Owens R A, Chen W, Hu Y, Hsu Y H
Plant Sciences Institute, United States Department of Agriculture/Agricultural Research Service, Beltsville, Maryland 20705, USA.
Virology. 1995 Apr 20;208(2):554-64. doi: 10.1006/viro.1995.1186.
Nucleotides within the pathogenicity domain of potato spindle tuber viroid (PSTVd) are known to play an important role in regulating symptom expression, but the underlying molecular mechanism is unknown. In order to determine more precisely how structural features within the pathogenicity domain regulate symptom expression, we have characterized a series of mutations that progressively stabilize premelting region 1 and the rest of the "virulence modulating" region. The structural effects of these mutations were monitored by temperature gradient gel electrophoresis of circularized RNA transcripts, and their biological effects were assessed by quantitative bioassays in tomato. Closure of a 4-nucleotide loop within the premelting region 1 virtually abolished PSTVd infectivity, especially when a nearby 2-nucleotide loop was also closed. Although RNA transcripts containing less stabilizing mutations were readily infectious, none of the four single and one double substitutions examined were stably maintained in vivo. The pattern of spontaneous, apparently compensatory sequence changes observed in the progeny suggests that PSTVd variants with less stable secondary structures enjoy a selective advantage. Mutations which stabilize the pathogenicity domain of PSTVd in vitro also suppressed symptom expression, but at least one other mutation having no obvious structural effects was associated with a similar phenotype. Conformational stability appears to be only one of several factors regulating PSTVd replication and pathogenicity.
已知马铃薯纺锤块茎类病毒（PSTVd）致病结构域内的核苷酸在调节症状表达中起重要作用，但其潜在分子机制尚不清楚。为了更精确地确定致病结构域内的结构特征如何调节症状表达，我们对一系列突变进行了表征，这些突变逐渐稳定了预熔区域1和“毒力调节”区域的其余部分。通过对环化RNA转录本进行温度梯度凝胶电泳监测这些突变的结构效应，并通过番茄中的定量生物测定评估其生物学效应。预熔区域1内一个4核苷酸环的封闭几乎消除了PSTVd的感染性，特别是当附近的一个2核苷酸环也被封闭时。尽管含有稳定性较低突变的RNA转录本易于感染，但所检测的四个单取代和一个双取代在体内均未稳定维持。在子代中观察到的自发的、明显的补偿性序列变化模式表明，具有较不稳定二级结构的PSTVd变体具有选择性优势。在体外稳定PSTVd致病结构域的突变也抑制了症状表达，但至少有一个没有明显结构效应的其他突变与类似表型相关。构象稳定性似乎只是调节PSTVd复制和致病性的几个因素之一。
