Studies on viroid-specific RNA silencing in relation to viroid pathogenicity and molecular evolution

类病毒是小的环状单链非编码rna，但一旦被感染到寄主植物细胞中，就会根据寄主的转录机制自主复制，并在多种作物上引起严重到轻度的疾病。为了研究防止植物感染病毒的新策略，我们分析了病毒诱导的RNA沉默与类病毒致病性和分子进化的关系。首先，利用下一代测序器对pstvd特异性小rna （srPSTVd）的生物发生进行大规模测序分析，发现来自基因组链的srPSTVd生物发生优于来自抗基因组链的srPSTVd生物发生，在Rutgers番茄中为9:1。是一个高度敏感的品种，但在摇钱树番茄中比例几乎为1:1。耐受性强的品种。在这两个品种中，srPSTVds一般来自两条链的整个分子，但也有一些特定的区域。热点。产生更丰富的srPSTVd。在基因组链上，两个品种都表现出相似的热点模式，而在抗基因组链上则表现出品种特异性的热点模式，即Moneymaker表现出更多的可变热点。结果表明，srPSTVd的生物发生具有寄主/品种依赖性。我们还比较了不同致病性的PSTVd变异，即中级（重度）和大丽花（轻度）菌株之间的热点模式，发现这两种菌株在罗格斯番茄的叶片和茎中都表现出相似的热点模式，并且根据个体热点的不同，热点的丰度不同。为了确定导致类病毒特异性疾病症状（如矮化和/或卷曲叶）的宿主基因，我们对感染或不感染PSTVd的番茄品种（Rutgers和Moneymaker）的基因表达进行了微阵列分析。与大多数已知植物激素（包括赤霉素和生长素）的生物合成相关的基因的表达或多或少地被特定疾病症状的发展所改变。其中，我们调查了含有一段与PSTVd同源的21个核苷酸序列的基因，发现编码赤霉素β-羟化酶的基因含有PSTVd特异性的21个核苷酸序列，并被PSTVd感染下调，表明该基因可能是PSTVd诱导的RNA沉默的假设靶标。一些番茄microrna如miR159也改变了它们的表达水平。我们已经创建了转基因番茄和烟草benthemiana系，它们组成性地表达发夹RNA，发夹RNA来源于PSTVd的各个区域或与RNA沉默相关的宿主基因，即DCL2和RDR6。目前，表达近全长发夹PSTVd （PSTVd-ΔTL）和srPSTVd热点之一（srPSTVd-257a）的转基因benthamiana T2株系成功表现出对PSTVd感染的抗性。为了研究类病毒与RNA沉默相关的分子进化，我们用从啤酒花、葡萄、李子和柑橘四种寄主物种中分离出来的天然类病毒（HpSVd）感染啤酒花。将这些植物保存15年，然后分析存在的HpSVd变体。结果，最初在栽培葡萄中发现的变异在25、26、54、193和281位点产生了各种突变组合，经过长时间的感染，这些变异经历了趋同进化，产生了有限数量的适应突变体。进一步分析表明，这五种突变对突变体在原始寄主（葡萄藤）、适应寄主（啤酒花）和第三寄主（黄瓜）中复制都没有好处。然而，该突变体在感染的啤酒花和黄瓜中积累的srHpSVd丰度较低，特别是含有5个突变的srHpSVd reads与总srHpSVd reads的相对比例显著下降，这表明该适应突变体通过产生5个突变成功地逃脱了类病毒诱导的RNA沉默。少

Viroids are small circular, single-stranded, non-coding RNAs, but, once infected into host plant cells, replicate autonomously depending on the host transcription machinery and caused severe to mild diseases on varieties of crop plants. To investigate the novel strategy to prevent plants from viorid infection, we have analyzed viroid-induced RNA silencing in relation to viroid pathogenicity and molecular evolution. First, by large-scale sequencing analysis using next generation sequencer on the biogenesis of PSTVd-specific small RNAs (srPSTVd), it was found that those derived from genomic strand was superior than those from anti-genomic strand, i.e., 9:1, in case of Rutgers tomato . a highly sensitive cultivar, but the ratio was almost 1:1 in Moneymaker tomato . a tolerant cultivar. In both cultivars, srPSTVds were derived in general from whole the molecule of both strands, but some specific regions . hotspots . produced more abundant srPSTVd. In the genomic strand, both cultivars show … More ed the similar hotspot pattern, however, the cultivar-specific pattern was observed in the anti-genomic strand, i.e., Moneymaker showed more variable hotspots. The results indicated that the srPSTVd biogenesis is host/cultivar-dependent. We have also compared the hotspot patterns among PSTVd variants with different pathogenicity, i.e., the Intermediate (severe) and the Dahlia (mild) strains, and found that both of the strains showed the similar hotspot pattern in Rutgers tomato in leaves and stem with distinct abundance depending on the individual hotspots.To identify the host genes responsible to exhibit viroid-specific disease symptoms such as dwarf and/or leaf curling, we have then performed microarray analysis of gene expression in tomato cultivars (Rutgers and Moneymaker) with or without infecting PSTVd. Expression of the genes related to biosynthesis of most of the known plant hormones including gibberellin and auxin have more or less been altered by development of specific disease symptoms. Among them, we have surveyed genes containing a stretch of 21-nucleotide sequence homologous to PSTVd, and found that the gene encoding gibberellin β-hydroxylase contains a PSTVd-specific 21-nucleotide sequence and was down-regulated by PSTVd infection, suggesting that the gene could be a hypothetical target by PSTVd-induced RNA silencing. Several of the tomato microRNAs such as miR159 have also changed their expression levels.We have created transgenic tomato and Nicotiana benthemiana lines constitutively expressing hairpin RNA derived from various regions of PSTVd or from host genes relating to RNA silencing, i.e., DCL2 and RDR6. At present, transgenic N. benthamiana T2 lines expressing near full-length hairpin PSTVd (PSTVd-ΔTL) and one of the srPSTVd hotspots (srPSTVd-257a) derived from lower central region of anti-genomic strand successfully exhibited resistance to PSTVd infection.To investigate molecular evolution of viroid in relation to RNA silencing, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus). These plants were maintained for 15 years, then analyzed the HpSVd variants present. As the result, the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281, and upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Further analysis revealed that these five mutations did not give a merit upon the mutant to replicate either in the original host (grapevine), the adapted host (hops), and the third host (cucumber). However, the mutant accumulated less abundant srHpSVd in infected hops and cucumber, and especially the relative ratio of srHpSVd reads containing the five mutations per total srHpSVd reads were significantly downed, suggesting that the adapted mutant successfully escaped from viroid-induced RNA silencing by giving rise to the five mutations. Less