Studies on viroid inducing RNA silencing from the view of plant pathology and agricultural impact

在番茄子叶期接种马铃薯纺锤块茎类病毒（Potato spindle tuber viroid，PSTVd），接种后14 d，第3片叶出现卷叶现象。PSTVd达到了除分生组织之外的所有叶子的可检测值。PSTVd特异性siRNA在第2 ~ 9叶也达到可检测水平。PSTVd和siRNA在卷叶严重的叶片中积累最多，但在卷叶严重的叶片中，PSTVd浓度在第9天下降，上部叶片从卷叶中恢复。结果表明，PSTVd特异性的RNA沉默被触发后不久，PSTVd达到其可检测的水平，PSTVd特异性的siRNA迅速蔓延到整个植物中，除了分生组织。在诱导RNA沉默后分化的叶片将被恢复，因为它们从分化的初始阶段就被置于RNA沉默之下，这导致PSTVd积累的抑制。在啤酒花矮化病毒感染的黄瓜和啤酒花、啤酒花潜伏病毒感染的啤酒花和苹果果实皱缩病毒感染的啤酒花中检测到约21和23个核苷酸的病毒特异性siRNA。在番茄感染黄瓜花叶病毒后，仅检测到约21个核苷酸的短siRNA，利用BLAST技术在DNA数据库中检索，鉴定出几个18-20个核苷酸的番茄EST序列。对它们进行PCR扩增、克隆和测序。以克隆的cDNA为探针，分析了这些基因的表达情况，但未检测到任何阳性信号。从健康和类病毒感染的果实中获得的番茄幼苗同样受到PSTVd攻击接种的感染，这意味着类病毒诱导的RNA沉默在番茄植株中的垂直传递不明显。另一方面，在从恢复阶段的PSTVd感染的番茄植株无性繁殖的插条中保持恢复。

When tomato plants were infected with Potato spindle tuber viroid (PSTVd) at the cotyledon stage, primary leaf curling appeared on the 3rd leaf 14 days after inoculation. PSTVd reached the detectable all the leaves but for the meristem. PSTVd-specific siRNA also reached the detectable level in the 2nd-9th leaves. Accumulation of PSTVd and the siRNA increased to the highest level in the leaves showing severe leaf curling, however, PSTVd concentration decreased in the 9th and the upper leaves recovered from severe leaf curling. The results indicated that PSTVd-specific RNA silencing was triggered soon after PSTVd reached its detectable level, and PSTVd-specific siRNA spread quickly throughout the plant except for the meristem. The leaves being differentiated after induction of RNA silencing are to be recovered, since they are placed under the RNA silencing from the initial stage of the differentiation which resulted in the suppression of PSTVd accumulation.Viroid-specific siRNAs of ca.21 and 23 nucleotides were detected in Hop stunt viroid-infected cucumber and hop, Hop latent viroid-infected hop and Apple fruit crinkle viroid-infected hop. On the other hand, only short siRNA of ca.21 nucleotides was detectable, in tomato infected with Cucumber mosaic virus.Using in silica analysis by BLAST search in DNA data base, several tomato EST sequences of 18-20 nucleotides were identified. They were PCR amplified, cloned and sequenced. Using the cloned cDNAs as probes, the expression of the genes were analyzed, however, any positive signal was not identified.Tomato seedlings obtained from healthy and viroid-infected fruits were equally infected with PSTVd challenge inoculation, which means that vertical transmission of viroid-induced RNA silencing was not apparent in tomato plants. On the other hand, the recovery was maintained in cuttings vegetatively propagated from PSTVd-infected tomato plants of the recovered stages.