MAP18 Regulates ROP2 Gtpase Activity During Root Hair Tip Growth In Arabidopsis Thaliana

Root hair is a tubular structure protruding from root epidermal cells of plants,and plays important roles in water,nutrient uptake and the interaction with the external environment.Tip growing root hair also serves as a model system to study cell polarity and morphogenesis.It has been shown that the growth of root hairs is regulated by many factors,such as Ca2-,vesicle trafficking,the cytoskeleton,small GTPase,and ROS.ROP GTPases are plant specific Rho like small GTPases which play crucial roles in almost every aspect of plant growth and development.As a binary molecular switch that transmits extracellular signals to intracellular biological events,the activity of ROP GTPases is precisely regulated.Previous studies had shown that several ROPs are involved in the regulation of root hair tip growth in Arabidopsis.ROP2 is localized at the root hair initiation site and the apical plasma membrane(PM)region of tip growing root hair,and takes part in regulating root hair initiation and elongation in Arabidopsis.Microtubule-Associated Protein 18(MAP 18)is a protein that is able to associate with both microtubules and micro filaments.MAP 18 regulated the polarized diffuse growth of leaf epidermal cells and hypocotyl epidermal cells,by destabilizing microtubules.Interestingly,MAP 18 involved in the regulation of pollen tube growth direction by severing F-actin in the presence of Ca2+.In the present study,MAP18 is found to be highly expressed in root hairs,which suggests a role of MAP 18 in the regulation of root hair tip growth.The observation of root hairs phenotypes in MAP 18 and ROP2 single mutants,double mutants,and overexpression plants indicates that MAP 18 may take action in ROP2 signaling pathway to regulate root hair tip growth.In addition,in vitro and in vivo analyses demonstrate a direct interaction between MAP 18 and ROP2.It is also shown that MAP 18 preferentially interacts with the inactive form of ROP2(including GDP-ROP2 and dorminant negative mutant DN-ROP2).In vitro Effector Binding assays and analysis of GFP-ROP2 localization in root hairs strongly suggest that MAP18 promotes ROP2 activities at the apical PM of the root hair.Further investigation revealed that MAP18 competes with AtRhoGDI1/SCN1(RhoGTPase GDP dissociation inhibitor 1/Supercentipede 1)for binding to ROP2,and results in a positive regulation of ROP2 activity,and subsequently involves in the regulation of root hair polairy and elongation.Furthermore,a truncated MAPI 8 protein lacking the N terminal 23 amino acids(MAP18?N23)is constructed.MAP18AN23 mutant failed to interact with the inactive form of ROP2,suggesting that the N23 domain of MAP 18 is essential for the interaction of MAP 18 with ROP2 in vitro.Additionally,MAP18?N23 can not affect the interaction between AtRhoGDIl/SCN1 and the inactive form of ROP2,indicating a failure of MAP18?N23 to compete with AtRhoGDI1/SCN1.Next,various MAP18 mutant proteins with mutations in VEEKK motifs are used to test their ROP2 binding activity.The results indicate that MAP 18 binding to ROP2 is unrelated to its Ca2--dependent F-actin-severing activity.Pharmacology analyses also demonstrate that the regulation of ROP2 activity by MAP 18 is independent on microtubules or actin filaments.In summary,the present study demonstrates that MAP18 is involved in ROP2 signaling pathway to regulate the root hair tip growth.MAP 18 acts as a GDI displacement factor,competing with RhoGDIl to interact with inactive form of ROP2,which facilitates the activation of ROP2.This study reveals a novel regulatory mechanism of ROP activity.It provides new insights in the knowledge of plant cell morphogenesis,and in the understanding of the Rho GTPases signal transduction.