The experimental verification of viroid-host adaptation

所有病原体都有宿主特异性。类病毒是已知最小的病原体，是分析RNA分子中哪一部分负责宿主特异性的绝佳模型。类啤酒花病毒（HSVd）在各种作物中广泛传播，并根据寄主种类发现其独特的序列变异。为了实验验证独特的变异是如何适应宿主的，(1)我们构建了由自然界中发现的各种突变组成的随机HSVd变异，将它们接种到几个宿主上，并分析它们如何适应每个宿主，(2)我们用天然HSVd分离物感染啤酒花，培养它们10年，并检查后代序列以分析每个分离物如何适应啤酒花。首先，利用随机cDNA洗牌，我们构建了随机hsv -变异。分别接种于黄瓜、葡萄藤、桃、柑橘、啤酒花；宿主为hsv和拟南螺旋体；HSVd的非宿主。HSVd在黄瓜、葡萄、桃、柑橘和啤酒花中积累，而在拟南芥中没有。对各种繁殖后代的核苷酸序列分析表明，只有hsvd -葡萄藤型能适应葡萄，而在其他寄主中，hsvd -葡萄藤与-柑橘的嵌合体以及hsvd -葡萄藤型均占优势。在啤酒花和桃花侵染早期发现了hsv -李子的变异特征，但在侵染后期大部分变异特征消失。二是用葡萄、啤酒花、李子和柑橘四种天然分离株感染啤酒花，培养10年。每年秋季提取所有分离株进行测序，分析序列变异率。持续侵染10年后，hsvd -柑橘和李子的变异率最高，而HSVd-hop和葡萄藤的变异率显著低于HSVd-hop。结果表明，4个菌株的跳适度不同。

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.