rop gtpases structure-function and signaling …...57 the establishment and maintenance of cell...

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ROP GTPases structure-function and signaling pathways 2

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Gil Feiguelmana, Ying Fub, and Shaul Yalovskya* 4

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aSchool of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv 6997801, Israel 6

bState Key Laboratory of Plant Physiology and Biochemistry College of Biological Sciences 7

China Agricultural University, Beijing, China 8

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ORCHID IDs: 0000-0002-1167-0849 (G.F.), 0000-0002-9436-212 (Y.F.), 0000-0003-3264-0005 10

(S.Y.) 11

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Running title: ROP function and signaling 13

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To whom correspondence should be addressed: [email protected] 15

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Author contributions 17

GF: wrote the paper and prepared the figures, YF: wrote the paper, SY: conceived the and 18

wrote the paper. 19

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Funding information 21

SY: the research was supported by Israel Academy of Sciences (grant nos. ISF 827/15; ISF–22

NCSF 1125/13) and by the Israel Center for Research Excellence on Plant Adaptation to 23

Changing Environment (grant no. I–CORE 757–12). 24

YF: The Natural Science Foundation of China (grant nos. 31325001; 31361140354). 25

26 Summary: Interactions between receptor like kinases and guanyl nucleotide exchange 27 factors together with identification of effector proteins reveal putative ROP GTPases 28 signaling cascades. 29

Plant Physiology Preview. Published on November 17, 2017, as DOI:10.1104/pp.17.01415

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Introduction 31

Rho of Plants (ROP) proteins also known as RACs are the plant specific subfamily of Rho 32

small GTP binding proteins, referred to here as small G proteins (Zheng and Yang, 2000; 33

Brembu et al., 2006; Elias and Klimes, 2012). Like other members of the Ras superfamily of 34

small G proteins, ROPs function as molecular switches due to changes in conformation upon 35

GTP binding and hydrolysis (Berken and Wittinghofer, 2008). The conformational 36

differences between the GTP- and GDP-bound states facilitate transient interactions with 37

effector and regulatory proteins that in turn result in periodic activation/inactivation cycles 38

of signaling cascades. Small G protein function is characterized by two central features: 1) 39

due to inefficient GTP hydrolysis these proteins remain in the GTP-bound active form for 40

extended periods of time and 2) due to the low dissociation coefficient of GDP its release is 41

inefficient and depends on enzymatic activity (Bourne et al., 1991; Vetter and Wittinghofer, 42

2001). Because of these features, the GTP-/GDP-dependent activation/inactivation cycles of 43

small G proteins are regulated in time and space by GDP/GTP Exchange Factors (GEFs) that 44

facilitate the release of GDP and GTPase Activating Proteins (GAPs) that enhance GTP 45

hydrolysis (Berken and Wittinghofer, 2008). 46

ROPs relay signaling from distinct plasma membrane domains; ROPs bind through post-47

translational lipid modifications and interaction with membrane lipids (Bloch and Yalovsky, 48

2013). Thus, ROPs function as molecular switches that transduce intracellular and 49

extracellular stimuli in spatially regulated fashion, resulting in localized regulation of 50

intracellular responses. 51

A third group of regulatory proteins, Rho GDP Dissociation Inhibitors (RhoGDIs) interact 52

with high and low affinity with GDP- and GTP-bound Rho proteins, respectively, remove 53

them from and the membrane and stabilizing them in the cytoplasm until their reactivation 54

by GEFs (Boulter and Garcia-Mata, 2010). The recycling induced by RhoGDIs is thought to 55

maintain Rho proteins in distinct plasma membrane domains and to play an essential role in 56

the establishment and maintenance of cell polarity (Johnson et al., 2011). As will be 57

discussed below, in plants RhoGDIs are critical for cell polarity maintenance. 58

Like Rho family proteins from fungi and mammalian cells, ROPs regulate organization and 59

dynamics of the actin and microtubule (MTs) cytoskeleton, endocytosis and exocytosis, and 60

activation of NADPH oxidase and intracellular kinase cascades. Through effects on actin, 61

MTs, vesicle trafficking, reactive oxygen species (ROS) production, and protein 62



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phosphorylation, ROPs regulate cell growth and shape, cytokinesis, subcellular protein 63

localization, and responses to pathogens (Gu et al., 2004; Basu et al., 2008; Yalovsky et al., 64

2008; Yang, 2008; Nagawa et al., 2010; Bloch and Yalovsky, 2013; Kawano et al., 2014; Oda 65

and Fukuda, 2014; Rivero et al., 2017). ROPs are also implicated in the regulation of abscisic 66

acid (ABA) and auxin signaling and transport (Wu et al., 2011; Liao et al., 2017) and in 67

regulation of mRNA transcription (Zhang et al., 2016) and protein translation (Schepetilnikov 68

et al., 2017) (Fig. 1). 69

In recent years, there have been a plethora of new studies on ROPs. Studies on pollen 70

tube and root hair development and plant immune responses have indicated that ROPGEFs 71

are activated by plasma membrane-bound Receptor Like Kinases (RLKs). The next steps will 72

be elucidation of ROP signaling pathways from identification of the upstream receptors to 73

mechanisms of ROPGEF and ROP activation to identification of downstream effectors and 74

their functions (Fig. 1). In this update, we aim to summarize our current understanding of 75

ROP structure-function and signaling with an emphasis on new findings that have not been 76

subject to previous reviews. Given to space limitations and focus, we are unable to cover all 77

published literature in the field including the proposed functions of ROPs in regulating 78

transcription and translation. We thus apologize to colleagues whose works are not cited. 79

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ROP evolution, structure and function 81

Evolution. ROPs have been identified in red and green algae and are ubiquitous in 82

embryophytes where they form multi-member protein families (Brembu et al., 2006; Elias 83

and Klimes, 2012). The number of ROP proteins varies: there are two in the lycophyte 84

Selaginella moellendorffii, four in the moss Physcomitrella patens, four in the gymnosperm 85

Pinus taeda (loblolly pine), seven in rice, nine in maize, nine in tomato, 11 in Arabidopsis, 86

and 13 in poplar (Fowler, 2010). Phylogenetic analyses indicate that the expansion of the 87

ROP family resulted from multiple gene duplication events (Winge et al., 2000; Fowler, 88

2010). Interestingly, ROPs and ROPGEFs or other Rho GTPases have not been identified in 89

some green algae, including Chlamydomonas reinhardtii and Volvox carteri, indicating loss 90

of the ROP/Rho signaling module in these organisms. Phylogenetic analyses indicated that 91

ROPs emerged prior to the diversification of the RAC and Cdc42 subfamilies and represent a 92

unique subfamily of Rho GTPases (Zheng and Yang, 2000; Vernoud et al., 2003; Brembu et 93

al., 2006; Boureux et al., 2007; Rojas et al., 2012). 94



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Analysis of Rho GTPases from divergent eukaryotes revealed that early phylogenetic 95

analyses of the Rho GTPases were biased by a focus on the fungal and animal members of 96

the family. For example, Cdc42, which plays a central role in regulation of cell polarity 97

(Johnson and Pringle, 1990; Woods and Lew, 2017), emerged with the Opisthokonts (fungi 98

and Metazoa) but is absent in other eukaryotes. The amoeba Dictyostelium discoideum has 99

proteins that have been historically called Racs but are divergent from each other as are 100

Rac, Cdc42, and Rho (Elias and Klimes, 2012). The analysis by Elias and Klimes further 101

confirmed that ROPs are unique to the plant kingdom (Elias and Klimes, 2012). Collectively, 102

the phylogenetic analyses carried by several groups justify the name “Rho of Plants” (Zheng 103

and Yang, 2000). As will be discussed in this update, ROPs combine functions of Rac, Cdc42, 104

and possibly other Rho proteins, as could be expected from their early divergence during 105

evolution. Plants also possess a unique family of ROPGEFs (Berken et al., 2005; Gu et al., 106

2006; Elias, 2008) and forms of Cdc42 Rac Interacting Binding (CRIB) domain containing 107

ROPGAPs (Wu et al., 2000). Furthermore, most of the ROP effectors identified to date are 108

unique to plants. Hence, it appears that many components of the ROP signaling module are 109

unique to plants. 110

Structure-Function. ROPs have molecular weights of 21-24 kDa. Like other members of 111

the Ras superfamily, ROPs are composed of an N-terminal catalytic G-domain where 112

nucleotide and effector binding take place and a C-terminal hypervariable domain (HVR), 113

which is responsible for subcellular targeting (Fig. 2A). The G-domain contains five 114

conserved sequence motifs known as the G-box motifs (G1-G5), where nucleotide binding, 115

