A novel ROP/RAC GTPase effector integrates plant cell form and pattern formation

ROPs/RACs are the only known signaling Ras superfamily small GTPases in plants. As such they have been suggested to function as central regulators of diverse signaling cascades. The ROP/RAC signaling networks are largely unknown, however, because only few of their effector proteins have been identified. In a paper that was published in the June 5, 2007 issue of Current Biology we described the identification of a novel ROP/RAC effector designated ICR1 (Interactor of Constitutive active ROPs 1). We demonstrated that ICR1 functions as a scaffold that interacts with diverse but specific group of proteins including SEC3 subunit of the exocyst vesicle tethering complex. ICR1-SEC3 complexes can interact with ROPs in vivo and are thereby recruited to the plasma membrane. ICR1 knockdown or silencing leads to cell deformation and loss of the root stem cells population, and ectopic expression of ICR1 phenocopies activated ROPs/RACs. ICR1 presents a new paradigm in ROP/RAC signaling and integrates mechanisms regulating cell form and pattern formation at the whole plant level.Key words: Rho, auxin, root development, vesicle trafficking, RAC, ROP, polarity, Arabidopsis, exocyst     

Structural Organization and Energy Transduction Mechanism of Na+,K+-ATPase Studied with Transition Metal-Catalyzed Oxidative Cleavage

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na⁺,K⁺-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H₂O₂, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe²⁺ bound at the cytoplasmic surface or by complexes of ATP–Fe²⁺, which directs the Fe²⁺ to the normal ATP–Mg²⁺ site. Fe²⁺- and ATP–Fe²⁺-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg²⁺ ions between E₁P and E₂P, which may be crucial in facilitating hydrolysis of E₂P. The pattern of domain interactions in E₁ and E₂ conformations, and role of Mg²⁺ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu²⁺ ions, bound at the extracellular surfaces, or a hydrophobic Cu²⁺-diphenyl phenanthroline (DPP) complex, which directs the Cu²⁺ to the membrane–water interface. Cu²⁺- or Cu²⁺-DPP-catalyzed cleavages are providing information on / subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.     

Fibroblast growth factor (FGF) homologous factors share structural but not functional homology with FGFs

Fibroblast growth factors (FGFs) interact with heparan sulfate glycosaminoglycans and the extracellular domains of FGF cell surface receptors (FGFRs) to trigger receptor activation and biological responses. FGF homologous factors (FHF1-FHF4; also known as FGF11-FGF14) are related to FGFs by substantial sequence homology, yet their only documented interactions are with an intracellular kinase scaffold protein, islet brain-2 (IB2) and with voltage-gated sodium channels. In this report, we show that recombinant FHFs can bind heparin with high affinity like classical FGFs yet fail to activate any of the seven principal FGFRs. Instead, we demonstrate that FHFs bind IB2 directly, furthering the contention that FHFs and FGFs elicit their biological effects by binding to different protein partners. To understand the molecular basis for this differential target binding specificity, we elucidated the crystal structure of FHF1b to 1.7-A resolution. The FHF1b core domain assumes a beta-trefoil fold consisting of 12 antiparallel beta strands (beta 1 through beta 12). The FHF1b beta-trefoil core is remarkably similar to that of classical FGFs and exhibits an FGF-characteristic heparin-binding surface as attested to by the number of bound sulfate ions. Using molecular modeling and structure-based mutational analysis, we identified two surface residues, Arg52 in the beta 4-beta 5 loop and Val95 in the beta 9 strand of FHF1b that are required for the interaction of FHF1b with IB2. These two residues are unique to FHFs, and mutations of the corresponding residues of FGF1 to Arg and Val diminish the capacity of FGF1 to activate FGFRs, suggesting that these two FHF residues contribute to the inability of FHFs to activate FGFRs. Hence, FHFs and FGFs bear striking structural similarity but have diverged to direct related surfaces toward interaction with distinct protein targets.     

Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication

We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.     

EHD1 Functions in Endosomal Recycling and Confers Salt Tolerance

Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain.     

The function of EHD2 in endocytosis and defense signaling is affected by SUMO

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.     

