Tan Ying, Bukys Alfredas, Molnár Attila, Hudson Andrew
Institute of Molecular Plant Sciences, University of Edinburgh, Max Born Crescent, Edinburgh, EH9 3BF UK.
College of Life Sciences, Hunan Normal University, 136 Lushan Road, Changsha, 410006 China.
Plant Methods. 2020 Oct 27;16:145. doi: 10.1186/s13007-020-00683-5. eCollection 2020.
(snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in and VIGS in and its relative
proved highly susceptible to systemic TRV infection. TRV carrying part of the () gene triggered efficient silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing and the trichome-suppressing () gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the mutant phenotype was also tested. TRV carrying the native coding sequence with to report infection failed to complement mutations and triggered VIGS of in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the mutant phenotype when expressed without a VIGS reporter.
We demonstrate an effective method for VIGS in the model genus and its relative that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.
金鱼草属物种是遗传和进化研究的模式植物，但对遗传转化具有抗性，限制了转基因方法在功能基因组学中的应用。病毒载体介导的瞬时基因表达和病毒诱导的基因沉默（VIGS）提供了无需转化的替代方法。在此，我们研究烟草脆裂病毒（TRV）在金鱼草属及其近缘物种中进行同源基因表达和VIGS的效用。
结果表明金鱼草对系统性TRV感染高度敏感。携带部分金鱼草（Am）基因的TRV引发了有效的Am沉默，表现为组织漂白，为VIGS的程度和位置提供了报告基因。VIGS最常起始于幼苗期，持续到花序和花朵，并且不受TRV基因组内同源序列方向的显著影响。通过降低在幼叶原基的原表皮或发育中的花朵中起作用的两个发育调节因子的表达，进一步证明了其效用。来自同一TRV基因组的Am和抑制毛状体形成的GLABRA（GL）基因共沉默的效果表明，组织漂白为在不同细胞层中起作用的第二个靶基因的VIGS提供了有用的标记。还测试了TRV编码的H蛋白互补Am突变体表型的能力。携带天然Am编码序列并带有CP以报告感染的TRV未能互补Am突变，并且在野生型植物中引发了Am的VIGS。然而，编码H蛋白的具有43%同义替换的序列在没有CP VIGS报告基因的情况下表达时能够互补Am突变体表型。
我们展示了一种在模式属金鱼草及其近缘物种中进行VIGS的有效方法，该方法在营养组织和生殖组织中均有效。我们还表明TRV可用于互补金鱼草中的功能缺失突变。这些方法使得在这些难以进行遗传转化的物种中快速测试基因功能成为可能，并开辟了使用依赖于转基因表达的其他细胞生物学和生化技术的可能性。