GTP hydrolysis, and the Mg2+ binding take place. The G2 and G3 motifs are also known as 116

the switch I and switch II loops; these regions have different conformations in GDP- and 117

GTP-bound states and are essential for the transient interaction with effector and 118

regulatory proteins. Within the G domain, ROPs also contain a helical domain unique to the 119

Rho family known as the “Insert Region” or i. The structural conservation of the G-domain 120

has been confirmed by three-dimensional crystal structures of the GDP-bound Arabidopsis 121

ROP9 (Sormo et al., 2006), ROP4 in complex with the catalytic PRONE (for Plant specific ROP 122

Nucleotide Exchanger) domain of ROPGEF3 (Thomas et al., 2007), ROP5 ((Thomas and 123

Berken, 2010) (PDB code: 3BWD)), the nucleotide-free ROP7 in complex with the PRONE 124

domain of ROPGEF8 (Thomas et al., 2009), and GTP-bound Rice RAC1 (Kosami et al., 2014) 125

(Fig. 2B-D). 126



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Based on structural conservation and known mutations in Ras (Feig, 1999), it was 127

predicted and subsequently shown that point mutations in a G1 glycine (G15 in Arabidopsis 128

ROPs1-7 and 9; G17 in ROP10 and 11; G27 in ROP8) or G3 glutamine (Q64 in Arabidopsis 129

ROPs1-7 and 9; Q66 in ROP10 and 11; Q76 in ROP8) would prevent GTP hydrolysis resulting 130

in GTP-locked, constitutively active (CA) mutants. Conversely, mutations in a G1 threonine 131

(T20 in Arabidopsis ROPs1-6, 7, and 9; T22 in ROP10 and 11; T32 in ROP8) or a G4 aspartate 132

(D121 in Arabidopsis ROP1-6, 7, and 9; D123 in ROP10 and 11; D133 in ROP8) are dominant 133

negative (DN), reducing affinity for nucleotides and stabilizing the interaction with GEFs 134

thereby preventing ROP activation (Berken and Wittinghofer, 2008). In addition to the 135

structural conservation of ROPs, the functional conservation of the constitutively active and 136

dominant-negative mutations was confirmed by biochemical assays (Berken et al., 2005; 137

Sorek et al., 2010; Kosami et al., 2014). The constitutively active and dominant-negative ROP 138

mutants have been used extensively for studying ROP function. 139

A comparison between the structure of the non-hydrolyzable GTP analog guanosine 5′-140

[β,γ-imido]triphosphate (GMP-PNP, Gpp(NH)p)-bound rice OsRAC1 and the GDP-bound 141

AtROP9 revealed the conformational differences between the GDP and GTP-bound states of 142

ROPs (Fig. 2B-D) (Kosami et al., 2014). The structural analysis showed conformational 143

differences in the switch I and switch II domains and the insert region (Fig. 2B-D). However, 144

the conformational changes in the switch I domain are the most critical for function. It was 145

shown that GTP-binding by OsRAC1 results in surface exposure of switch I glutamic acid 146

(D45) and a tyrosine (Y39); both are required for its interaction with rice Respiratory Burst 147

Oxidase Homolog (OsRBOHB) NADPH oxidase (Kosami et al., 2014). D45 and Y39 are highly 148

conserved, and it will be interesting to examine whether they play central roles in the 149

interaction of ROPs with other effectors. The structure of ROPs solved so far reveal 150

differences in the structures of the switch II and insert regions (Berken and Wittinghofer, 151

2008; Thomas and Berken, 2010; Kosami et al., 2014). It is not known yet whether these 152

differences also imply different substrate and regulatory specificities. Collectively, the 153

sequence, structural, and functional conservation of ROPs indicate that regardless of their 154

early divergence they function in similar fashion to the well characterized Rho GTPases from 155

fungi and metazoa. 156

The C-terminal HVRs of ROPs are composed of cysteine-containing sequence motifs, 157

which undergo post-translational lipid modifications, and proximal arginine-lysine rich poly 158



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basic regions (PBRs). Type I ROPs (in Arabidopsis ROP1-8) terminate with a canonical CaaL 159

box motif (C-Cys, a1 and a2 are aliphatic residues, L-Leu) in which the Cys residue is modified 160

by the C20 isoprenyl lipid geranylgeranyl. Type II ROPs terminate with GC-CG boxes (G-Gly, 161

C-Cys) in which the two cysteines are separated by 5 or 6 aliphatic residues and undergo S-162

acylation by the C16 palmitate or C18 stearate fatty acids. Type II ROPs are likely not 163

regulated by RhoGDIs since gernaylgeranylation is required for the interaction between Rho 164

protein and RhoGDI. The PBRs in animal Rhos, RACs, and Cdc42 enhance membrane 165

attachment by interaction with phosphatydilphosphoinositide (4,5) diphosphate (PIP2) and 166

PI(3,4,5) P3 (PIP3) (Heo et al., 2006). The PBR is required for plasma membrane attachment 167

of type II ROPs (Lavy and Yalovsky, 2006), possibly by facilitating interaction with PIPs. In 168

addition, ROPs undergo activation-dependent transient S-acylation on conserved G-domain 169

Cys residues (C21 and C156 in AtROP6). The transient S-acylation stabilizes the interaction of 170

ROPs with the plasma membrane, leads to their accumulation in lipid rafts, and is required 171

for their function (Sorek et al., 2010; Sorek et al., 2011; Sorek et al., 2017). 172

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ROP regulators: GEFs, GAPs and RhoGDI 174

GEFs. Plants have two major types of ROP GEFs : 1) a plant-unique family known as 175

ROPGEFs that contain the PRONE catalytic domain (Berken et al., 2005; Gu et al., 2006) and 176

2) distantly related homologs of the animal Ced5, Dock180, Myoblast city (CDM) Zizimin 177

Homology (CZH) RhoGEFs. The CZH RhoGEFs share three tandem Doc Homology Regions 178

(DHR) of which DHR2 has GEF activity. A single CZH RhoGEF homolog called SPIKE1 (SPK1) 179

was identified in Arabidopsis (Qiu et al., 2002), and it was shown that either the full-length 180

protein or its DHR2 domain can catalyze ROP nucleotide exchange (Basu et al., 2008). The 181

rice OsSWAP70A and OsSWAP70B bear homology to Diffuse B cell Lymphoma (Dbl) type 182

RhoGEFs that are prevalent in yeast and mammalian cells. OsSWAP70A and OsSWAP70B 183

have GEF activity and were suggested to function as GEFs for ROPs (Yamaguchi et al., 2012). 184

ROPGEFs. ROPGEFs catalyze nucleotide exchange on ROPs but not on non-plant Rho 185

GTPases (Berken et al., 2005). The specificity for ROPs is likely associated with the presence 186

of a small glycine residue in the insert regions of ROPs at a position which in non-plant ROPs 187

is occupied by a large arginine residue (Thomas et al., 2007; Berken and Wittinghofer, 2008). 188

Structural studies revealed that ROPGEFs function as dimers and interact with two ROP 189

molecules. Interestingly, it was found that the structure of ROPGEFs is different from the 190



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Dbl RhoGEFs, but the mechanism of guanyl nucleotide exchange is similar (Thomas et al., 191

2007). Furthermore, based on the co-crystal structure of ROP4 and PRONE8 it was predicted 192

that that the interaction between ROPs and ROPGEFs could be regulated by 193

phosphorylation (Thomas et al., 2007; Berken and Wittinghofer, 2008; Thomas and Berken, 194

2010). The catalytic PRONE domain of ROPGEFs is constitutively active and its function is 195

regulated by the ROPGEF C-terminal domain (Berken et al., 2005; Gu et al., 2006). Studies in 196

pollen tubes indicated that ROPGEFs interact with membrane-associated RLKs, which 197

phosphorylate the C-terminal domain and relieve the auto-inhibition of the PRONE domain 198

leading to ROPGEF activation (Kaothien et al., 2005; Zhang and McCormick, 2007; Chang et 199

al., 2013). Similarly, studies in root hairs have demonstrated that ROPGEFs function 200

downstream of the RLK FERONIA (FER) (Duan et al., 2010; Yu et al., 2012; Huang et al., 201

