Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Halle, Germany.
Molecular Phytopathology, Karlsruhe Institute of Technology, Karlsruhe, Germany.
Methods Mol Biol. 2020;2146:185-196. doi: 10.1007/978-1-0716-0603-2_14.
The complexity of the obligate symbiotic interaction of arbuscular mycorrhizal (AM) fungi and their host roots requires sophisticated molecular methods. In particular, to capture the dynamic of the interaction, cell-specific methods for gene expression analysis are required. In situ hybridization is a technique that allows to determine the location of transcript accumulation within tissues, being of special interest for these fungi that cannot be genetically modified. The method requires proper fixation and embedding methods as well as specific probes for the hybridization allowing detection of specific transcripts. In this chapter, we present a method to prepare roots, which have established a symbiosis with an arbuscular mycorrhizal fungus for the detection of fungal transcripts. This includes chemical fixation, subsequent embedding in a suitable medium, sectioning and pretreatment of sections, the hybridization procedure itself, as well as the immunological detection of RNA-RNA hybrids.
丛枝菌根（AM）真菌与其宿主根系之间的专性共生相互作用非常复杂，这需要复杂的分子方法。特别是，为了捕捉相互作用的动态，需要用于基因表达分析的细胞特异性方法。原位杂交是一种可以确定组织内转录物积累位置的技术，对于那些不能进行基因改造的真菌来说，这种技术特别有意义。该方法需要适当的固定和包埋方法以及用于杂交的特定探针，以检测特定的转录物。在本章中，我们介绍了一种为检测真菌转录本而准备与丛枝菌根真菌建立共生关系的根的方法。这包括化学固定、随后在合适的介质中包埋、切片和切片预处理、杂交程序本身以及 RNA-RNA 杂交体的免疫检测。
