ROP GTPases Structure-Function and Signaling Pathways

Feiguelman, Gil; Fu, Ying; Yalovsky, Shaul

doi:10.1104/pp.17.01415

Cited by 158 publications

(197 citation statements)

References 200 publications

(270 reference statements)

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“…ROP GTPases (RHO of Plants, also called RAC for rat sarcoma–related C3 botulinum toxin substrate) are small monomeric G-proteins that form a RHO subfamily, which is exclusively present in plants. ROPs can cycle between an actively signaling GTP-bound state and an inactive GDP-bound state and are crucial for polarity of diverse types of plant cells (Feiguelman et al 2018). ROPs seem to fulfill different functions depending on the interacting downstream factors called ROP-effectors.…”

Section: Introductionmentioning

confidence: 99%

ROP INTERACTIVE PARTNER b interacts with RACB and supports fungal penetration into barley epidermal cells

McCollum

Engelhardt

Weiß

et al. 2019

Preprint

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10 RHO of Plants (ROP) G-proteins are key components of cell polarization 11 processes in plant development. The barley (Hordeum vulgare) ROP protein 12 RACB, is a susceptibility factor in the interaction of barley with the barley 13 powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh). RACB also 14 drives polar cell development, and this function might be coopted during 15 formation of fungal haustoria in epidermal cells of barley. In order to 16 understand RACB signaling during the interaction of barley with Bgh, we 17 searched for potential downstream interactors of RACB. Here, we show that 18 ROP INTERACTIVE PARTNER b (RIPb) directly interacts with RACB in 19 yeast and in planta. Over-expression of RIPb supports susceptibility of 20 barley to Bgh. RIPb further interacts with itself at microtubules. However, 21 the interaction with activated RACB takes place at the plasma membrane.22 Both, RIPb and RACB are recruited to the site of fungal attack around the 23 neck of developing haustoria suggesting locally enhanced ROP activity. We 24 further assigned different functions to different domains of the RIPb protein.25 The N-terminal coiled-coil CC1 domain is required for microtubule 26 localization, while the C-terminal coiled-coil CC2 domain is sufficient to 27 interact with RACB and to fulfill a function in susceptibility at the plasma 28 membrane. Hence, RIPb appears to locate at microtubules and is then 29 recruited by activated RACB for a function at the plasma membrane during 30 formation of the haustorial complex. 31 32 33 90 Domain1) plays a key role in xylem cell development in Arabidopsis. During 91 the formation of the secondary cell wall in progenitor cells, RIP3 interacts 92 with ROP11 and the kinesin KIN13A, which leads to local microtubule 93 depletion and the formation of secondary wall pits (Mucha et al. 2010, Oda 94 et al. 2010, Oda and Fukuda 2012, Oda and Fukuda 2013). 95 ROP GTPases also play a role as signaling components in plant defense 96 (Ono et al. 2001, Chen et al. 2010). For instance, upon chitin perception, 97 the receptor kinase CERK1 phosphorylates RacGEF1, a ROP guanosine 98 nucleotide exchange factor that in turn activates RAC1, which supports 99 immunity to Magnaporthe oryzae (Akamatsu et al. 2013). 100 The barley ROP protein RACB is involved in root hair outgrowth and controls 101 asymmetric cell division of subsidiary cells in stomata development (Scheler 102 et al. 2016). RACB and RACB-associated proteins influence arrays and 103 stability of filamentous actin and the microtubule cytoskeleton (Opalski et 104 al. 2005, Hoefle et al. 2011, Huesmann et al. 2012). Next to its function in 105 polar cell development, RACB is also a susceptibility factor in the interaction 106 with the powdery mildew fungus Bgh. Over-expression of constitutively 107 activated RACB (CA RACB) enhances the penetration success of Bgh into 108 barley epidermal cells, silencing of RACB leads to a decreased penetration 109 rate (Schultheiss et al. 2002, Schultheiss et al. 2003, Hoefle et al. 2011). 11...

