Laser Microdissection of Plant–Fungus Interaction Sites and Isolation of RNA for Downstream Expression Profiling

Abstract

The molecular mechanisms that mediate the intimate interaction of an adapted obligate biotroph, such as the powdery mildew Golovinomyces orontii, on its host plant are spatially and temporally distinct. As G. orontii exclusively infects epidermal cells with a dominant host response occurring in the underlying mesophyll cells, we sought to develop a method to accurately and reproducibly perform global expression profiling on Arabidopsis thaliana leaf epidermal and mesophyll cells at the site of infection. Specific stages of G. orontii disease progression on Arabidopsis are visible by microscopy thus allowing distinct phases of the interaction to be studied. Tissue preparation, laser microdissection, and RNA isolation protocols that allow for temporally and spatially defined global expression profiling are described. By using these procedures to examine the growth and reproduction phase (5 days postinfection) of G. orontii on Arabidopsis, we identified known and novel processes, process components, and putative regulators of these processes that mediate the sustained growth and reproduction of this adapted obligate biotroph.