The fungal plant pathogen Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein) Petch) is the causal agent of Fusarium head blight (FHB) of small grain cereals and cob rot of maize worldwide. Trichothecene toxins produced by the fungus e.g. nivalenol (NIV) and deoxynivalenol (DON) contaminate cereal products and are harmful to humans, animals, and plants. We demonstrated recently, that F. graminearum forms toxin producing infection structures during infection of wheat husks, so called infection cushions (Boenisch and Schäfer, 2011). Structural characteristics of infection cushions were visualized by 3D images following laser scanning microscopy. We observed multiple penetration events underneath infection cushions by scanning electron microscopy. To understand the molecular basis of initial colonization of the leaf surface followed by infection cushion development, a laser capture microdissection (LCM) approach was established to isolate separately epiphytically growing runner hyphae and infection cushions. Several hundred runner hyphae and infection cushions grown on wheat glumes were isolated and RNAseq perfomed. Quantitative expression analysis show marked differences in gene expression patterns between runner hyphae and infection cushions. We will discuss specifically differences in carbohydrate active enzymes including cell wall degrading enzymes, putative effector proteins, and secondary metabolites. Thereby new insights in the initial infection process of FHB disease are gained. To our knowledge, we provide the first transcriptome data of runner hyphae and infection cushions from a fungal plant pathogen obtained under in planta conditions.