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Section: Introductionmentioning

confidence: 99%

ROP INTERACTIVE PARTNER b interacts with RACB and supports fungal penetration into barley epidermal cells

McCollum

Engelhardt

Weiß

et al. 2019

Preprint

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“…The Rho of Plants (ROP) GTPases are conserved molecules that play diverse roles in cell growth and signaling (Feiguelman et al, 2018). In P. patens, four ROP family members have been identified; however, no strong hypomorphic mutants are available (Eklund et al, 2010; Burkart et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

Goshima

2019

Preprint

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12Recent years, the bryophyte moss Physcomitrella patens has become an emerging 13 model organism for studying conserved signaling pathways and developmental 14 processes during plant evolution. Its short life cycle, ease of cultivation, and high rate of 15 homologous recombination have made it an ideal system for genetic analysis. However, 16 the presence of highly redundant genes and the difficulty of isolating hypomorphic 17 mutants have limited its broader use. Here we developed a simple, fast, and efficient 18 method to generate customized mutants in P. patens. We show that transient 19 cotransformation of CRISPR/Cas9 and oligonucleotide templates enables microindel 20 knock-in with high efficiency and accuracy. Using this method, we generated strains 21 carrying various types of mutations, including amino acid substitution, out-of-frame 22 deletion/insertion, splice site alteration, and small tag integration. We also demonstrate 23 that multiplex gene editing can be efficiently achieved to generate putative null and 24 hypomorphic mutants of redundant genes in one step. Thus our method will not only 25 simplify multiple-gene knockout, but also allows the generation of hypomorphic mutants 26 of genes of interest, especially those that are essential for viability. 27 28 Keywords 29 Bryophyte, Physcomitrella patens, CRISPR/Cas9, Oligonucleotide, Knock-in 30 4 2017a, 2017b). By forming a complex, its components, the endonuclease Cas9 and the 62 single guide RNA (sgRNA), are capable of introducing double-strand breaks (DSBs) at 63 predicted genomic loci once the sgRNA is customized with a targeting sequence (Jiang 64 and Doudna, 2017). These DSBs are repaired through the non-homologous end joining 65 or homology directed repair pathways, which leads to subsequent nucleotide changes 66 (Jiang and Doudna, 2017). In P. patens, CRISPR/Cas9 has been exploited to generate 67 heritable mutants (Nomura et al., 2016; Lopez-Obando et al., 2016; Collonnier et al., 68 2017a). However, it is unclear whether precise mutation knock-in can be achieved to 69 generate hypomorphic mutants. In addition, as microhomology-mediated end joining 70 (MMEJ) pathway is active in P. patens (Kamisugi et al., 2006; Collonnier et al., 2017a), 71 template-free editing can generate specific deletions at a high frequency, which may not 72 necessarily introduce out-of-frame mutations. For instance, a previous study reported 73 that 74% of the mutations generated by one CRISPR target are identical in-frame 74 deletions (Collonnier et al., 2017a).75 76Here we report a fast and efficient method to generate customized mutants in P. patens. 77 We show that transient cotransformation of CRISPR/Cas9 and oligonucleotide 78 templates induces microindel knock-in at a high efficiency and accuracy. This allows us 79 to introduce various types of mutations into the genome including amino acid 80 substitution, out-of-frame insertion/deletion, splice site alteration, or small tag integration. 81 We also demonstrate that multiplex gene editing can be effici...

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“…To translate signalling cues into downstream-signalling ROP-GTP interacts with so-called ROP-effectors that either perform a direct function or serve as scaffolds for recuitment of other downstream factors in higher order complexes. The knowledge on plant ROP-effectors is constantly increasing but still very incomplete and for many ROP-effectors, we lack knowledge about the molecular mechanism, by which they control cellular organization [3]. Therfore and because ROP signaling is involved in plant resistance and susceptibility to diseases, we are interested in finding further ROP-effectors.…”

