Liu Liming, Peng Bin, Zhang Zhenwei, Wu Yang, Miras Manuel, Aranda Miguel A, Gu Qinsheng
First, second, third, fourth, and seventh authors: Henan Provincial Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, Henan, China; and fifth and sixth authors: Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, Apdo. Correos 164, 30100 Espinardo, Murcia, Spain.
Phytopathology. 2017 Sep;107(9):1080-1086. doi: 10.1094/PHYTO-03-17-0107-R. Epub 2017 Jul 20.
Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus (family Virgaviridae) that causes serious economic losses in cucurbit crops. A possibility for CGMMV control is the use of cross-protection, for which stable attenuated isolates are required. In this study, an infectious clone was constructed for the hn isolate of CGMMV. Unexpectedly, this clone carried a nonconserved mutation involving a single nucleotide change resulting in the replacement of Arg by Cys at residue 284 of the replicase protein; this mutation correlated with delayed symptom induction and RNA accumulation, as shown in time-course experiments. Sequencing of the viral progeny showed that restoration of wild-type symptoms and increased RNA accumulation correlated with reversion of the mutation to the wild-type sequence, a phenomenon that occurred at approximately 7 to 10 days postinoculation. Thus, Arg284 seems to be crucial but not strictly necessary for virus infection. Subsequently, four other mutants in the triplet encoding Arg284 were constructed and assayed. Results showed that symptoms and their timing were diverse for the different mutants, with enhanced pathogenicity and RNA accumulation always correlating with reversion to Arg284. Therefore, the nature of the mutation strongly influenced the genetic stability of the mutant. At least two mutants were identified for which reversion did not occur by 30 days postinoculation, and these were defined as good candidates to attain stable symptom attenuation that could be useful in cross-protection.
黄瓜绿斑驳花叶病毒（CGMMV）是烟草花叶病毒属（病毒科）的成员，会给葫芦科作物造成严重的经济损失。控制CGMMV的一种可能性是利用交叉保护，为此需要稳定的减毒株。在本研究中，构建了CGMMV hn分离株的感染性克隆。出乎意料的是，该克隆携带了一个非保守突变，涉及单个核苷酸变化，导致复制酶蛋白第284位残基的精氨酸被半胱氨酸取代；如时间进程实验所示，这种突变与症状诱导延迟和RNA积累相关。病毒子代的测序表明，野生型症状的恢复和RNA积累的增加与突变回复到野生型序列相关，这种现象发生在接种后约7至10天。因此，精氨酸284似乎对病毒感染至关重要，但并非严格必需。随后，构建并检测了编码精氨酸284的三联体中的其他四个突变体。结果表明，不同突变体的症状及其出现时间各不相同，致病性增强和RNA积累总是与回复到精氨酸284相关。因此，突变的性质强烈影响突变体的遗传稳定性。鉴定出至少两个在接种后30天内未发生回复突变的突变体，这些被定义为实现稳定症状减弱的良好候选者，可用于交叉保护。
