A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells  

Abstract

Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.

ROPGAPs of Arabidopsis limit susceptibility to powdery mildew

“…13 Heterologous Y2H assays with barley HvRAC/ROPs revealed also interaction of AtROPGAPs with several barley RAC/ROPs in yeast with AtROPGAP4 interacting with HvRACB and CA HvRACB (data not shown). This may support conserved functions of AtROPGAPs and HvMAGAP1 although they are differently localized in epidermal leaf cells.…”

“…This may support conserved functions of AtROPGAPs and HvMAGAP1 although they are differently localized in epidermal leaf cells. 13 In contrast to HvMAGAP1, which is associated with microtubules in barley epidermal cells, GFP-fusions of AtROPGAP1 and AtROPGAP4 show mainly cytoplasmic and nuclear localization in Arabidopsis epidermal cells. 13 This can be explained because the C-terminus of HvMAGAP1, responsible for association of the protein with microtubules, greatly differs in sequence from that of Arabidopsis ROPGAPs.…”

“…13,14 HvRACB is hence a common factor of surface expansion both in epidermal root cells during formation of hairs and in epidermal leaf cells during formation of the haustorial complex. This includes formation of the extrahaustorial membrane, which is in continuum with the host plasma membrane, and of the extrahaustorial matrix a new apoplastic compartment with cell wall like constituents.…”

“…HvMAGAP1 belongs to the family of ROPGAPs containing a CRIB domain for G-protein interaction. 13,15 Transient-induced gene silencing of HvMAGAP1 resulted in enhanced susceptibility of barley epidermal cells to Bgh. A similar effect was observed when a mutated version of HvMAGAP1, MAGAP-R185G, was expressed, which can still interact with HvRACB in planta but apparently exerts a dominant negative effect due to mutation of the catalytic residue.…”

“…Similarly, Arabidopsis Atropgap1 and Atropgap4 knockout mutants display enhanced susceptibility in a compatible interaction with the powdery mildew fungus Erysiphe cruciferarum. 13 Although many RAC/ROP-regulated processes have been identified in plants, only little is known about physiological processes controlled via RAC/ROP-regulating ROPGAP proteins. CRIB containing NtRHOGAP1 is an important factor for restriction of RAC/ROP activity to the apex of the growing pollen tube tip by lateral control of RAC/ROPs in tobacco.…”