d-变异体，将它们接种到几种宿主中，并分析它们如何适应每种宿主，（2）我们用天然HSV d分离物感染啤酒花，培养它们10年，首先，我们利用随机cDNA改组技术构建了随机的HSV d变异体。接种黄瓜、葡萄藤、桃、柑桔、啤酒花等植物，分别接种HSVd和A. thaliana; HSVd的非宿主。HSVd子代在黄瓜、葡萄藤、桃、柑橘和啤酒花中积累，而在A. thaliana.对每个种繁殖后代的核苷酸序列分析表明，只有HSVd-葡萄藤型适合于葡萄，但HSVd-葡萄藤和柑橘之间的几种嵌合体以及HSVd-葡萄藤型在其他宿主中占主导地位。在啤酒花和桃中，HSVd-李的变异在感染初期就已出现，但在感染后期大部分变异消失。其次，啤酒花被HSVd-葡萄、啤酒花、李和柑橘4个天然分离物感染，并进行了10年的栽培试验。每年秋季提取所有分离株进行测序，分析序列变异率。持续感染10年后的变化率是最高的HSVd柑橘和李，但显着较低的HSVd啤酒花和葡萄藤。结果表明，4个菌株对啤酒花的适合度不同。

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.