Janik Katrin, Schlink Katja
Department of Molecular Biology - Functional Genomics, Laimburg Research Centre;
Department of Molecular Biology - Functional Genomics, Laimburg Research Centre.
J Vis Exp. 2017 Jan 20(119):55150. doi: 10.3791/55150.
Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called "effectors," it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant's molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.
在植物科学中，揭示疾病表现的分子机制对于理解病理学和症状发展至关重要。细菌已经进化出不同的策略来操纵宿主代谢以使其自身受益。这种细菌操纵通常伴随着严重的症状发展或受影响植物的死亡。确定负责宿主操纵的特定细菌分子已成为微生物学研究的一个重要领域。在鉴定出这些被称为“效应子”的细菌分子后，阐明它们的功能很重要。确定效应子功能的一种直接方法是通过酵母双杂交（Y2H）筛选在其天然宿主中鉴定其蛋白质结合伴侣。通常，宿主含有许多潜在的结合伴侣，任何计算机算法都无法充分预测。因此，用假设的效应子针对整个表达的宿主蛋白文库进行筛选是最佳选择。如果病原体像植原体一样不可培养，这尤其具有挑战性。本方案提供了从感染植原体的木本宿主植物中纯化DNA、扩增潜在效应子以及随后通过Y2H筛选鉴定植物分子相互作用伴侣的分步说明。尽管Y2H筛选很常用，但有一种趋势是将这项技术外包给以一定成本提供Y2H服务的生物技术公司。本方案提供了关于如何在任何装备良好的分子生物学实验室中使用标准实验室技术进行Y2H的说明。
