A Novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking

ROP/RAC GTPases are master regulators of cell polarity in plants, implicated in the regulation of diverse signaling cascades including cytoskeleton organization, vesicle trafficking, and Ca(2+) gradients [1-8]. The involvement of ROPs in differentiation processes is yet unknown. Here we show the identification of a novel ROP/RAC effector, designated interactor of constitutive active ROPs 1 (ICR1), that interacts with GTP-bound ROPs. ICR1 knockdown or silencing leads to cell deformation and loss of root stem-cell population. Ectopic expression of ICR1 phenocopies activated ROPs, inducing cell deformation of leaf-epidermis-pavement and root-hair cells [3, 5, 6, 9]. ICR1 is comprised of coiled-coil domains and forms complexes with itself and the exocyst vesicle-tethering complex subunit SEC3 [10-13]. The ICR1-SEC3 complexes can interact with ROPs in vivo. Plants overexpressing a ROP- and SEC3-noninteracting ICR1 mutant have a wild-type phenotype. Taken together, our results show that ICR1 is a scaffold-mediating formation of protein complexes that are required for cell polarity, linking ROP/RAC GTPases with vesicle trafficking and differentiation.     

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.     

RAC/ROP GTPases: 'hubs' for signal integration and diversification in plants

RAC/ROP GTPases are a family of plant-specific signaling molecules solely representing the Ras and Rho family of Ras-related G proteins in plants. RAC/ROPs potentially interact with cell surface-associated signal perception apparatus for a broad range of extracellular stimuli, including hormones, pathogen elicitors and abiotic stress, and mediate diverse cellular pathways in response to these signals. They are also known to interact with multiple effectors, affecting cellular and biochemical systems that regulate actin dynamics, reactive oxygen species production, proteolysis, and gene expression. RAC/ROPs are, thus, ideally suited as integrators for multiple signals and as coordinators of diverse cellular pathways to control growth, differentiation, development and defense responses. Recent findings that suggest how RAC/ROP signaling activity is regulated and how functional specificity can be achieved are discussed here.     

Formation of self-organizing functionally distinct Rho of plants domains involves a reduced mobile population

Rho family proteins are central to the regulation of cell polarity in eukaryotes. Rho of Plants-Guanyl nucleotide Exchange Factor (ROPGEF) can form self-organizing polar domains following co-expression with an Rho of Plants (ROP) and an ROP GTPase-Activating Protein (ROPGAP). Localization of ROPs in these domains has not been demonstrated, and the mechanisms underlying domain formation and function are not well understood. Here we show that six different ROPs form self-organizing domains when co-expressed with ROPGEF3 and GAP1 in Nicotiana benthamiana or Arabidopsis (Arabidopsis thaliana). Domain formation was associated with ROP–ROPGEF3 association, reduced ROP mobility, as revealed by time-lapse imaging and Fluorescence Recovery After Photobleaching beam size analysis, and was independent of Rho GTP Dissociation Inhibitor mediated recycling. The domain formation depended on the ROPs’ activation/inactivation cycles and interaction with anionic lipids via a C-terminal polybasic domain. Coexpression with the microtubule-associated protein ROP effector INTERACTOR OF CONSTITUTIVELY ACTIVE ROP 1 (ICR1) revealed differential function of the ROP domains in the ability to recruit ICR1. Taken together, the results reveal mechanisms underlying self-organizing ROP domain formation and function.

Plasma membrane self-organizing polarity domains of small GTP-binding proteins form upon their co-expression together with their activator and suppressor due to restriction of protein mobility.     

Targeting and signaling of Rho of plants guanosine triphosphatases require synergistic interaction between guanine nucleotide inhibitor and vesicular trafficking

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle‐carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle‐mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP‐ribosylation factor 1‐mediated post‐Golgi trafficking. Trafficking of vesicle‐carried ROPs between the plasma membrane and the trans‐Golgi network is mediated through adaptor protein 1 and sterol‐mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.     

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.     

Toxoplasma gondii manipulates host cell signaling pathways via its secreted effector molecules

The obligate intracellular parasite Toxoplasma gondii secretes a vast variety of effector molecules from organelles known as rhoptries (ROPs) and dense granules (GRAs). ROP proteins are released into the cytosol of the host cell where they are directed to the cell nucleus or to the parasitophorous vacuole (PV) membrane. ROPs secrete proteins that enable host cell penetration and vacuole formation by the parasites, as well as hijacking host-immune responses. After invading host cells, T. gondii multiplies within a PV that is maintained by the parasite proteins secreted from GRAs. Most GRA proteins remain within the PV, but some are known to access the host cytosol across the PV membrane, and a few are able to traffic into the host-cell nucleus. These effectors bind to host cell proteins and affect host cell signaling pathways to favor the parasite. Studies on host–pathogen interactions have identified many infection-altered host signal transductions. Notably, the relationship between individual parasite effector molecules and the specific targeting of host-signaling pathways is being elucidated through the advent of forward and reverse genetic strategies. Understanding the complex nature of the host–pathogen interactions underlying how the host-signaling pathway is manipulated by parasite effectors may lead to new molecular biological knowledge and novel therapeutic methods for toxoplasmosis. In this review, we discuss how T. gondii modulates cell signaling pathways in the host to favor its survival.     

