The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs)

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.     

The crystal structure of Arabidopsis thaliana RAC7/ROP9: the first RAS superfamily GTPase from the plant kingdom

Arabidopsis thaliana RAC/ROP GTPases constitute a plant specific Rho GTPase family in the RAS superfamily, which has been implicated in numerous pivotal signalling cascades in plants. Research has shown that plants in some cases have evolved different modes of regulating Rho GTPase activity as compared to the equivalent systems in animals and yeast. In order to gain structural insight into plant signaling at the molecular level, we have determined the first crystal structure of a RAC-like GTPase belonging to the RAS superfamily from the plant kingdom. The structure of AtRAC7/ROP9 bound to GDP was solved at a resolution of 1.78 A. We have found that the structure of plant Rho GTPases is based upon a conserved G-domain architecture, but structural differences were found concerning the insert region and switch II region of the protein.     

A RHOse by any other name： a comparative analysis of animal and plant Rho GTPases

Rho GTPases are molecular switches that act as key regulators of a many cellular processes,including cell movement,morphogenesis,host defense,cell division and gene expression.Rho GTPases are found in all eukaryotic kingdoms.Plantslack clear homologs to conventional Rho GTPases found in yeast and animals;instead,they have over time developeda unique subfamily,ROPs,also known as RAC.The origin of ROP-like proteins appears to precede the appearance ofland plants.This review aims to discuss the evolution of ROP/RAC and to compare plant ROP and animal Rho GTPases,focusing on similarities and differences in regulation of the GTPases and their downstream effectors.     

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules.     

Structure and function of Rho-type molecular switches in plants.

Molecular switches of the Rho family, in concert with their associated regulators and effectors are well known as important control elements of vital signaling pathways in eucaryotic organisms. Yet, this knowledge has so far been established mainly from animal and fungal studies. However, during the recent years, the Rho switch has gone increasingly green as well, and it turned out that the homologous system in plants holds some distinctive features regarding structures, functions and molecular mechanisms for signal transduction. In this review, we give an overview about the structural characteristics of the Rho proteins of plants, termed ROP, highlighting some exciting differences to their animal and fungal counterparts. We further address the unique regulators and effectors of the ROPs and discuss the structural basis for the function and interaction of those proteins in ROP controlled reaction cascades. We finally intend to stimulate the demand for future three-dimensional structures that advance our understanding of the ROP switch in plants.     

Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus

Small monomeric G-proteins of the plant ras (rat sarcome oncogene product) related C3 botulinum toxin substrate (RAC)/Rho of plants (ROP) family are molecular switches in signal transduction of many cellular processes. RAC/ROPs regulate hormone effects, subcellular gradients of Ca2+, the organisation of the actin cytoskeleton and the production of reactive oxygen intermediates. Therefore, we followed a genetic bottom-up strategy to study the role of these proteins during the interaction of barley (Hordeum vulgare L.) with the fungal biotrophic pathogen Blumeria graminis f.sp. hordei (Bgh). We identified six barley RAC/ROP proteins and studied their gene expression. Five out of six Rac/Rop genes were expressed constitutively in the leaf epidermis, which is the site of interaction with Bgh. None of the genes showed enhancement of mRNA abundance after inoculation with Bgh. After microprojectile mediated transformation of single barley epidermal cells with constitutively activated mutant RAC/ROP proteins, we found an RAC/ROP-specific enhancement of pathogen accessibility, tagging HvRACB, HvRAC3 and HvROP6 as host proteins potentially involved in the establishment of susceptibility to Bgh. Confocal laser scanning microscopy (CLSM) of green fluorescent protein (GFP):HvRAC/ROP-transformed cells revealed varying strengths of plasma membrane association of barley RAC/ROPs. The C-terminal CAAX motif for presumable prenylation or the C-terminal hypervariable region (HVR), respectively, were required for membrane association of the RAC/ROPs. Proper intracellular localisation was essential for HvRACB and HvRAC3 function. Together, our data support the view that different paths of host signal transduction via RAC/ROP G-proteins are involved in processes supporting parasitic entry into epidermal host cells.     

SWAP70 functions as a Rac/Rop guanine nucleotide-exchange factor in rice

Rho family small GTPases are involved in diverse signaling processes including immunity, growth, and development. The activity of Rho GTPases is regulated by cycling between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms, in which guanine nucleotide exchange factors (GEFs) predominantly function to promote activation of the GTPases. In animals, most Rho GEFs possess a Dbl (diffuse B-cell lymphoma) homology (DH) domain which functions as a GEF-catalytic domain. However, no proteins with the DH domain have been identified in plants so far. Instead, plant-specific Rho GEFs with the PRONE domain responsible for GEF activity have been found to constitute a large family in plants. In this study, we found rice homologs of human SWAP70, Oryza sativa (Os) SWAP70A and SWAP70B, containing the DH domain. OsSWAP70A interacted with rice Rho GTPase OsRac1, an important signaling factor for immune responses. The DH domain of OsSWAP70A exhibited the GEF-catalytic activity toward OsRac1 as found in animal Rho GEFs, indicating that plants have the functional DH domains. Transient expression of OsSWAP70A enhanced OsRac1-mediated production of reactive oxygen species in planta. Reduction of OsSWAP70A and OsSWAP70B mRNA levels by RNA interference resulted in the suppression of chitin elicitor-induced defense gene expression and ROS production. Thus, it is likely that OsSWAP70 regulates immune responses through activation of OsRac1.     