2013). Thus, the activation of at least some ROPGEFs depends on intercellular signaling that 202

involves phosphorylation by membrane bound RLKs (Fig. 3). 203

ROPGEFs have only been identified in red algae (Rhodophyta) and green plants 204

(Chloroplastida) and appear to be a family of protein unique to the plant kingdom 205

(archeaplstida/plantae) that are not of a cyanobacterial origin (Elias, 2008). They form 206

protein families with numbers ranging from two in Selaginalla moellendorfii, to six in 207

Physcomitrella patens, to fourteen in Arabidopsis (Berken et al., 2005; Gu et al., 2006; 208

Eklund et al., 2010). Arabidopsis ROPGEF mutants often display mild phenotypes (Li and Liu, 209

2012; Chang et al., 2013; Huang et al., 2013; Zhao et al., 2015; Wang et al., 2017) indicating 210

that they likely function redundantly. Two reports indicated that ROPGEF1 and ROPGEF7 211

have specific functions during embryo development. ROPGEF7 RNAi plants displayed 212

compromised stem cell maintenance in embryo and roots (Chen et al., 2011). The ropgef1 213

mutants are characterized by defective embryo development and embryo auxin distribution 214

and a mild gravitropic response phenotype (Liu et al., 2017), suggesting that the ROPGEF1 215

function in embryos is unique. However, ROPGEF1 might function in conjunction with other 216

ROPGEFs in root gravitropic responses. 217

ROPGEFs are soluble proteins that are however, observed in the plasma membrane, 218

suggesting that they interact with components at the plasma membranes that facilitate 219

their attachment to the membrane. Deletion of the AtROPGEF12 C-terminal domain 220

abolished membrane association following transient expression in tobacco pollen tubes. 221

Upon transient expression in tobacco pollen tubes, several ROPGEF-GFP fusion proteins are 222



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detected at the pollen tube tip (Gu et al., 2006). In embryos, the AtROPGEF1-GFP fusion 223

protein, driven by the ROPGEF1 prompter was localized in a polarized manner (Liu et al., 224

2017). Taken together these data indicate that ROPGEFs likely activate ROPs in the plasma 225

membrane and that their spatial distribution influences the site or sites of ROP activation. 226

SPIKE1 (SPK1). SPK1 was discovered in a forward genetic screen in Arabidopsis for 227

mutants with altered trichome development. spk1 mutants are seedling lethal and display a 228

pleotropic phenotype that includes stunted plant size and abnormal leaf pavement cells and 229

trichome growth, cotyledon shape, and cytoskeleton organization (Qiu et al., 2002). In in 230

vitro pull-down assays, SPK1 interacts weakly with GDP-bound and nucleotide-free type I 231

and type II ROPs but not with human RAC1 (Basu et al., 2008), and similar results were 232

obtained in yeast two-hybrid assays (Uhrig et al., 2007). Co-immunoprecipitation and 233

protein interaction assays indicated that SPK1 is associated with the Suppressor of cAMP 234

Receptor (SCAR) / Wiskot-Aldrich syndrome protein-family Verprolin homology protein 235

(WAVE) complex, which activates actin nucleation-branching by the Actin Related Protein 236

2/3 (Arp2/3) complex (Uhrig et al., 2007; Basu et al., 2008). GTP-bound type I ROPs interact 237

with the SCAR/WAVE complex subunit SRA1 (also known as PIROGI/KLUNKER) (Basu et al., 238

2004; Basu et al., 2008) or with other subunits of the complex (Uhrig et al., 2007). 239

Importantly, analysis of SPK1, SRA1, and Arp2/3 mutants demonstrated that they function in 240

the same pathway (Basu et al., 2008). Taken together, the genetic and biochemical analyses 241

demonstrate that SPK1, type I ROPs (possibly ROP2 and ROP4), SCAR/WAVE, and Arp2/3 242

complexes regulate actin nucleation (Figs. 4 and 5). 243

In developing trichomes, nucleation of actin at the tip depends on WAVE-dependent 244

function of Arp2/3 (Yanagisawa et al., 2015), which is likely locally regulated by SPK1. Thus, 245

the combinatorial regulation of SPK1, ROPs, and WAVE subcellular distribution is very likely 246

responsible for localized actin nucleation. It is not known how SPK1 function is regulated; 247

like ROPGEFs, it may be activated by upstream signaling. It is also not known whether SPK1 248

regulates additional pathways. In Physcomitrella patens there are six SPK1 homologs, 249

whereas in Selaginella moellendorfii and Arabidopsis SPK1 is encoded by a single gene 250

(Eklund et al., 2010). Sequence analysis of SPK1 homologs from additional plant species will 251

be required before it can be determined whether the reduction in SPK1 homologs followed 252

an evolutionary trend. Genetic analysis in Physcomitrella patens should reveal whether SPK1 253



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function in regulation of actin nucleation is evolutionarily conserved and whether it had 254

additional functions that were lost during evolution. 255

OsSWAP70. The rice proteins OsSWAP70A and OsSWAP70B contain Pleckstrin Homology 256

(PH) and Diffuse B cell Homology (DH) domains similar to many animal RhoGEFs and display 257

similarity to human SWAP70 RhoGEF (Shinohara et al., 2002; Yamaguchi et al., 2012). 258

OsSWAP70A displays GEF activity toward OsRAC1 and enhances OsRAC1-dependent ROS 259

production. Surprisingly, however, OsSWAP70A interacts with both constitutively active and 260

dominant-negative OsRAC1 and with Osrac7CA mutants but not with the dominant-negative 261

form of Osrac7DN. Furthermore, OsSWAP70B does not interact with any rice ROP 262

(Yamaguchi et al., 2012). SWAP70 homologs have been identified in other plants 263

(Yamaguchi et al., 2012). However, given that OsSWAP70A interacts with GTP-bound RAC1, 264

which contradicts a long held dogma regarding RhoGEF activity (Berken and Wittinghofer, 265

2008; Thomas and Berken, 2010), and in the absence of loss-of-function mutants to support 266

the biochemical analysis, more work is required to determine whether the plant SWAP70 267

homologs are indeed ROPGEFs. 268

ROP GAPs. Fungal and metazoan RhoGAPs share a conserved GAP domain that contains 269

an invariable arginine residue required for the catalytic activity. In addition to the GAP 270

domain RhoGAPs usually contain additional domains that are required for activity and 271

specificity (Lamarche and Hall, 1994; Rittinger et al., 1997; Graham et al., 1999). In plants, 272

there are two types of GAPs for ROPs known as ROPGAPs and RENGAPs (ROP1 Enhancer 273

GAPs) (Wu et al., 2000; Hwang et al., 2008; Eklund et al., 2010). 274

ROPGAPs. ROPGAPs are characterized by a Cdc42 RAC Interacting Binding (CRIB) domain 275

in addition to the GAP domain. The CRIB domain is found in Rho effectors such as WASP in 276

animal cells and ROP Interacting CRIB containing proteins (RICs) in plants and not in fungal 277

and metazoan RhoGAPs. In ROPGAPs, the CRIB domain is required for activity as it enhances 278

the binding between ROPs and ROPGAPs (Wu et al., 2000; Schaefer et al., 2011). The CRIB 279

domain is also required for subcellular localization of ROPGAPs (Wu et al., 2000; Klahre and 280

Kost, 2006). ROPGAPs function as dimers in a 2:2 stoichiometry with ROPs (Schaefer et al., 281

2011). Arabidopsis AtROPGAP2 has differential binding affinities for various ROPs (Schaefer 282

et al., 2011), and phosphorylation regulates the interaction of tobacco RhoGAP1 and an 283

interacting 14-3-3 protein (Klahre and Kost, 2006), suggesting how ROPGAP function might 284

be regulated. 285



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Following transient expression of NtRhoGAP1 in tobacco pollen tubes, the protein was 286

localized at the plasma membrane below the tip (Klahre and Kost, 2006; Sun et al., 2015). In 287

agreement with this finding, ectopic expression of Arabidopsis AtROPGAP1 in tobacco pollen 288

tubes restricts the active ROP domain (Hwang et al., 2010) (Fig. 4). ROPGAPs are also 289

important rheostats of ROP signaling. In Arabidopsis, O2 deprivation induces ROP-regulated 290

production of ROS. In turn, ROS induce the expression of ROPGAP4, which downregulates 291