Section: Discussionmentioning

confidence: 99%

“…However, not all ROP-effectors directly fulfill a function in cellular organization but instead are suggested to be scaffolds or adapter proteins that link activated ROPs with downstream factors. RIPs ( R OP-Interactive P artner, also called Interactor of Constitutive Active ROPs [ICR]) and RICs ( R OP-Interactive C RIB motif-containing proteins) are such ROP-effectors without known biochemical but potential ROP-scaffolding function [3].…”

Section: Introductionmentioning

confidence: 99%

Barley ROP-INTERACTIVE PARTNER-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains

Hoefle

McCollum

Hückelhoven

2019

Preprint

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8Small ROP (also called RAC) GTPases are key factors in polar cell development and in 9 interaction with the environment. ROP-Interactive Partner (RIP) proteins are predicted scaffold 10 or ROP-effector proteins, which function downstream of activated GTP-loaded ROP proteins 11 in establishing membrane heterogeneity and cellular organization. Grass ROP proteins 12 function in cell polarity, resistance and susceptibility to fungal pathogens but grass RIP proteins 13 are little understood. 14 We found that the barley (Hordeum vulgare L.) RIPa protein can interact with barley ROPs in 15 yeast. Fluorescent-tagged RIPa, when co-expressed with the constitutively activated ROP 16 protein CA RAC1, accumulates at the cell periphery or plasma membrane. Additionally, RIPa, 17 locates into membrane domains, which are laterally restricted by microtubules, when co-18 expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING 19 PROTEIN 1. Both structural integrity of MICROTUBULE-ASSOCIATED ROP-GTPASE 20 ACTIVATING PROTEIN 1 and microtubule stability are key to maintenance of RIPa-labeled 21 membrane domains. In this context, RIPa also accumulates at the interface of barley and 22 invading hyphae of the powdery mildew fungus Blumeria graminis f.sp. hordei. 23 Data suggest that barley RIPa interacts with barley ROPs and specifies RAC1 activity-24 associated membrane domains with potential signaling capacity. Lateral diffusion of this RAC1 25 signaling capacity is restricted the resulting membrane heterogeneity requires intact 26 microtubules and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. 27 Focal accumulation of RIPa at sites of fungal attack may indicate locally restricted ROP activity 28 at sites of fungal invasion. 29 30 Keywords: Arabidopsis thaliana, Hordeum vulgare, interactor of constitutive active ROPs, 31 membrane asymmetry, microtubule, RAC GTPase, ROP GTPase, susceptibility, resistance 32 In plants, ROP (RHO of plants) small GTPases are the only members of the RHO protein 35 family, which consists of several subfamilies (RHO, RAC, CDC42, Rnd und RhoBTB) in 36 mammals [1, 2]. ROPs organize a bunch of cellular processes as signaling GTPase. Among 37 the most prominent ROP-regulated events are the subcellular organization of the cytoskeleton 38 and vesicular traffic [3]. ROP-regulated cellular organization is crucial for normal plant 39 development e.g. in polar cell growth or asymmetric cell division but also in interaction with the 40 environment e.g. in regulation of stomata aperture or in interaction with pathogens. ROP 41 activity is tightly regulated via proteins that facilitate hydrolysis and exchange of ROP-bound 42 nucleotides. ROP-GDP is the signaling-inactive form of ROP and can be further controlled by 43 ROP-GDIs (ROP-guanine nucleotide dissociation inhibitors) that bind to ROP-GDP. ROP-44GDIs support cytosolic localization of ROPs most likely by direct binding of isoprenyl-residues 45 at the C-terminus of type I ROPs, which carry a CAAX-box prenylation motif. ROP-GDP further 46 can in...

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ROP GTPases Structure-Function and Signaling Pathways

Cited by 158 publications

References 200 publications

ROP INTERACTIVE PARTNER b interacts with RACB and supports fungal penetration into barley epidermal cells

ROP INTERACTIVE PARTNER b interacts with RACB and supports fungal penetration into barley epidermal cells

Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

Barley ROP-INTERACTIVE PARTNER-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains

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