Barley C2-Domain Abscisic Acid-Related protein CARa supports susceptibility to Blumeria hordei and localizes to the extrahaustorial membrane

Abstract

Rho GTPases are key regulators of cellular signalling processes in eukaryotes. In plants, Rho of plants (ROP) proteins function in cell polarization, hormone signalling, and plant immunity or susceptibility to diseases. The barley (Hordeum vulgare) ROP protein RACB is involved in susceptibility towards the biotrophic ascomycete fungus Blumeria hordei (Bh) and supports the accommodation of fungal haustoria. In healthy barley, RACB plays a role in cell development, a function that the pathogen may co-opt for cellular ingrowth of its haustorium into intact epidermal cells of barley. The majority of identified functions of GTP-bound activated RACB appear to be mediated by scaffold proteins. One of them, the ROP Interactive Partner b (RIPb, synonym: Interactor of Constitutive Active ROP b, ICRb) binds to RACB-GTP and co-localizes with RACB at the site of fungal host cell entry. RIPb interacts with RACB-GTP via its C-terminal coiled-coil CC2 domain (RIPbCC2) at the plasma membrane. Here, we show that a barley Calcium-Dependent Phospholipid Binding 2 (C2)-Domain Abscisic Acid-Related protein (CARa) interacts with RIPb and its RIPbCC2 domain in planta. Transient knockdown of CARa renders barley epidermal cells less susceptible to invasion by Bh, whereas overexpression supports fungal haustorium accomodation. Upon fungal attack, CARa is recruited to the site of fungal attack and localizes around the haustorial neck and at the extrahaustorial membrane. These findings further support that Bh profits from host ROP signalling components and CARa. CARa might connect RACB-RIPb-signalling to membrane organization for support of fungal infection structures in barley epidermal cells.