ROP activity and contributes to plant tolerance under anoxic conditions (Baxter-Burrell et 292

al., 2002). Gene expression studies in apple showed that ethylene coordinately 293

downregulates ROPs, ROPGAPs, and ROPGEFs leading to altered apoplastic H2O2 levels, 294

suggesting that regulation of ROP activation status has an important function in regulation 295

of different abiotic stress and physiological responses (Zermiani et al., 2015). 296

RENGAPs. RENGAPs were initially identified by a mutant screen in pollen overexpressing 297

ROP1. RENGAPs contain a PIP-binding PH domain in addition to the GAP domain (Hwang et 298

al., 2008). In pollen tubes, REN1 (also known as RENGAP1) is localized in subapical 299

cytoplasmic vesicles and its PH domain is not required for this localization. ren1-mutant 300

pollen tubes are swollen indicating that REN1 functions to restrict the active ROP domain to 301

the pollen tube tips (Hwang et al., 2008) (Fig. 4). 302

RENGAP2 and RENGAP3 interact with the mitosis-specific kinesin 12 POK1. During 303

interphase RENGAP2 and RENGAP3 are cytoplasmic, but during mitosis they are localized to 304

the cortical division zone (CDZ). The rengap2 rengap3 double mutant displays mild cell 305

division reorientation, indicating that rapid inactivation of ROPs at the CDZ is required for 306

correct orientation of cell divisions (Stockle et al., 2016). The division of labor between 307

ROPGAPs and RENGAPs is currently unknown, and in some cases, such as during mitosis, 308

they might function redundantly (Stockle et al., 2016). 309

Formation of active ROP domains by the combinatorial function of ROPGEFs and 310

ROPGAPs. During metaxylem development, simultaneous expression of ROP11, ROPGEF4, 311

and ROPGAP3 results in the formation of active ROP domains. The formation of active 312

ROP11 domain was recapitulated by transient co-expression of ROP11, the PRONE domain 313

of ROPGEF4 (ROPGEF4PRONE), and ROPGAP3. Within the domain, ROP11 and ROPGEF4 are 314

localized in the center and ROPGAP3 in the circumference (Oda and Fukuda, 2012). These 315

experiments demonstrated that ROP-ROPGEF-ROPGAP modules have self-organization 316



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properties, suggesting that coordinated expression and localized activation of ROPGEFs can 317

together result in cellular patterning (Figs. 3 and 4). 318

RhoGDIs. Plant RhoGDIs are structurally conserved (Bischoff et al., 2000). Structural and 319

biochemical analyses have established that the interaction between Rho proteins and 320

RhoGDI under physiological conditions depends on prenylation by geranylgeranyl of the 321

GTPases and involves a hydrophobic pocket in the RhoGDI (Hoffman et al., 2000; 322

DerMardirossian and Bokoch, 2005). In plants, type I ROPs are geranylgeranylated (Sorek et 323

al., 2011; Sorek et al., 2017), but type II ROPs do not contain a conserved 324

geranylgeranylation CaaL box motif and are S-acylated but not prenylated (Lavy et al., 2002; 325

Lavy and Yalovsky, 2006). It is therefore likely that type II ROPs are not regulated by 326

RhoGDIs. 327

Transient expression of NtRhoGDI1 in tobacco pollen tubes indicated that that it 328

functions in recycling of ROPs and in their focusing at the pollen tube tip (Klahre et al., 2006; 329

Sun et al., 2015). Analysis of RhoGDI mutants in Arabidopsis showed their involvement in 330

the maintenance of ROP activation domains and ROP stability (Carol et al., 2005; Feng et al., 331

2016). There are three RhoGDIs in Arabidopsis called RhoGDI1, RhoGDI2a, and RhoGDI2b. 332

RhoGDI1 mutants, also known as SUPERCENTIPIDE 1 (SCN1), develop multiple root hair (RH) 333

initials instead of a single RH in each trichoblast. In scn1 mutants, GFP-ROP2 was observed 334

along the trichoblast membrane and ROS production was detected at multiple sites, in 335

contrast to wild-type plants where ROP2 accumulation and ROS formation are detected 336

specifically at the RH initiation sites (Carol et al., 2005). Interestingly, the phenotype of scn1 337

fits a mathematical model predicting that a loss of RhoGDI would result in “traveling waves” 338

instead of a focal polar domain (Jilkine et al., 2007). 339

Silencing of RhoGDI2a by RNAi resulted in development of shorter pollen tubes and 340

expansion of ROP distribution; however, possible redundancy with other RhoGDIs was 341

noted (Hwang et al., 2010). rhogdi1 rhogdi2a rhogdi2b triple mutants develop shorter and 342

wider pollen tubes with higher levels of ROPs in the pollen tube tip plasma membranes and 343

expanded ROP activation domains. In addition, protein immunoblots indicated that the 344

overall ROP levels are lower in the RhoGDI triple mutants (Feng et al., 2016). In mammalian 345

cells, reduction in RhoGDI1 levels by siRNA resulted in higher levels of plasma membrane 346

associated Rho proteins concomitantly with reduction in their levels (Boulter et al., 2010). It 347

appears therefore that RhoGDIs have evolutionary conserved function in regulation of 348



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homeostasis of Rho GTPases. Notably, although the RhoGDI triple mutant plants are less 349

fertile than wild-type plants they produce viable seeds and their sporophytes do not seem 350

to be significantly different from wild-type controls (Feng et al., 2016). This indicates that 351

type I ROPs are functional and possibly there is redundancy between type I and type II ROPs. 352

353

ROP function in tip and diffuse growth 354

Regulation of pollen tubes tip growth Pollen tubes are characterized by highly polarized 355

oscillatory tip growth that depends on differential cell wall composition tip focused vesicle 356

transport, specialized actin organization, and Ca2+, ROS, and pH gradients (Cardenas et al., 357

2008; Cheung and Wu, 2008; Chebli et al., 2012; Bloch et al., 2016; Michard et al., 2017). 358

Actin organizes into multiple forms in different regions of pollen tubes, including thick long 359

actin cables that are axially aligned in the shank, a mesh ring or funnel-like F-actin structure 360

known as actin fringe in the subapical region and a group of fine and less abundant F-actin 361

in the pollen tube apex. Various form of F-actin structures participate in cytoplasmic 362

streaming and vesicle trafficking to and from the tip and accumulation of exocytotic vesicles 363

at the pollen tube apex (Cardenas et al., 2008; Cheung and Wu, 2008; Qin and Yang, 2011; 364

Rounds et al., 2014; Fu, 2015; Qu et al., 2017). ROP signaling regulates pollen growth and 365

was shown to affect actin organization, Ca2+ levels, and vesicle trafficking. 366

LUREs are ovule-derived small peptides that attract the pollen tube toward ovules 367

(Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015). Several LURE1 368

receptors have been identified (Takeuchi and Higashiyama, 2016; Wang et al., 2016). It was 369

shown that Pollen-specific Receptor Kinase 6 (PRK6) is a LURE1 receptor that localizes to 370

pollen tube tip and interacts with the regulatory C-terminal domain of pollen-expressed 371

ROPGEF 8/9/12/13 (Takeuchi and Higashiyama, 2016) (Fig. 3). In agreement, 372

ROPGEF8/9/12-GFP fusion proteins are localized at the pollen tube tip following transient 373

expression in tobacco pollen tubes and AtROPGEF9 has high specific activity toward the 374

pollen-expressed AtROP1 (Gu et al., 2006). Tomato pollen PRK1 and PRK2 interact with a 375

ROPGEF, originally called Kinase Protein Partner (KPP) (Kaothien et al., 2005). Arabidopsis 376

PRK2a interacts with ROPGEF12 in yeast through the C-terminal regulatory domain. Co-377

expression of PRK2a and ROPGEF12 in tobacco pollen tube induce isotropic growth, and a 378

phospho-mimicking mutation at the C-terminal domain activates ROPGEF12 (Zhang and 379

McCormick, 2007). Taken together, the results from studies described in this section 380