Dbl family guanine nucleotide exchange factors

The Dbl family of guanine nucleotide exchange factors are multifunctional molecules that transduce diverse intracellular signals leading to the activation of Rho GTPases. The tandem Dbl-homology and pleckstrin-homology domains shared by all members of this family represent the structural module responsible for catalyzing the GDP–GTP exchange reaction of Rho proteins. Recent progress in genomic, genetic, structural and biochemical studies has implicated Dbl family members in diverse biological processes, including growth and development, skeletal muscle formation, neuronal axon guidance and tissue organization. The detailed pictures of their autoregulation, agonist-controlled activation and mechanism of interaction with Rho GTPase substrates, have begun to emerge.     

ROPs:植物细胞内多种信号通路的分子开关

植物RHO相关蛋白GTPases(RHO-related GTPases of plants, ROPs)是广泛存在于植物中的一类信号转导G蛋白(又称GTP结合蛋白),其通过结合GDP或GTP在非活性和活性状态间进行切换,进而在细胞极性控制、形态发育、激素水平调控、逆境反应等诸多植物生命活动的信号转导过程中扮演重要的分子开关角色。本文对ROP蛋白的结构域及基于蛋白质结构分类进行了介绍,并对拟南芥、玉米、水稻和大麦中的ROP家族蛋白质进行了系统进化分析。分析结果表明,这些植物中的ROP蛋白根据蛋白质结构域组成可分为Ⅰ类(typeⅠ)和Ⅱ类(typeⅡ)两种类型,而根据蛋白质序列的保守性可将其在植物中的ROP蛋白划分为4个进化枝。本综述不但对ROP蛋白作为分子开关在细胞内调控各种信号通路的机制进行了叙述,还对ROP在花粉管、根毛及植物表皮铺盖细胞极性发育,以及其他抗逆反应中的具体作用和机制及研究进展进行了阐述。本文还对ROP蛋白在ABA、IAA、BR等植物激素信号传导过程中的调控作用及研究进展进行了阐述。本文对植物ROP蛋白研究过程中尚未解决的问题,例如不同的ROP蛋白在同一个信号通路中的作用为何如此不同,以及ROP是如何协调不同的信号通路以共同调控一个植物发育或者生理过程等问题进行了总结,并在此基础上对未来的研究方向进行了展望。     

The unique plant RhoGAPs are dimeric and contain a CRIB motif required for affinity and specificity towards cognate small G proteins

Plant Rho proteins (ROPs) are inactivated by specific GTPase activating proteins, called RopGAPs. Many of these comprise the exclusive combination of a classic, catalytic Arg-containing RhoGAP domain, and a Cdc42/ Rac interactive binding (CRIB) motif which in animal and fungi has been identified in effectors for Cdc42 and Rac1, but never in any GAP protein. Both elements are required for an efficient RopGAP activity. Here, we analyzed the effect of the CRIB motif on the complex formation and the binding reaction with plant and human Rho proteins by using kinetic and equilibrium methods. We show that RopGAP2 from Arabidopsis thaliana dimerizes via its GAP domain and forms a 2:2 complex with ROP. The CRIB effector motif mediates high affinity and specificity in binding. The catalytic Arg in the context of the CRIB motif is inhibitory for binding. The unusually slow association and dissociation reactions suggest a major conformational change whereby the CRIB motif functions as a lid for binding and/or release of ROP. We propose a two-site interaction model where ROP binds to the CRIB motif as described for the human CRIB effectors and to the catalytic GAP domain as described for animal RhoGAPs.     

小G蛋白ROP的研究进展

小G蛋白(small GTPases)是近年来研究细胞信号转导过程的热点问题,包括Ras、Rab、Rho、Arf和Ran5个亚家族,其中ROP蛋白是Rho家族成员,为植物特有,在调控细胞生长、发育及调节植物对环境响应等各方面起重要作用.对ROP蛋白的活性调节和功能进行了重点介绍.     

Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica

The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engages a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.     

Mutation of an N-terminal acidic-rich region of p115-RhoGEF dissociates alpha13 binding and alpha13-promoted plasma membrane recruitment

The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the alpha(13) heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. Alpha(13) binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of alpha(13) activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated alpha(13), and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated alpha(13). However, alpha(13)-interacting deficient N-terminal mutants of p115-RhoGEF retain alpha(13)-dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to alpha(13) and dissociate two activities of p115-RhoGEF: binding to activated alpha(13) and translocation to the PM in response to activated alpha(13).     

RhoGDIs revisited: novel roles in Rho regulation

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.     

RAC/ROP GTPases and auxin signaling

Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin-RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch.     

Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure

C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD⁺ to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3.     

Differential effects of prenylation and s-acylation on type I and II ROPS membrane interaction and function

Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.     

Molecular mechanisms controlling pavement cell shape in Arabidopsis leaves

Pavement cells have an interlocking jigsaw puzzle-shaped leaf surface pattern. Twenty-three genes involved in the pavement cell morphogenesis were discovered until now. The mutations of these genes through various means lead to pavement cell shape defects, such as loss or lack of interdigitation, the reduction of lobing, gaps between lobe and neck regions in pavement cells, and distorted trichomes. These phenotypes are affected by the organization of microtubules and microfilaments. Microtubule bands are considered corresponding with the neck regions of the cell, while lobe formation depends on patches of microfilaments. The pathway of Rho of plant (ROP) GTPase signaling cascades regulates overall activity of the cytoskeleton in pavement cells. Some other proteins, in addition to the ROPs, SCAR/WAVE, and ARP2/3 complexes, are also involved in the pavement cell morphogenesis.