Anionic phospholipids connect barley ROP signaling with resistance and susceptibility to fungal cell entry

Rho-of-plant small GTPases (ROPs) are regulators of cell polar growth processes and of plant-pathogen interactions. The barley ROP protein RACB is involved in susceptibility towards infection by the barley powdery mildew fungus Blumeria hordei, but little is known about the cellular pathways that connect RACB-signaling to disease susceptibility. We now used an untargeted co immunoprecipitation-liquid chromatography-coupled mass spectrometry screening from Bh-infected epidermal layers of barley plants, which expressed green fluorescing protein-tagged constitutively activated (CA) RACB. This uncovered novel RACB interaction partners of plant and fungal origin. Three of those proteins, a plant phosphoinositide phosphatase, a plant phosphoinositide phospholipase and one putative Bh-effector protein appear to be involved in the barley-Bh-pathosystem and function in disease resistance or susceptibility, respectively. RACB and its plant interactors bind to overlapping anionic phospholipid species in vitro, and in case of RACB, this lipid-interaction is mediated by its carboxy-terminal polybasic region (PBR). Four diverse fluorescent plant anionic phospholipid-markers show altered subcellular distribution during Bh-attack. Phosphatidyl-4-phosphate and phosphatidylserine show a distinct enrichment at the haustorial neck region, suggesting a connection to subcellular targeting of RACB at this site. We suggest that the interplay of ROPs with anionic phospholipids and phospholipid–metabolizing enzymes may steer subcellular enrichment of components that are pivotal for fungal penetration success or failure.