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

Abstract

8Small ROP (also called RAC) GTPases are key factors in polar cell development and in 9 interaction with the environment. ROP-Interactive Partner (RIP) proteins are predicted scaffold 10 or ROP-effector proteins, which function downstream of activated GTP-loaded ROP proteins 11 in establishing membrane heterogeneity and cellular organization. Grass ROP proteins 12 function in cell polarity, resistance and susceptibility to fungal pathogens but grass RIP proteins 13 are little understood. 14 We found that the barley (Hordeum vulgare L.) RIPa protein can interact with barley ROPs in 15 yeast. Fluorescent-tagged RIPa, when co-expressed with the constitutively activated ROP 16 protein CA RAC1, accumulates at the cell periphery or plasma membrane. Additionally, RIPa, 17 locates into membrane domains, which are laterally restricted by microtubules, when co-18 expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING 19 PROTEIN 1. Both structural integrity of MICROTUBULE-ASSOCIATED ROP-GTPASE 20 ACTIVATING PROTEIN 1 and microtubule stability are key to maintenance of RIPa-labeled 21 membrane domains. In this context, RIPa also accumulates at the interface of barley and 22 invading hyphae of the powdery mildew fungus Blumeria graminis f.sp. hordei. 23 Data suggest that barley RIPa interacts with barley ROPs and specifies RAC1 activity-24 associated membrane domains with potential signaling capacity. Lateral diffusion of this RAC1 25 signaling capacity is restricted the resulting membrane heterogeneity requires intact 26 microtubules and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. 27 Focal accumulation of RIPa at sites of fungal attack may indicate locally restricted ROP activity 28 at sites of fungal invasion. 29 30 Keywords: Arabidopsis thaliana, Hordeum vulgare, interactor of constitutive active ROPs, 31 membrane asymmetry, microtubule, RAC GTPase, ROP GTPase, susceptibility, resistance 32 In plants, ROP (RHO of plants) small GTPases are the only members of the RHO protein 35 family, which consists of several subfamilies (RHO, RAC, CDC42, Rnd und RhoBTB) in 36 mammals [1, 2]. ROPs organize a bunch of cellular processes as signaling GTPase. Among 37 the most prominent ROP-regulated events are the subcellular organization of the cytoskeleton 38 and vesicular traffic [3]. ROP-regulated cellular organization is crucial for normal plant 39 development e.g. in polar cell growth or asymmetric cell division but also in interaction with the 40 environment e.g. in regulation of stomata aperture or in interaction with pathogens. ROP 41 activity is tightly regulated via proteins that facilitate hydrolysis and exchange of ROP-bound 42 nucleotides. ROP-GDP is the signaling-inactive form of ROP and can be further controlled by 43 ROP-GDIs (ROP-guanine nucleotide dissociation inhibitors) that bind to ROP-GDP. ROP-44GDIs support cytosolic localization of ROPs most likely by direct binding of isoprenyl-residues 45 at the C-terminus of type I ROPs, which carry a CAAX-box prenylation motif. ROP-GDP further 46 can in...

Arabidopsis pavement cell shape formation involves spatially confined ROPGAP regulators

“…Intriguingly, the distantly related barley MICROTUBULE-ASSOCIATED GAP 1 (MAGAP1), which associates with the entity of microtubules within a cell, has been implicated in the organization of microtubule-limited membrane domains in transient expression. 33,55 Regardless, PHGAPs associate only with a subset of microtubules.…”