Morcillo Rafael J L, Zhao Achen, Tamayo-Navarrete María I, García-Garrido José M, Macho Alberto P
Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences;
Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences; University of Chinese Academy of Sciences.
J Vis Exp. 2020 Mar 11(157). doi: 10.3791/60302.
Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.
青枯雷尔氏菌是一种极具破坏力的土传维管束病原体，可感染多种植物物种，对农业构成重大威胁。然而，与其他涉及细菌性植物病原体的模型（如拟南芥中的丁香假单胞菌）相比，雷尔氏菌模型的研究还相当不足。旨在了解雷尔氏菌与作物植物之间相互作用的研究对于开发对抗青枯病的可持续解决方案至关重要，但目前由于缺乏直接的实验方法来表征天然寄主植物中相互作用的不同组成部分而受到阻碍。在这种情况下，我们开发了一种对番茄（雷尔氏菌的天然寄主）进行雷尔氏菌感染基因分析的方法。该方法基于发根农杆菌介导的番茄根转化，然后对所得植物进行雷尔氏菌土壤浇灌接种，这些植物含有表达感兴趣构建体的转化根。根转化试验的多功能性允许进行基因过表达或RNAi介导的基因沉默。作为概念验证，我们使用该方法表明番茄根中SlCESA6的RNAi介导沉默赋予了对雷尔氏菌的抗性。在此，我们详细描述了该方法，使遗传方法能够在相对较短的时间内，以对设备和植物生长空间的小需求来了解青枯病。
