Plett Jonathan M, Montanini Barbara, Kohler Annegret, Ottonello Simone, Martin Francis
INRA-Nancy, Champenoux, France.
Methods Mol Biol. 2011;722:249-81. doi: 10.1007/978-1-61779-040-9_19.
Given recent technological advances, we are in a golden era of cell and whole organism research. With the availability of so many sequenced genomes, and the data that has been mined there-in, it is easy to gain the impression that all our work as scientists is complete. Instead, such work and results have now provided oceans of data, but with minimal functional information. We also do not have a full grasp on the working relationships within a number of different plant developmental pathways. This is especially true in the study of the symbiotic interaction between ectomycorrhizal fungi and their plant hosts. One of the current interests in symbiotic and pathogenic interactions between plants and fungi is the role of small, secreted proteins. What makes fungal small secreted proteins so interesting is that only a few of them share sequence homology to any other known proteins, but some may act as effectors modulating plant metabolism and development. Therefore, it is difficult to make predictions as to the action of these proteins without functional analysis. For this reason, we created a pipeline to analyze the role and function of these proteins. Typically, this involves transcriptional analysis of genes followed by protein localization, identification of protein-protein interactions, and functional analysis of the protein through heterologous expression in yeast among many other different procedures. Due to the physiology of mycorrhizal root tips, there are a number of unique challenges that must be overcome to properly study a fungal effector. Here, we outline some of the methods, and hopefully helpful tips, that we are currently using to pursue the study of different effectors in the Laccaria-Populus interaction.
鉴于最近的技术进步,我们正处于细胞和整个生物体研究的黄金时代。随着众多已测序基因组的可得性以及从中挖掘出的数据,很容易让人觉得我们科学家的所有工作都已完成。相反,这样的工作和结果现在提供了海量的数据,但功能信息却极少。我们也尚未完全掌握许多不同植物发育途径中的工作关系。在研究外生菌根真菌与其植物宿主之间的共生相互作用时尤其如此。当前对植物与真菌之间共生和致病相互作用的兴趣之一是小的分泌蛋白的作用。真菌小分泌蛋白如此有趣的原因在于,它们中只有少数与任何其他已知蛋白具有序列同源性,但有些可能作为调节植物代谢和发育的效应子。因此,在没有功能分析的情况下很难预测这些蛋白的作用。出于这个原因,我们创建了一个流程来分析这些蛋白的作用和功能。通常,这涉及基因的转录分析,随后是蛋白质定位、蛋白质 - 蛋白质相互作用的鉴定,以及通过在酵母中异源表达等许多其他不同程序对蛋白质进行功能分析。由于菌根根尖的生理学特性,要正确研究真菌效应子必须克服许多独特的挑战。在这里,我们概述了一些我们目前用于研究乳牛肝菌 - 杨树相互作用中不同效应子的方法以及希望有用的提示。