Barley ROP-INTERACTIVE PARTNER-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains

Abstract

Small ROP (also called RAC) GTPases are key factors in polar cell development and in interaction with the environment. ROP-Interactive Partner (RIP) proteins are predicted scaffold or ROP-effector proteins, which function downstream of activated GTP-loaded ROP proteins in establishing membrane heterogeneity and cellular organization. Grass ROP proteins function in cell polarity, resistance and susceptibility to fungal pathogens but grass RIP proteins are little understood.\n\nWe found that the barley (Hordeum vulgare L.) RIPa protein can interact with barley ROPs in yeast. Fluorescent-tagged RIPa, when co-expressed with the constitutively activated ROP protein CA RAC1, accumulates at the cell periphery or plasma membrane. Additionally, RIPa, locates into membrane domains, which are laterally restricted by microtubules, when co-expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Both structural integrity of MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1 and microtubule stability are key to maintenance of RIPa-labeled membrane domains. In this context, RIPa also accumulates at the interface of barley and invading hyphae of the powdery mildew fungus Blumeria graminis f.sp. hordei.\n\nData suggest that barley RIPa interacts with barley ROPs and specifies RAC1 activity-associated membrane domains with potential signaling capacity. Lateral diffusion of this RAC1 signaling capacity is restricted the resulting membrane heterogeneity requires intact microtubules and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Focal accumulation of RIPa at sites of fungal attack may indicate locally restricted ROP activity at sites of fungal invasion.