14

strongly suggest that upon LURE perception, PRKs activate pollen-specific ROPGEFs that in 381

turn activate pollen specific ROPs. It should be noted that ROPGEFs-dependent ROP 382

activation by LUREs has yet to be demonstrated. 383

Ectopic expression of constitutively active ROP mutants results in isotropic pollen tube 384

growth, whereas expression of dominant-negative ROP mutant or ROPGAPs inhibit pollen 385

tube growth and are associated with changes in actin organization (Fu et al., 2001; Klahre et 386

al., 2006; Klahre and Kost, 2006). Several studies addressed the mechanisms by which ROPs 387

regulate actin organization. Co-expression studies in tobacco pollen tube suggest that the 388

tobacco ROP NtRAC5 can lead to phosphorylation and suppression of actin 389

depolymerization activity of Actin Depolymerizing Factor (ADF; also known as Cofilin) (Chen 390

et al., 2003). 391

Three RIC proteins were implicated in regulation of pollen tube growth. RIC3 function is 392

associated with increase in intracellular Ca2+ levels, leading to actin depolymerization, 393

whereas RIC4 is implicated in actin polymerization. The antagonistic functions of RIC3 and 394

RIC4 are suggested to regulate actin dynamics in pollen tubes (Fig. 5) (Gu et al., 2005). The 395

mechanisms of RIC3 and RIC4 function remain to be elucidated. RIC1 regulates pollen 396

growth through its F-actin severing and capping activities. RIC1 displays oscillatory 397

localization at the apical plasma membrane of pollen tube tip, which depends on an intact 398

CRIB domain. The severing activity of plasma membrane-localized RIC1 mediates the release 399

of F-actin from the pollen tube apical plasma membrane into the cytoplasm. RIC1 also 400

contributes to severing F-actin and capping the barbed ends in the cytoplasm (Zhou et al., 401

2015). 402

An effector of the tobacco ROP called RAC5 Interacting Subapical Pollen tube protein 403

(RISAP) was identified in a yeast two-hybrid screen. RISAP-XFP fusion proteins localize to the 404

trans-Golgi network and interact with actin and with the globular tail domain of a tobacco 405

myosin XI through a protein called Domain of Unknown Function 593 (DUF593). RISAP 406

overexpression interferes with apical membrane trafficking and blocks tip growth. It was 407

suggested that RISAP functions in tip-directed vesicle trafficking in a ROP-dependent fashion 408

(Stephan et al., 2014). It would be of interest to analyze RISAP loss-of-function mutants to 409

further examine its function. An Arabidopsis protein (At1g18990) is 43% identical to RISAP 410

and homologs have also been identified in other plant species. Thus, it should be possible to 411

examine the conservation of RISAP function. 412



15

Using RabA4d as an exocytotic marker in transient expression assays in tobacco pollen 413

tubes, it was shown that its accumulation displays an oscillatory pattern that precedes the 414

growth phase and is regulated by F-actin (Lee et al., 2008). Analysis of the exocyst Sec3a 415

subunit mutant demonstrated that polarized exocytosis is required for both the selection of 416

the pollen tube initiation site and polarized growth (Bloch et al., 2016). It is not known 417

whether ROPs are directly involved in the regulation of polarized exocytosis in pollen. 418

Analysis of multiple pollen expressed ROP mutants has not yet been described, and it is not 419

known whether they are required for selection of the pollen tube germination sites, similar 420

to the function of Cdc42 in the selection of the bud site in Saccharomyces cerevisiae 421

(Johnson and Pringle, 1990; Woods and Lew, 2017). 422

Regulation of root hair tip growth. RH tip growth is characterized by actin/myosin-423

dependent vesicle trafficking toward the growing tip and cell wall relaxation at the tip 424

(Mendrinna and Persson, 2015). RHs form close to the rootward side of trichoblasts and 425

their initiation and growth require tip focused cell wall relaxation that depends on ROS-426

dependent pH fluctuations (Monshausen et al., 2007) and the activity of cell wall expansins 427

(Cho and Cosgrove, 2002). During RH growth, F-actin forms fine meshwork at the tip and 428

longitudinally filaments oriented axially along the RH shank. When growth ceases, the actin 429

filaments are also detected at the RH tip (Jones et al., 2002; Bloch et al., 2005; Bloch et al., 430

2011). ROPs regulate both RH initiation and growth. Prior to RH initiation, ROP2/4/6 431

accumulate at the site of future RH formation in response to a local auxin gradient 432

(Molendijk et al., 2001; Jones et al., 2002; Fischer et al., 2006; Ikeda et al., 2009) (Fig. 4). The 433

accumulation of ROPs at future RH formation sites was found to be altered in procuste 1 434

(prc1) cellulose synthase mutant, in clasp and sabre mutants that affect MTs organization 435

and in actin 7 (act7) and actin interacting protein 1-2 (aip1-2) mutants, suggesting 436

involvement of the cell wall MTs and F-actin in the regulation of the RH initiation sites (Singh 437

et al., 2008; Pietra et al., 2013; Kiefer et al., 2015). 438

ROP2 is expressed in RHs and its overexpression induces ectopic RH initiations, resulting 439

in higher RH density; expression of dominant-negative ROP2 mutant reduces RH density. 440

Importantly, overexpression of the activated ROP2CA also induces formation of additional 441

RHs, but the numbers were significantly lower than observed for wild-type ROP2 442

overexpressors (Jones et al., 2002). This suggests that ROP2 recycling between active and 443

inactive states is essential for RH initiation. The rop2 null mutant develops shorter RHs, 444



16

indicating that ROP2 is required for RH growth but that its function is redundant with other 445

ROPs. Interestingly, ectopic expression of ROP6/7/11 did not increase RH initiations, 446

suggesting that ROP2 and possibly ROP4 signaling in RH initiation is specific (Molendijk et 447

al., 2001; Jones et al., 2002; Bloch et al., 2005). Interestingly, RHs developed in pluripetala 448

(plp) mutant plants, which lack protein prenylation, suggesting that type II ROPs possibly 449

also contribute to RH initiation (Running et al., 2004; Sorek et al., 2011; Chai et al., 2016). 450

ROP2 activity is also regulated by Microtubule Associated Protein 18 (MAP18), which 451

interacts preferentially with GDP-bound ROP2 and competes with RhoGDI (Kang et al., 452

2017). 453

Expression of either type I or type II constitutively active ROP mutants results in RHs 454

swelling, which is associated with disorganized actin filaments at the RHs tips, inhibition of 455

endocytosis, and non-polar ROS distribution (Molendijk et al., 2001; Jones et al., 2002; Bloch 456

et al., 2005; Sorek et al., 2010). ROPs activate respiratory burst oxidase homolog (RBOH)-457

type NADPH oxidases during immune responses (Wong et al., 2007; Nagano et al., 2016). 458

The SCN1/RhoGDI1-dependent and RHD2/AtRBOHC-dependent ROS accumulation at RH 459

formation sites strongly suggest that ROP2/4 activates RBOHC during RH formation and 460

growth (Carol et al., 2005). Consistent with the link between ROPs, ROS formation and pH 461

regulation, it was found that the ROP11CA-induced RH swelling was suppressed by omission 462

of nitrogen containing compounds (either NH4+ or NO3

-) from the growth medium. The 463

omission or addition of NH4+ and NO3

- affected intracellular and extracellular pH fluctuations 464

and the overall pH at RH tips, implying a close correlation between ROP signaling and the pH 465

at the RH tip (Bloch et al., 2011; Bloch et al., 2011). 466

Upon activation, at least some ROPs undergo activation-dependent transient S-acylation 467

on one or two G domain cysteine residues and consequently partition into detergent 468

resistant membrane, which is required for their function (Sorek et al., 2009; Sorek et al., 469

2010; Sorek et al., 2011; Sorek et al., 2017). Consistently, ROP2 association with the plasma 470

membrane is compromised in Protein S-acyl Transferase 4 (PAT4) mutants. pat4 RHs are 471

shorter and the multiple RH phenotype of scn1/rhogdi1 mutant is suppressed in the pat4 472

background. Furthermore, ROPs activate rice RBOHB/H by recruiting them into lipid 473

microdomains (Nagano et al., 2016). At the future RH initiation sites, ROP2/6 co-localize 474

with PIP 4 phosphate 5 kinase 3 (PIP5K3), Dynamin Related Protein 1A (DRP1A), DRP2B, and 475

the AGCVIII kinase D6 Protein Kinase (D6PK) in sterol enriched domains (Stanislas et al., 476



17

2015). Auxin has been implicated in ROP activation (Tao et al., 2002; Wu et al., 2011; 477

