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，但是一旦感染到宿主植物细胞中，依赖于宿主转录机制自主复制，并在多种作物植物上引起严重到轻度的疾病。为了探索植物抗类病毒感染的新策略，我们分析了类病毒诱导的RNA沉默与类病毒致病性和分子进化的关系。首先，通过使用下一代测序仪对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那些优于来自反基因组链的那些，即，9：1，如果是罗格斯番茄。而Moneymaker番茄的比例接近1：1。一种耐药的品种。在两个品种中，srPSTVds一般都来自两条链的整个分子，但也有一些特定的区域。热点产生了更丰富的srPSTVd。在基因组链中，两个品种都显示 ...更多信息 艾德发现了类似的热点模式，然而，在反基因组链中观察到了培养物特异性模式，即，Moneymaker显示了更多的可变热点。结果表明，srPSTVd的生物发生是宿主/品种依赖的。我们还比较了具有不同致病性的PSTVd变体之间的热点模式，即，中间（严重）和大丽花（轻度）菌株，并发现这两种菌株在罗格斯番茄的叶和茎中显示出相似的热点模式，根据各个热点具有不同的丰度。然后，我们在有或没有感染PSTVd的番茄栽培品种（Rutgers和Moneymaker）中进行了基因表达的微阵列分析。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，我们研究了一段与PSTVd同源的21个核苷酸的基因，发现编码赤霉素β-羟化酶的基因含有PSTVd特异的21个核苷酸的序列，并且在PSTVd感染后表达下调，表明该基因可能是PSTVd诱导的RNA沉默的一个假想靶基因。我们已经建立了转基因番茄和本氏烟草品系，它们组成性地表达来自PSTVd的各个区域或来自与RNA沉默相关的宿主基因的发夹RNA，即，DCL2和RDR6。目前，转基因N.表达近全长发夹PSTVd的本塞姆氏T2系为了研究类病毒RNA沉默与类病毒分子进化的关系，我们用来自啤酒花、葡萄、李和柑橘4种植物的天然啤酒花矮化类病毒（HpSVd）感染啤酒花，研究了类病毒RNA沉默与类病毒进化的关系。将这些植物保持15年，然后分析存在的HpSVd变体。结果，最初在栽培葡萄藤中发现的变体在位置25、26、54、193和281处产生了各种突变组合，并且在长时间感染后，这些变体经历了趋同进化，导致有限数量的适应突变体。进一步的分析表明，这五个突变并没有给一个优点后，突变体复制无论是在原始主机（葡萄），适应主机（啤酒花），和第三个主机（黄瓜）。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读段/总srHpSVd读段的相对比率显著下降，表明适应的突变体通过产生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