Cryo-Fixation and Gene Product Localization in Fungi

Abstract

There are many strategies for documenting the distribution of gene products or other molecules within cells or tissues. Depending on the required information, techniques can range from cell fractionation to methods that employ electron microscopy: some preclude examination of living cells, and thus can not provide dynamic temporal information. Time sequence data are paramount when gene expression is transient, as is often the case during fungal-pathogen plant-host interactions. Fluorescent dyes and protein tags (e.g., green fluorescent protein, GFP) allow monitoring of specific molecules, organelles or cells over time, for simultaneous recording of both spatial and temporal information. in addition, fluorescent markers are amenable to powerful 3-D analysis using confocal or multi-photon imaging methods. We have achieved good expression of fluorescent proteins in the fungal blast pathogen Magnaporthe grisea, and have generated a functional fusion protein between GFP and P-tubulin (Figs. 1,2).