Miyawaki and Yang, 2014), yet the underlying mechanisms remain to be elucidated. PIN 478

phosphorylation by D6PK regulates auxin transport (Barbosa et al., 2014; Zourelidou et al., 479

2014). Hence, the association of ROPs and D6PK in the RH initiation domain may provide a 480

link between ROP signaling and auxin (Fig. 4 and Box 2). 481

Signaling between the cell wall extensibility and mechanisms that regulate cell growth is 482

mediated in RHs by the RLK FER. FER is a member of the CrRLK1 (Catharanthus roseus RLK1) 483

family that are thought to sense the cell though extracellular malectin-like domains that are 484

presumed to interact with carbohydrates (Hofte, 2015; Li et al., 2016; Voxeur and Hofte, 485

2016). RHs collapse or burst in fer mutant alleles. FER interacts with ROPGEF1 in yeast two-486

hybrid and in bimolecular fluorescence complementation assays and active ROP2 levels and 487

ROS production are reduced in fer mutant plants (Duan et al., 2010). Hence FER appears to 488

regulate ROP function during the RH growth phase (Fig. 3). 489

To summarize, local auxin concentration gradients direct the formation of sterol-490

enriched ROP domains. The formation of single rather than multiple ROP domains depends 491

of RhoGDI-dependent recycling of ROPs to and from the plasma membrane as well as 492

cycling of ROPs between active and inactive states. During RH growth ROP function is 493

regulated by FER, which coordinates and adjusts cell wall composition in response to 494

intracellular growth signals. Auxin signaling is also known to regulate RH growth (Knox et al., 495

2003), but it is unknown yet whether or not it signals through ROPs. Activated ROPs likely 496

activate RBOHC leading to ROS production and associated changes in intracellular and 497

extracellular pH and intracellular Ca2+ levels (Foreman et al., 2003; Takeda et al., 2008). 498

Through effectors, which are yet to be identified, ROPs regulate MTs and F-actin 499

organization and dynamics (Fig. 3). 500

ROP function during diffuse growth. With the exception of RHs and pollen tubes, plant 501

cells grow by diffuse growth; this implies that their growth proceeds along the cell and is not 502

limited to a single domain as in tip growth. Three model systems have been extensively used 503

for studying ROP function during diffuse growth: trichomes, leaf epidermal pavement cells, 504

and petal epidermal cells. Trichomes grow faster at the region closer to the tip where the 505

cell wall is thinner than that of the basal side. Dense transverse MT arrays exist at the upper 506

half but are absent from the tip and correspond to higher cell wall pressure and tension. 507

Arp2/3 driven actin nucleation takes place at the MTs clear zone at the tip and depends on 508



18

active WAVE complex (Yanagisawa et al., 2015). It is likely that ROPs are locally activated by 509

SPK1 to promote WAVE activation. Expression of constitutively active rop2CA resulted in 510

development of trichomes with thicker stalks and branches and altered branch position 511

while expression of dominant negative ROP mutants did not alter trichome growth (Fu et 512

al., 2002). However, expression of the Clostridium difficile toxin B catalytic domain, which 513

glycosylates and inhibits ROP function, alters trichome morphogenesis (Singh et al., 2013). It 514

is expected that genetic analysis of loss-of-function ROP mutants will reveal which ROPs are 515

involved in trichome development. 516

Pavement cells are interdigitated, characterized by their lobes and indentations. Mutual 517

stresses operating between neighboring cells affect the orientation of the MTs and in turn 518

the patterning of cell growth (Sampathkumar et al., 2014). Compromised isotropic 519

pavement cells with reduced interdigitation are observed in plants with gain- and loss-of-520

function mutants in genes encoding ROP, ROP effectors, SPK1, and PLP and in transgenic 521

plants expressing anti-ROP toxins (Fu et al., 2002; Qiu et al., 2002; Bloch et al., 2005; Fu et 522

al., 2005; Lavy et al., 2007; Fu et al., 2009; Sorek et al., 2010; Sorek et al., 2011; Poraty-523

Gavra et al., 2013; Singh et al., 2013). Based on analysis of Arabidopsis ROP2, ROP4, and 524

ROP6 loss-of-function and constitutively active mutants it was proposed that these proteins 525

co-regulate pavement cell growth by affecting the organization of both cortical MTs and F-526

actin, via different ROP effectors. ROP6 interacts with RIC1, a ROP effector and MTs binding 527

protein. MT-associated RIC1 interacts with the P60 subunit of MT severing protein katanin 528

(KTN1). RIC1 promotes the MT severing activity of KTN1, and the formation of the 529

transverse cortical MTs (Fig. 5) (Fu et al., 2005; Fu et al., 2009; Lin et al., 2013). 530

Overexpression of RIC1 induced highly ordered transverse MTs that result in isomorphic cell 531

growth. ric1 loss-of–function pavement cells exhibited more randomly oriented cortical MTs 532

and wider neck/indentation region than the wild type (Fu et al., 2009). ROP2 and possibly 533

ROP4 are were shown to promote formation of actin microfilaments via another ROP 534

effector, RIC4. ROP2 was also shown to recruit RIC1 to the plasma membrane and away 535

from MTs thereby inhibiting its action on MTs (Fu et al., 2005). As ric1 loss-of-function 536

pavement cells are interdigitated (Fu et al., 2009), RIC1 function is likely redundant during 537

pavement cell growth. Treating cotyledons with cellulase results in development of 538

isomorphic non-interdigitated pavement cells. Interestingly, the cellulase-induced 539

isomorphic pavement cell growth phenotype was suppressed in a ric1 loss-of-function 540



19

background (Higaki et al., 2017), suggesting that RIC1 could be part of a pathway that senses 541

cell wall integrity. 542

The physical and signaling interaction between FER and ROPGEF1 established a 543

connection between cell wall sensing and ROP signaling. It was recently reported that 544

ROPGEF4 interacts with the cell wall sensing CrRLK THESEUS1 during the defense response 545

to Botrytis cinerea (Qu et al., 2017). It will be interesting to study the function of RIC1 in 546

these signaling cascades. Further work will be required to elucidate how ROP signaling is 547

integrated with cell wall pressure and strain and what are the signals that activate and 548

inactivate ROPs during pavement cell growth. 549

The abaxial petal epidermal cells are interdigitated. The interdigitation of the cells is 550

compromised in the spk1 mutant and in the rop2 rop6 rop4RNAi mutant, and cells display 551

transverse MT arrays. Furthermore, the levels of active ROP2 and ROP6 are lower in spk1 552

than wild-type plants (Ren et al., 2016). Taken together, these results indicated that SPK1 is 553

responsible for activating ROP2, ROP6, and likely ROP4, which together regulate growth of 554

petal epidermal cells. The adaxial petal epidermis cells have cone-like structures with sharp-555

angled tips and transverse MT arrays. In ktn1 mutants the cone-shaped cells have wider tip 556

angles and less ordered MT arrays. Similarly, spk1 and rop2 rop6 rop4RNAi mutants develop 557

cells with wider tip angles. It was proposed that ROP2/6 and 4 regulate MTs via KTN1 (Ren 558

et al., 2017). These new finding also suggest that ROP signaling networks play important and 559

general roles in the regulation of cell expansion and cell shape, however, the precise 560

regulatory mechanisms might be distinct in specific organs. 561

562

ROP function in secondary wall patterning, cytokinesis and nuclei migration 563

Regulation of secondary wall patterning by ROPs. During vascular differentiation, the ROP 564

effector Microtubule Depletion Domain 1 (MIDD1) also known as Interactor of 565

Constitutively Active ROP 5 (ICR5) or ROP Interacting Partner 3 (RIP3) is a regulator of 566

secondary cell wall deposition in metaxylem. MIDD1 is an MT binding protein that interacts 567

with the MT-destabilizing protein KINESINE13A (KIN13A) (Mucha et al., 2010). During 568

metaxylem differentiation, MIDD1 promotes depolymerization of cortical microtubules, 569

leading to the formation of secondary wall pits (Oda et al., 2010). During metaxylem 570

differentiation, co-expression of ROP11, ROPGEF4, and ROPGAP3 results in formation of 571

activated ROP11 domains. The active ROP11 in turn recruits MIDD1, which in turn recruits 572



20

KIN13A leading to local MT breakdown (Oda and Fukuda, 2012). It has recently been 573

demonstrated that the size of the active ROP domains in the metaxlyem is limited by a 574

plasma membrane-associated protein designated IQD13, which affects MT orientation and 575

dynamics (Fig. 5) (Sugiyama et al., 2017). 576

The function of ROPs in regulation of cytokinesis. Mutant and protein localization 577

studies have implicated ROPs in the regulation of cytokinesis. In dividing BY2 cells, GFP-578

