Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the “master” sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid’s accumulation by inducing the RNA silencing of the host’s defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid’s interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.

类病毒是非编码RNA植物病原体，其特征在于具有很高的突变水平。尽管已经很好地理解了类病毒感染植物中的序列异质性，但是由于感染过程中的突变导致的类病毒种群动态变化仍然知之甚少。在这项研究中，通过高通量测序鉴定了在感染PSTVd的番茄植株中在不同时间间隔表达的马铃薯纺锤块茎类病毒RG1（PSTVd）的十个最丰富的序列变异体。对形成准物种的序列变体进行识别有利突变的区域以及突变对类病毒二级结构和类病毒衍生小RNA（vd-sRNA）的影响。在PSTVd感染的第1周，有25％的序列变体与“主”序列相似（即，用于接种的顺序）。PSTVd感染后第2周，人群中主序列的频率增加至70％，然后在疾病周期的其余部分（即第3和第4周）稳定下来。虽然PSTVd感染后第1周有一些序列变异，但随着时间的推移，它们的频率趋于下降。例如，在复制处和类病毒感染过程中显示出重要作用的在253位或254位插入，可能影响环E以及亚稳发夹I结构的变体导致频率降低。数据来自 尽管PSTVd感染后第1周有一些序列变异，但随着时间的推移，它们的频率趋于下降。例如，在复制处和类病毒感染过程中显示出重要作用的在253位或254位插入，可能影响环E以及亚稳发夹I结构的变体导致频率降低。数据来自 虽然PSTVd感染后第1周有一些序列变异，但随着时间的推移，它们的频率趋于下降。例如，在复制处和类病毒感染过程中显示出重要作用的在253位或254位插入，可能影响环E以及亚稳发夹I结构的变体导致频率降低。数据来自在计算机上还分析了类病毒衍生的小RNA（vd-sRNA）的分析。一些突变体具有通过诱导宿主防御相关基因的RNA沉默而积极影响类病毒积累的潜力。还鉴定了具有可能对类病毒丰度产生负面影响的突变的变体，因为其衍生的vd-sRNA不再能够靶向任何宿主mRNA或将其靶序列从宿主防御基因更改为一些其他不重要的宿主基因。总之，这些发现为理解序列变体的生物学作用，该类病毒与宿主成分的相互作用，突变体在感染过程中产生的稳定和亚稳态结构以及序列变体对稳定类病毒种群动态的影响开辟了道路。