Constitutively activated barley ROPs modulate epidermal cell size, defense reactions and interactions with fungal leaf pathogens

RHO-like monomeric G-proteins of plants (ROPs, also called RACs), are involved in plant development and interaction with the environment. The barley (Hordeum vulgare) ROP protein HvRACB has been shown to be required for entry of the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) into living host cells. To get a deeper insight into evolutionarily conserved functions of ROPs in cell polarity and pathogen responses, we stably expressed constitutively activated (CA) mutant variants of different barley ROPs (HvRACB, HvRAC1, HvRAC3) in barley. CA HvROPs induced epidermal cell expansion and/or abolished polarity in tip growing root hairs. All three CA HvROPs enhanced susceptibility of barley to penetration by Bgh whereas only CA HvRAC1 supported whole cell H₂O₂ production in non-penetrated cells. Despite increasing penetration by Bgh, CA HvRAC1 promoted callose deposition at sites of fungal attack and resistance to penetration by Magnaporthe oryzae. The data show an involvement of ROPs in polar growth processes of the monocot barley and in responses to fungal pathogens with different life style.     

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。     

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.     

Molecular basis for the substrate specificity of plant guanine nucleotide exchange factors for ROP

Plant G proteins of the ROP/RAC family regulate cellular processes including cytoskeletal rearrangement in polar growth. Activation of the ROP molecular switch is triggered by guanine nucleotide exchange factors. Plant-specific RopGEFs are exclusively active on ROPs despite their high homology to animal Rho proteins. Based on a sequence comparison of ROPs vs. animal Rho proteins together with structural data on distinct ROPs, we identified unique substrate determinants of RopGEF specificity by mutational analysis: asparagine 68 next to switch II, arginine 76 of a putative phosphorylation motif and the Rho insert are essential for substrate recognition by RopGEFs. These data also provide first evidence for a function of the Rho insert in interactions with GEFs.     

Cell polarity: ROPing the ends together

Cell polarity plays an important role in plant development, but the mechanisms that first establish polarity cues remain obscure. By contrast, a flurry of information has recently emerged on the elaboration of cell shape from such unknown initial cell-polarity cues. Recent studies suggest that Rho-related GTPases in plants (ROPs), and their effector targets among the ROP-interactive CRIB motif-containing proteins (RICs), mediate two antagonistic pathways that have opposing action on cell polarization. ROP proteins appear to interact directly with upstream regulators of the ARP2/3 complex, which are conserved modulators of the actin cytoskeleton. ROP function is dependent on the class 1 ADP-ribosylation factors (ARFs), which are core components of the vesicle transport machinery that are also involved in the polar localization of PIN-FORMED (PIN) family auxin efflux facilitators.     

Plant small monomeric G-proteins (RAC/ROPs) of barley are common elements of susceptibility to fungal leaf pathogens,cell expansion and stomata development