Some sweet and bitter tastants stimulate inhibitory pathway of adenylyl cyclase via melatonin and alpha 2-adrenergic receptors in Xenopus laevis melanophores

The sweeteners saccharin, D-tryptophan, and neohesperidin dihydrochalcone (NHD) and the bitter tastant cyclo(Leu-Trp) stimulated concentration-dependent pigment aggregation in a Xenopus laevis melanophore cell line similar to melatonin. Like melatonin, these tastants inhibited (by 45-92%) cAMP formation in melanophores; pertussis toxin pretreatment almost completely abolished the tastant-induced cAMP inhibition, suggesting the involvement of the inhibitory pathway (Gi) of adenylyl cyclase. The presence of luzindole (melatonin receptor antagonist) almost completely abolished the inhibition of cAMP formation induced by saccharin, D-tryptophan, and cyclo(Leu-Trp) but only slightly affected the inhibitory effect of NHD. In contrast, the presence of an alpha2-adrenergic receptor antagonist, yohimbine, almost completely abolished the inhibition of cAMP formation induced by NHD but had only a minor effect on that induced by the other tastants. Thus saccharin, D-tryptophan, and cyclo(Leu-Trp) are melatonin receptor agonists whereas NHD is an alpha2-adrenergic receptor agonist, but both pathways lead to the same transduction output and cellular response. Formation of D-myo-inositol 1,4,5-trisphosphate (IP3) in melanophores was reduced (15-58%, no concentration dependence) by saccharin, D-tryptophan, and cyclo(Leu-Trp) stimulation but increased by NHD stimulation. Tastant stimulation did not affect cGMP. Although some of the above tastants were found to be membrane permeant, their direct activation of downstream transduction components in this experimental system is questionable. MT1 and MT2 melatonin receptor mRNAs were identified in rat circumvallate papilla taste buds and nonsensory epithelium, suggesting the occurrence of MT1 and MT2 receptors in these tissues. Melatonin stimulation reduced the cellular content of cAMP in taste cells, which may or may not be related to taste sensation.     

The intracellular nucleotide‐binding leucine‐rich repeat receptor (SlNRC4a) enhances immune signalling elicited by extracellular perception

Plant recognition and defence against pathogens employs a two‐tiered perception system. Surface‐localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide‐binding leucine‐rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB‐LRR required for hypersensitive response‐associated cell death‐4). Co‐immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR‐mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled‐coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.     

The response of an aphid tending ant to artificial extra-floral nectaries on different host plants

Sap-feeding homopterans, which reduce the fitness of their host plants, are often tended by ants that feed on their honeydew. The composition of the honeydew varies with both the aphid and the host plant. Extra-floral nectaries (EFNs) are believed to have evolved to attract attending ants, protecting the hosts, but it is unknown if EFNs on different plants have the same impact on the relations between an aphid species feeding on those plants and its tending ant. Experimental research was conducted to examine the attraction of Tapinoma erraticum scout ants to honeydew from the aphid Aphis gossypii feeding on two different plants, Prunus amygdalus and Mentha piperita, negligence of tending the aphids, and survival of the aphids in the presence of artificial EFNs. The scout ants were significantly more attracted to artificial nectar dispensed on P. amygdalus leaves than on M. piperita, or aphids on both plants and water. They neglected aphids in the presence of artificial EFNs on M. piperita but not on P. amygdalus. The aphid population on M. piperita did not statistically change in the presence of artificial EFNs during the 8 days of the third experiment. On P. amygdalus, the aphids succeeded in developing fully to winged form. In conclusion, the responses of the ants tending aphids to the presence of artificial EFNs were influenced by the host plant.     

Formation of oxidised (OX) proteins in Entamoeba histolytica exposed to auranofin and consequences on the parasite virulence

Metronidazole (MNZ), the first line drug for amoebiasis and auranofin (AF), an emerging antiprotozoan drug, are both inhibiting Entamoeba histolytica thioredoxin reductase. The nature of oxidised proteins (OXs) formed in AF‐ or MNZ‐treated E. histolytica trophozoites is unknown. In order to fill this knowledge gap, we performed a large‐scale identification and quantification of the OXs formed in AF‐ or MNZ‐treated E. histolytica trophozoites using resin‐assisted capture coupled to mass spectrometry (MS). We detected 661 OXs in MNZ‐treated trophozoites and 583 OXs in AF‐treated trophozoites. More than 50% of these OXs were shared, and their functions include hydrolases, enzyme modulators, transferases, nucleic acid binding proteins, oxidoreductases, cytoskeletal proteins, chaperones, and ligases. Here, we report that the formation of actin filaments (F‐actin) is impaired in AF‐treated trophozoites. Consequently, their erythrophagocytosis, cytopathic activity, and their motility are impaired. We also observed that less than 15% of OXs present in H₂O₂‐treated trophozoites are also present in AF‐ or MNZ‐treated trophozoites. These results strongly suggest that the formation of OXs in AF‐ or MNZ‐treated trophozoites and in H₂O₂‐treated trophozoites occurred by two different mechanisms.