AtROP4 is associated with the cell plate (Molendijk et al., 2001). During stomata 579

development in maize, partial knockout of type I ROPs (rop2-/- rop9+/-) or treatment with a 580

RAC inhibitor results in weak cell division polarization defects. ROP2/9 accumulate at the 581

junctions between Subsidiary Mother Cells (SMC) and Guard Mother Cells (GMC) and their 582

accumulation at these sites depends on the membrane RLK PANGLOS 1 (PAN1). When 583

expressed at high levels, GFP-ZmROP2 is not polarly localized in SMCs, and cell division 584

polarization defects are observed. Furthermore, ROP accumulation in detergent-resistant 585

membranes is lower in the pan1 mutant than in wild-type plants, suggesting that PAN1 is 586

required for ROP activation. The accumulation of ROPs and PAN at the SMC/GMC junctions 587

precedes formation of actin patches at these locations (Humphries et al., 2011). 588

The first component known to accumulate at the SMC/GMC junctions is the SCAR/WAVE 589

complex. PAN1 and PAN2 polarization and accumulation at the SMC/GMC junction is 590

compromised in a brk1 SCAR/WAVE subunit mutant. Based on these results and the 591

interaction between SCAR/WAVE subunits and ROPs and SPK1, it was proposed that the 592

SCAR/WAVE complex recruits both PAN1 and ROP2/9 and that resulting ROP activation 593

leads to the activation of SCAR/WAVE and in turn Arp2/3 activation and formation of the 594

actin patch (Facette et al., 2015). It is not yet known whether SPK1 is associated with the 595

SCAR/WAVE complex in maize, whether the PAN1-dependent ROP activation is associated 596

with catalytic activity of PAN1 on ROPs or ROPGEFs, or whether it is required for the 597

recruitment of ROPs to the SMC/GMC junctions through a protein-protein interaction. 598

A role for ROP signaling in the regulation of cytokinesis polarity in the Arabidopsis root 599

has been recently established (Stockle et al., 2016). Two PH domain containing ROPGAPs 600

designated PHGAP1/2 interact the PHRAGMOPLAST ORIENTING KINESIN 1 (POK1). phgap1 601

phgap2 double mutants display weak cell wall positioning phenotype, and during 602

cytokinesis PHGAP1 and PHGAP2 proteins localize to the CDZ in a POK1-dependent fashion 603

(Stockle et al., 2016). Taken together, these data suggest that the down-regulation of ROPs 604



21

at specific sites at the cell division zone is required for proper orientation of the cell division 605

planes. 606

ROP signaling and nuclear migration. The case ROP8 and nuclei migration in the ovule 607

central cell. The central cell contains actin filaments that grow from the plasma membrane 608

toward the nucleus. Upon entry of the of the sperm nucleus it is engulfed by actin filaments 609

that make an astral-like structure that pull it toward the central cell nucleus. AtROP8 is 610

uniquely expressed in the central cell and is localized at the plasma membrane. Expression 611

of rop8DN disrupted the organization of the astral-like actin filaments and the migration of 612

the sperm nuclei. Interestingly expression of rop8CA did not affect actin organization or 613

pollen nucleus migration, suggesting that ROP8 is maintained in an active state in the 614

central cell (Kawashima et al., 2014). Nuclei migration is a vital process in the differentiation 615

of different cell types in plants, and it would be of interest to examine whether other ROPs 616

have a broad role in other cell types. 617

The regulation of nuclei migration during root hair formation by ROP signaling. Prior to 618

RH emergence trichoblasts nuclei are localized close to the inner cell membrane at the 619

center of the long cell axis. Consequently, the nuclei migrate into the growing RHs. It has 620

recently been reported that the positioning of the nuclei close to the inner membrane and 621

their migration are regulated by ROP and auxin signaling that synergistically regulate actin 622

organization and dynamics (Nakamura et al., 2017). 623

624

ROP function in biotic and abiotic stress responses 625

Biotic responses. The roles of ROP GTPases in plant pathogen and biotic responses has 626

been subject of recent detailed reviews (Bhagatji et al., 2010; Kawano et al., 2014; Rivero et 627

al., 2017). This section will briefly highlight certain relevant aspects. Class VI Receptor-like 628

Cytoplasmic Kinases (RLCKs) belong to the large superfamily of receptor-like kinases, which 629

are involved in a variety of cellular processes including plant growth, development, and 630

immune responses (Jurca et al., 2008; Lin et al., 2013). RLCKs of the VI class appear to be 631

downstream ROP effectors that regulate the pathogen response (Molendijk et al., 2008; 632

Huesmann et al., 2012; Reiner et al., 2015). Barley HvRBK1 is a type VI_A RLCK that is 633

activated by HvRACB upon infection by the fungal pathogen Blumeria graminis f.sp. hordei 634

(Bgh) (Huesmann et al., 2012). Transient knockdown of HvRBK1 influences the stability of 635

the cortical microtubules in barley epidermal cells. HvRACB also regulates a RIC family 636



22

member, RIC171, in the pathogen defense response. Overexpression of RIC171, similar to 637

overexpression of constitutively activated RACB, renders epidermal cells more susceptible 638

to penetration by Bgh. Additionally, RIC171 accumulates at sites of fungal attack, suggesting 639

an enhanced ROP activity at sites of attempted fungal penetration (Schultheiss et al., 2008). 640

HvRACB signaling is regulated by the barley MICROTUBULE-ASSOCIATED ROPGAP1 641

(HvMAGAP1) limiting the susceptibility of barley to Bgh (Hoefle et al., 2011). 642

In rice, OsRAC1 plays a critical role in defense responses induced by various Microbe 643

Associated Molecular Patterns (MAMPs) including chitin. OsRAC1 is activated by chitin 644

perception mediated by OsCERK1, a receptor-like kinase that forms a complex with OsCEBiP, 645

a chitin binding protein (Shimizu et al., 2010). In the presence of chitin, OsCERK1 646

phosphorylates and activates OsRACGEF. In turn, OsRAC1 activation activates lignin 647

biosynthesis by activating Cynnamoyl CoA Reductase (CCR) and ROS production by 648

activation of NADPH oxydase (Wong et al., 2007; Akamatsu et al., 2013; Nagano et al., 649

2016). 650

In Medicago trancatula and Lotus japonicus, ROPs, MtROP10 and LjROP6 interact with 651

RLKs MtNFP and LjNFR5 in the membrane, respectively, and are required for rhizobium-652

induced nodule formation and for tip growth (Ke et al., 2012; Lei et al., 2015; Ke et al., 2016; 653

Rivero et al., 2017). It is not known yet whether ROPGEFs co-localize with the respective 654

ROPs and RLKs. 655

Expression of dominant negative Atrop6DN under regulation of the ROP6 promoter 656

induced salicylic acid (SA)-dependent systemic acquired resistance (SAR) response 657

characterized by constitutive expression of Pathogenesis Related (PR) genes. The rop6DN 658

plants are small, have twisted leaves with rectangular pavement cells, and have altered 659

actin and microtubule organization. rop6DN plants with mutations in SA biosynthesis and 660

signaling factors demonstrated that the developmental and SAR phenotypes are separable. 661

Interestingly, regardless of the constitutive SAR response, the rop6DN plants did not display 662

enhanced resistance to Golovinomyces orontii powdery mildew (Poraty-Gavra et al., 2013). 663

Possibly, the rop6DN-induced changes in cytoskeleton organization and cell wall influence 664

the SAR response. In the future, it will be important to carry out gene expression analyses 665

because in these mutants as this analysis could potentially reveal “hidden” phenotypes that 666

affect the plant that would otherwise remain undetectable. 667



23

ROPs and Abiotic signaling responses. Accumulating evidence indicates that ROPs and 668

ABA form negative feedback loops in which ABA signaling suppresses ROP activation and 669

ROP signaling suppresses ABA responses (Lemichez et al., 2001; Zheng et al., 2002; Xin et al., 670

2005; Li et al., 2012; Li et al., 2012; Li et al., 2012; Li and Liu, 2012; Yu et al., 2012; Li et al., 671

