Study of DNA methylation changes upon PSTVd infection of N. benthamiana

Viroids are small circular RNAs infecting a wide range of plants. They do not encode for any protein or peptide therefore they rely in their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study global DNA methylation upon infection. The study indicated that although a small number of targets are differentially methylated upon PSTVd infection, the overall methylation status did not significantly change. These results enhance our understanding on the epigenetic host changes as a result of Pospiviroid infection.

类病毒（viroids）是一类侵染多种植物的小型环状RNA分子。它们不编码任何蛋白质或多肽，因此其生命周期完全依赖自身结构完成。目前认为，类病毒侵染植株所表现出的表观表型，是通过宿主转录/翻译水平的改变所引发的。介导此类改变的关键机制之一是RNA指导的DNA甲基化（RNA-directed DNA methylation, RdDM）。迄今为止，学界针对类病毒对RdDM的干扰作用所开展的研究结论尚存分歧。
本研究以本氏烟（Nicotiana benthamiana）为实验材料，探究类病毒侵染所引发的宿主表观遗传效应。本研究以马铃薯纺锤块茎类病毒（potato spindle tuber viroid, PSTVd）为侵染病原，通过生物信息学分析，筛选出在PSTVd侵染植株与健康植株中积累量存在差异的24 nt宿主小干扰RNA（small interfering RNA, siRNA）所靶向的内源基因启动子与转座元件。随后，研究人员通过甲基化敏感限制性内切酶联合PCR扩增技术，以及亚硫酸氢盐测序法，对上述靶向位点的甲基化状态进行了检测。此外，本研究还采用甲基化敏感扩增多态性（Methylation Sensitive Amplification Polymorphism, MSAP）结合测序技术（MSAP-seq），分析了侵染后宿主的全基因组DNA甲基化水平变化。
研究结果显示，尽管仅有少量靶向位点在PSTVd侵染后出现了甲基化水平的差异变化，但宿主整体的甲基化状态并未发生显著改变。本研究结果有助于加深学界对马铃薯纺锤块茎类病毒属（Pospiviroid）侵染所引发的宿主表观遗传改变的认知。