Phytopathogenic fungi are a huge threat to agriculture, especially in present times where the world’s population is rising and so is the need for more resources. The extensive use of pesticides has caused several health and environmental hazards over the years and therefore, there is high demand for new and durable solutions. To achieve that, an extensive and comprehensive analysis of the highly complex and still often elusive plant-pathogen interactions is required. This thesis focuses on Blumeria hordei (Bh), the barley powdery mildew fungus which is among the most severe pathogens for the crop. As an obligate biotrophic fungus it can only survive on living host tissue and it has been used as a model system to unravel biotrophic plant-pathogen interactions.

Specific types of plant immune responses are activated upon attack by cell-penetrating pathogens like Bh, characterized by the formation of defence structures called papillae and encasements, which are essential for preventing fungal entry and haustorial function, respectively. Both contain extracellular vesicles (EV) carrying various cargo, several antimicrobial compounds and callose. However, they do not rely on the same pathway for EVsecretion, as papillae recruit EVs through an unconventional recycling mechanism, dependent on the ARF-GEF GNOM and independent from Rab5 and Rab7 GTPases, that regulate

secretion to encasements. The secretory mechanism of EVs to the papilla is poorly characterized and has been the main question of the current study.

To address this question, I focused on the PM syntaxin PEN1/ROR2 that is present in EVs secreted to both papillae and encasements. Here, I present data that show that PEN1/ROR2 secretion to the papillae remains unaffected by a dysfunctional ESCRT-machinery and is regulated by an alternative pathway that requires the sphingolipid ceramide. Moreover, secretion of ROR2 to the papillae was compromised in barley cells that overexpressed the Bh effector CSEP0254. Subsequent Split Ubiquitin Y2H studies revealed several potential interactors of CSEP0254 in barley. Two of these proteins were selected for further investigation – cytochrome B5 and a papain-like cysteine protease – for their role in lipid metabolism and plant immunity, respectively. Finally, Arabidopsis ceramide synthase mutants and autophagy deficient mutants were tested for their involvement in penetration immunity.