Small monomeric RAC/ROP GTPases act as molecular switches in signal transduction processes of plant development and stress responses. They emerged as crucial players in plant-pathogen interactions either by supporting susceptibility or resistance. In a recent publication, we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs regulate susceptibility to barley fungal leaf pathogens of different life style in a contrasting way. This illustrates the distinctive signalling roles of RAC/ROPs for different plant-pathogen combinations. We also reported the involvement of RAC/ROPs in plant epidermis development in a monocotyledonous plant. Here we further discuss a failure of CA HvRAC/ROP-expressing barley to normally develop stomata.Key words: Hordeum vulgare, G-proteins, RAC, ROP, cell expansion, stomata, transpirationMembers of the RHO family of small G-proteins in plants (RAC/ROPs) regulate signal transduction processes at the plasma membrane.¹ They act as multifunctional signalling switches in plant development and a variety of stress responses. RAC/ROP GTPases play regulatory roles in polar growth and cell morphogenesis in several cell systems including pollen tubes, developing root hairs and leaf pavement cells.²In a recent publication,³ we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs support susceptibility to the barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). CA HvRAC1 supported susceptibility to biotrophic Bgh but resistance to hemibiotrophic Magnaporthe oryzae in barley at the penetration level in both cases. Additionally, CA HvRAC1 supported local callose deposition at sites of attack from Bgh and a secondary H₂O₂ burst in whole non-penetrated epidermal cells. This supports a regulatory function of RAC/ROPs in plant defence¹ and the potential corruption of defence pathways in susceptibility to Bgh. Because the rice ortholog of HvRAC1, OsRAC1, can regulate an H₂O₂ burst via activation of the plasma membrane NADPH oxidase OsRBOHB,⁴ one can speculate that the secondary H₂O₂ burst in CA HvRAC1 barley could also be caused by over-activation of an NADPH oxidase. However, CA HvRAC1 barley was also more susceptible to fungal penetration, and penetrated cells did not show an H₂O₂ burst. Hence, CA HvRAC1 did not contribute to penetration resistance, and the H₂O₂ burst might have been suppressed by Bgh after successful penetration. Interestingly, Bgh secretes a catalase during interaction with the plant.⁵The involvement of RAC/ROPs in plant development has been widely studied in the dicots Arabidopsis and tobacco. In Arabidopsis, CA AtRAC/ROPs disturb root hair tip growth and epidermal cell morphogenesis.^6,7 We showed similar developmental aberrations as a result of CA HvRAC/ROP expression in monocotyledonous barley. Root hair polarity disruption and enhanced leaf epidermal cell expansion was observed in CA HvRAC/ROP expressing barley. Here, we further report on reduced or abnormal development of stomata as an effect of CA HvRAC/ROP expression.In barley, stomata and short epidermal cells alternate in a row of leaf epidermal cells (Fig. 1A). The number of stomata number was significantly reduced in three CA HvRAC/ROP (CA HvRACB, CAHvRAC3, CA HvRAC1) expressing barley genotypes when compared to azygous controls (barley siblings that lost the transgene due to segregation) (Fig. 1E). In part, this could be explained by enhanced length of epidermal cells intercalated between stomata (Fig. 1B). The presence of longer epidermal cells in all CA HvRAC/ROP-barleys further supports that RAC/ROPs are operating in epidermal cell expansion.³Open in a separate windowFigure 1Stomatal abnormalities observed in CA HvROPexpressing transgenic barley leaves. (A) Wild type leaf adaxial epidermis with alternating stomata complexes (arrows) and short epidermal cells (asterisk). (B) Presence of more than one short epidermal cell in between two stomata. Arrows point the stomata. Double headed arrows highlight intercalated cells with enhanced cell length (C) Two stomata lacking an intercalated short epidermal cell. (D) Stoma failed to develop and left an abnormal blank cell. (E) Average number of stomata present in 5 cm of a stomatal row in transgenic plants expressing distinct CA barley CA HvRAC/ROPs. For all samples, stomatal rows present on either side of the mid rib were counted in the leaf upper epidermis. Fully expanded leaves of 3-weeks-old barley plants were used for counting stomata. Error bars show 95% confidence intervals. Repetition of the experiment led to similar results. Scale bars = 50 µm.Previously, we carried out porometry experiments to measure stomata conductivity in CA HvRACB expressing barley leaves.⁸ The CA HvRACB leaves showed up to 50% less transpiration than azygous controls without any treatment. Additionally, CA HvRACB leaves were less responsive to abscisic acid (ABA) and subsequently they could not effectively reduce transpiration when treated with ABA or when cut-off from water supply.⁸ Our data on numbers of stomata per leaf segment could now explain the lower rates of transpiration in non-stressed CA HvRACB barley when compared to wild type.Apart from the stomata number, developmental abnormalities were observed in the arrangement of epidermal cells. Generally, the shape of epidermal cells was less regular in CA HvRAC/ROP barley.³ We also observed the presence of more than one short epidermal cell in between two stomata (Fig. 1B) or two stomata lacking an intercalated short epidermal cell (Fig. 1C), or stomata failed to develop, which ended up in an abnormally short epidermal cell (Fig. 1D). Although such abnormalities were also rarely observed in wild type plants, all three CA HvRAC/ROP-barley leaves exhibited a clearly higher frequency of abnormalities in a given length of a stomata row. Together, CA HvRAC/ROPs had an effect on both the number and development of stomata. These observations suggest that RAC/ROPs are not only operating in cell expansion but also in barley cell differentiation for stomata development.     

Barley RIC171 interacts with RACB in planta and supports entry of the powdery mildew fungus

RHO-like GTPases of plants (ROPs, also called RACs) are involved in plant development and interaction with the environment. The barley ROP protein RACB is involved in susceptibility to the fungal pathogen Blumeria graminis f.sp. hordei ( Bgh ) . By screening barley sequence databases for potential protein interactors of plant RHO-like proteins, we identified a ROP-interactive CRIB (CDC42/RAC interactive binding) motif containing protein of 171 amino acids (RIC171). The protein interacted with constitutively activated RACB in a targeted yeast two-hybrid assay. By use of split yellow fluorescing protein fusions, we demonstrated that RIC171 interacts with constitutively activated (CA) RACB-G15V but not with dominant negative RACB-T20N in planta . Transient overexpression of RIC171, similar to overexpression of CA RACB-G15V, rendered epidermal cells more susceptible to penetration by Bgh . In contrast, expression of a 46-amino-acid RIC171-CRIB peptide, which was sufficient to interact with CA RACB-G15V, had a dominant negative effect and reduced susceptibility to Bgh . A red fluorescing DsRED–RIC171 fusion protein colocalized with green fluorescing GFP–RACB-G15V at the cell periphery. Coexpression with CA RACB-G15V but not with RACB-T20N increased peripheral localization of DsRED–RIC171. Additionally, DsRED–RIC171 accumulated at sites of fungal attack, suggesting enhanced ROP activity at sites of attempted fungal penetration.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.     

Activation status-coupled transient S acylation determines membrane partitioning of a plant Rho-related GTPase

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His₆-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His₆-GFP-Atrop6^CA mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His₆-GFP-Atrop6^CAmS¹⁵⁶ in which cysteine¹⁵⁶ was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.