2016). ROP10 and ROP11 inhibit ABA signaling by physically interacting with ABA negative 672

regulators, ABA Insensitive 1 (ABI1) and ABI2 PP2C phosphatases. It was postulated that the 673

interactions with ROPs prevent ABI1/2 ABA-dependent inactivation by PYR/PIL ABA 674

receptors (Li et al., 2012; Yu et al., 2012). ABA inhibits ROP activation by promoting the 675

degradation of several ROPGEFs. ROPGEF1 interacts with ABI1 in yeast and in vitro and in 676

response to ABA. In the abi1 abi2 hab1 pp2CA quadruple mutant it is localized to prevacuolar 677

compartments and undergoes degradation (Li et al., 2016). Thus, ABI1 and possibly other 678

phosphatases may form regulatory hubs that interact with both ROPGEFs and ROPs forming 679

a positive feedback loop that suppresses ABA responses. Upon increase in ABA levels, PP2C 680

is sequestered by PYR1/PYL ABA receptors, leading to ROPGEF degradation and ROP 681

inactivation. Oxygen deprivation induces expression of ROPGAP4, and ropgap4 mutant 682

plants displayed lower oxygen deprivation tolerance due to ROS-induced over production of 683

alcohol dehydrogenase (Baxter-Burrell et al., 2002). Hence ROP activation/inactivation loops 684

play an important function in abiotic stress tolerance and ABA signaling. 685

A recent report indicates that ROPGEF2 is activated directly or indirectly by phyB and in 686

turn activates ROP2 and ROP7 to suppress red light-induced stomata opening (Wang et al., 687

2017). Nucleotide exchange assays presented in this work suggest that ROPGEF2 catalyzes 688

nucleotide activity on Cdc42. Yet, it is well documented that ROPGEFs are specific to plant 689

ROPs and cannot catalyze GDP/GTP exchange on non-plant Rho GTPases (Berken et al., 690

2005; Thomas et al., 2007; Thomas and Berken, 2010). Hence, some of the data presented 691

in this work might have to be revisited. 692

ROP signaling is involved in regulation of abiotic stress via regulation of the cytoskeleton. 693

The biphasic reorganization of MTs (rapid MT disassembly followed by reassembly of new 694

MTs) induced by high salinity is required for plant tolerance to salt stress. ROP2-RIC1 695

signaling pathway was found to modulate microtubule reorganization in response to salt 696

stress in Arabidopsis (Li et al., 2017). During salt stress, activation of ROP2 leads to removal 697

of RIC1 from MTs leading to enhance MTs reassembly and in turn an increase plant 698

tolerance to the salt stress (Li et al., 2017). 699



24

700

701

Figure legends 702

Figure 1: ROP signaling pathways. A, ROP activation/deactivation cycles. ROPs cycle 703

between a GTP-bound active state and a GDP-bound inactive state. Activation is regulated 704

by ROP specific GEFs, inactivation is enhanced by ROP-specific GAPs and recycling of type I 705

ROPs between the cytosol and the plasma membrane is facilitated by RhoGDIs. Some GEFs 706

are activated by peptide-activated, plasma membrane-associated RLKs. ROPs are also 707

upregulated by auxin by an as yet unknown pathway (dashed arrow) and inactivated by 708

ABA, which enhances degradation of ROPGEFs. B, The Dock family GEF homolog SPK1 is 709

associated with the WAVE complex. Upon activation ROP-GTP interacts with the WAVE 710

SRA1 subunit and possibly other subunits resulting in WAVE activation. Activated WAVE 711

activates actin nucleation/branching induced by Arp2/3. C, ROP downstream signaling. ROP 712

regulated pathways and known effectors. Dashed arrow indicates uncertainty as to the 713

cellular targets of ROP. CPL1 is regulated by ROP signaling but does not physically interact 714

with ROPs. TOR interacts weakly with GTP-bound ROP2 and more strongly with the GDP-715

bound form. 716

717

Figure 2: The structures of ROPs. A, A schematic model highlighting the conserved G-box 718

motifs (G1–G5), switch I and II domains, insert region, and the HVR. The positions of the 719

conserved residues mutated in the constitutively active and dominant negative mutations 720

are highlighted by green and magenta arrows, respectively. C22 and C156 are conserved G 721

domain Cys residues that undergo activation-dependent transient S-acylation (black 722

arrows). The HVR terminates with a canonical CaaX box geranylgeranylation motif and PBR 723

in type I ROPs or with a GC-CG box and a proximal PBR in type II ROPs. B, The three-724

dimensional structure of GTP-bound OsRAC1 (OsRAC1 (GMPPNP)). C, The three-dimensional 725

structure of the GDP-bound AtROP9 (AtROP9 (GDP)). In panels B and C, the switch I, switch 726

II, and insert regions are colored green. GMPPNP and GDP are shown as stick models (red, 727

oxygen; blue, nitrogen; orange, phosphorus). The Mg2+ ions are shown as yellow spheres. D, 728

Superimposed structures of OsRAC1(GMPPNP) and AtROP9(GDP). The main chains of 729

OsRAC1(GMPPNP) and AtROP9(GDP) (chain B, PDB code 2J0V) were superimposed using 730

PyMOL. Regions of the OsRAC1 and AtROP9 proteins are colored blue and pink, respectively. 731



25

The side chains of four key Switch I residues in OsRAC1 (Val43, Phe44, Asp45, and Tyr39) and 732

equivalent residues in AtROP9 (Val39, Phe40, Asp41, and Tyr35) are shown in stick 733

representation. Panels B-D were prepared and contributed by Izuru Ohki and Chojiro Kojima 734

and are based on their paper describing the structure of GTP-bound OsRAC1 (Kosami et al., 735

2014). 736

737

Figure 3: ROP signaling pathways. Schematic overview of identified ROP signaling 738

pathways. Components of the signaling pathways mentioned in the text are noted, and their 739

relationships to each other are indicated by arrows. Question marks note areas of 740

insufficient data. From top to bottom in each section are listed the upstream signal, the 741

receptors that relay the signal into the cell, regulation of ROP activity, and the downstream 742

effectors that modulate the cellular response. 743

744

Figure 4: Mechanisms of spatial ROP activity regulation. A, Local activation of ROPs in 745

pollen tube. ROPs are activated at the tip by the activation of ROPGEFs by associated RLKs. 746

CRIB-domain ROPGAPs inactivate ROPs at the shank to maintain polar growth. PH domain 747

GAPs are localized in subapical cytoplasmic vesicles, restricting ROP active domains. 748

RhoGDIs recycle ROPs from the membrane. B, The formation of active ROP domains in 749

differentiating metaxylem. During metaxylem differentiation ROP11 is co-expressed with 750

ROPGEF4 and ROPGAP3 resulting in formation of active ROP11 domains and global 751

inactivation. The size of the ROP11 domains is restricted by microtubules and the 752

microtubules and plasma membrane-associated protein IQD13. C, Activation of ROPs by 753

SPK1 and formation of active actin nucleation/branching domains. SPK1 is associated with 754

the WAVE complex. Activation of ROPs by PSK1 causes their association with the SRA1 755

subunit of WAVE complex and its activation. Active WAVE can then activate actin 756

nucleation/branching by Arp2/3. 757

758

Figure 5: ROP regulation of cytoskeleton organization. A, Cortical MT reordering by ROP6, 759

RIC1, and KTN1. RIC1 physically interacts with and promotes the MT severing by KTN1, 760

leading to MT reordering. B, Localized cortical MT depolymerization. In developing 761

metaxylem MT depolarization takes place by the recruitment of MIDD1 and Kinesin13A to 762

active ROP11 domains. C, Actin dynamics in tip growing pollen tubes. ROP regulates actin 763



26

assembly by two counteracting pathways. RIC4 promotes assembly whereas RIC3 causes 764

disassembly by stimulating Ca2+ influx into the cytoplasm. D, Actin nucleation/branching in 765

trichomes and pavement cells. ROPs and the ROPGEF SPK1 interact with the SCAR/WAVE 766

complex and promote actin filament nucleation/branching. 767

768

Acknowledgements 769

The research was supported by Israel Academy of Sciences (grant nos. ISF 827/15; ISF–NCSF 770

1125/13) and by the Israel Center for Research Excellence on Plant Adaptation to Changing 771

Environment (grant no. I–CORE 757–12) to SY; and the Natural Science Foundation of China 772

to Y. F. (grant nos. 31325001; 31361140354). 773

774

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