The use of surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and Ms-Rac4 with GTP

Brecht, Martina; Sewald, Katherina; Schiene, Karin; Keen, G.Anne; Fricke, Marc; Sauer, Markus; Niehaus, Karsten

doi:10.1016/j.jbiotec.2004.04.030

Cited by 7 publications

(5 citation statements)

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“…1b). Similar models have been computed for two ROP proteins from Medicago sativa in which a shortened helix αi is present in MsRac4, while the corresponding region in MsRac1 shows no helix at all [20]. In the AtROP4 model certain amino acids that fundamentally differ between plant and non-plant Rho GTPases are orientated towards the surface of the molecule, making them possible candidates to add to ROP-specific interactions.…”

Section: Introductionmentioning

confidence: 74%

ROPs in the spotlight of plant signal transduction

Berken

2006

Cell. Mol. Life Sci.

103

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Small guanine nucleotide binding proteins of the Rho family called ROP play a crucial role as regulators of signal transduction in plants. They participate in pathways that influence growth and development, and the adaptation of plants to various environmental situations. As members of the Ras superfamily, ROPs function as molecular switches cycling between a GDP-bound 'off' and a GTP-bound 'on' state in a strictly regulated manner. Latest research provided fascinating new insights into ROP regulation by novel guanine nucleotide exchange factors, unconventional GTPase activating proteins, and guanine nucleotide dissociation inhibitors, which apparently organize localized ROP activation. Important progress has also been made concerning signaling components upstream and downstream of the ROP cycle involving receptor-like serine/threonine kinases and effectors that can manipulate cytoskeletal dynamics, intracellular calcium levels, H2O2 production and further cellular targets. This review outlines the fast developing knowledge on ROP GTPases highlighting their specific features, regulation and roles in a cellular signaling context.

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Section: Introductionmentioning

confidence: 74%

ROPs in the spotlight of plant signal transduction

Berken

2006

Cell. Mol. Life Sci.

103

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“…When expressed as an antisense construct in transgenic tobacco, the plants failed to develop necrotic lesions upon elicitor infiltration, suggesting an involvement in ROS signaling (Schiene et al, 2000). Heterologously expressed MsRac1 then was shown to interact with GTP (Brecht et al, 2004). In this study, we have utilized the previously investigated MsRac1 sequence from M. sativa for RNA interference (RNAi)-mediated gene silencing in the model legume M. truncatula.…”

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confidence: 99%

Silencing of the Rac1 GTPaseMtROP9inMedicago truncatulaStimulates Early Mycorrhizal and Oomycete Root Colonizations But Negatively Affects Rhizobial Infection

et al. 2012

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RAC/ROP proteins (r-related GTPases of plants) are plant-specific small G proteins that function as molecular switches within elementary signal transduction pathways, including the regulation of reactive oxygen species (ROS) generation during early microbial infection via the activation of NADPH oxidase homologs of plants termed RBOH (for respiratory burst oxidase homolog). We investigated the role of Medicago truncatula Jemalong A17 small GTPase MtROP9, orthologous to Medicago sativa Rac1, via an RNA interference silencing approach. Composite M. truncatula plants (MtROP9i) whose roots have been transformed by Agrobacterium rhizogenes carrying the RNA interference vector were generated and infected with the symbiotic arbuscular mycorrhiza fungus Glomus intraradices and the rhizobial bacterium Sinorhizobium meliloti as well as with the pathogenic oomycete Aphanomyces euteiches. MtROP9i transgenic lines showed a clear growth-reduced phenotype and revealed neither ROS generation nor MtROP9 and MtRBOH gene expression after microbial infection. Coincidently, antioxidative compounds were not induced in infected MtROP9i roots, as documented by differential proteomics (two-dimensional differential gel electrophoresis). Furthermore, MtROP9 knockdown clearly promoted mycorrhizal and A. euteiches early hyphal root colonization, while rhizobial infection was clearly impaired. Infected MtROP9i roots showed, in part, extremely swollen noninfected root hairs and reduced numbers of deformed nodules. S. meliloti nodulation factor treatments of MtROP9i led to deformed root hairs showing progressed swelling of its upper regions or even of the entire root hair and spontaneous constrictions but reduced branching effects occurring only at swollen root hairs. These results suggest a key role of Rac1 GTPase MtROP9 in ROS-mediated early infection signaling.

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“…FRET was used to assess the nucleotide exchange activity of Trio variants ( 13 , 43 ). In a 384-well black low-volume round-bottom microplate (Corning 4514), 2 μM RhoA·GDP or Rac1·GDP was incubated with 50 nM GEF for 5 min at room temperature in freshly prepared nucleotide exchange buffer containing 20 mM HEPES pH 8.0, 200 mM NaCl, 2 mM DTT, and 10 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%