Control of Tobacco mosaic virus Movement Protein Fate by CELL-DIVISION-CYCLE Protein48

Like many other viruses, Tobacco mosaic virus replicates in association with the endoplasmic reticulum (ER) and exploits this membrane network for intercellular spread through plasmodesmata (PD), a process depending on virus-encoded movement protein (MP). The movement process involves interactions of MP with the ER and the cytoskeleton as well as its targeting to PD. Later in the infection cycle, the MP further accumulates and localizes to ER-associated inclusions, the viral factories, and along microtubules before it is finally degraded. Although these patterns of MP accumulation have been described in great detail, the underlying mechanisms that control MP fate and function during infection are not known. Here, we identify CELL-DIVISION-CYCLE protein48 (CDC48), a conserved chaperone controlling protein fate in yeast (Saccharomyces cerevisiae) and animal cells by extracting protein substrates from membranes or complexes, as a cellular factor regulating MP accumulation patterns in plant cells. We demonstrate that Arabidopsis (Arabidopsis thaliana) CDC48 is induced upon infection, interacts with MP in ER inclusions dependent on the MP N terminus, and promotes degradation of the protein. We further provide evidence that CDC48 extracts MP from ER inclusions to the cytosol, where it subsequently accumulates on and stabilizes microtubules. We show that virus movement is impaired upon overexpression of CDC48, suggesting that CDC48 further functions in controlling virus movement by removal of MP from the ER transport pathway and by promoting interference of MP with microtubule dynamics. CDC48 acts also in response to other proteins expressed in the ER, thus suggesting a general role of CDC48 in ER membrane maintenance upon ER stress.Plant viruses are obligate intracellular pathogens that replicate in association with host membranes (Laliberté and Sanfaçon, 2010) and subvert host intra- and intercellular trafficking pathways to achieve cell-to-cell and systemic spread (Harries and Ding, 2011; Niehl and Heinlein, 2011). In the case of the well-studied Tobacco mosaic virus (TMV), viral replication factories form on membranes of the endoplasmic reticulum (ER; Heinlein et al., 1995, 1998). As the plant ER is continuous between cells through plasmodesmata (PD; Ding et al., 1992), this membrane network provides a direct pathway for the spread of replicated virus from the replication sites in infected cells into the ER network of noninfected cells. The spread of plant viruses depends on virus-encoded movement proteins (MPs; Deom et al., 1987; Lucas, 2006). The MP of TMV facilitates the cell-to-cell passage of the infectious particle by forming a ribonucleoprotein complex with the viral RNA (Citovsky et al., 1990) and by increasing the size exclusion limit of PD (Wolf et al., 1989).During the course of infection, as well as when ectopically expressed, the MP associates with PD, the ER/actin network, and microtubules (Heinlein et al., 1995, 1998; Reichel and Beachy, 1998; Wright et al., 2007; Sambade et al., 2008; Hofmann et al., 2009; Boutant et al., 2010; Peña and Heinlein, 2012; Supplemental Fig. S1). Shortly after infection of a new cell, the MP localizes to small, mobile, ER-associated particles proposed to play a role in PD targeting of the viral RNA (Boyko et al., 2007; Sambade et al., 2008). Similar small, mobile MP particles are observed early upon ectopic expression of the protein. These particles colocalize with RNA and undergo stop-and-go movements in association with the ER (Sambade et al., 2008). The particle movements pause at microtubule proximal sites and their detachment requires microtubule polymerization (Sambade et al., 2008). These observations suggest that the interaction with the microtubule system plays a critical role in the maturation and ER-mediated delivery of infectious viral RNA particles to PD during early infection stages. Consistently, tobacco (Nicotiana tabacum) mutants with reduced microtubule dynamics exhibit reduced TMV movement (Ouko et al., 2010). Following virus movement, the previously infected cell further accumulates MP at the ER, a process that coincides with the formation of large ER inclusions that contain viral replicase and viral RNA in addition to MP and likely function as virus factories (Heinlein et al., 1998; Más and Beachy, 1999). In mature form, these inclusions may represent the so-called viroplasms or X-bodies described in the classical literature (Bawden and Sheffield, 1939; Esau and Cronshaw, 1967; Hills et al., 1987). Their formation is associated with rearrangements of the ER membrane and likely mediated by the accumulated MP since the inclusions diminish and reconstitute a native ER structure when MP becomes degraded by the 26S proteasome (Reichel and Beachy, 1998, 2000). Transfected cells accumulate MP in similar inclusions as those formed during infection, indicating that accumulated MP is indeed necessary and sufficient to form inclusions in association with the ER (Reichel and Beachy, 1998; Supplemental Fig. S1). Following accumulation of MP in virus factories, the infected cells accumulate the MP also along microtubules (Heinlein et al., 1998). The accumulation of MP in virus factories and on microtubules in cells behind the leading front of infection is dispensable for virus movement (Heinlein et al., 1998; Boyko et al., 2000a). At these late infection stages, the virus factories may enable the virus to produce high virion titers (Laliberté and Sanfaçon, 2010; Tilsner et al., 2012), and the subsequent accumulation along microtubules may play a role in withdrawing MP from the cell-to-cell communication pathway (Curin et al., 2007) and in stockpiling MP prior to degradation (Padgett et al., 1996; Gillespie et al., 2002).The molecular mechanisms that guide the MP to the ER and subsequently to microtubules during infection are not known. The MP is a hydrophobic protein that behaves like a membrane-integral or tightly membrane-associated protein in differential fractionation experiments and contains two predicted transmembrane domains (Reichel and Beachy, 1998; Brill et al., 2000, 2004) involved in ER association (Fujiki et al., 2006). The association with microtubules depends on MP amino acids 1 to 213 required for MP function (Kahn et al., 1998; Boyko et al., 2000b,Boyko et al., 2000c, 2002; Kotlizky et al., 2001). Moreover, certain amino acid exchange mutations known to affect the function of MP in virus movement in a temperature-sensitive manner also affect the ability of MP to interact with microtubules (Boyko et al., 2007,Boyko et al., 2000b). Interestingly, these mutations cluster together in a short domain of 25 amino acids showing a structural similarity with the M-loop of tubulin involved in tubulin-tubulin interactions (Boyko et al., 2000b; Waigmann et al., 2007). Importantly, this M-loop similarity domain overlaps with the predicted transmembrane domain (Brill et al., 2000, 2004) thus suggesting that the association of MP with membranes or microtubules is an alternative event that may depend on specific posttranslational modifications or specific folds of MP. However, although the different subcellular localizations of MPs during the course of infection indicate directional transport of MP from the ER to microtubules and may indicate different folds and functions of the protein when associated with these different subcellular components, the mechanism that controls the subcellular localization and, thus, the fate and function of MP is not known.Here, we identify CELL-DIVISION-CYCLE protein48 (CDC48), named p97/VCP (Valosin-containing protein) in mammals and Cdc48p in yeast (Saccharomyces cerevisiae), as a cellular factor regulating MP subcellular accumulation patterns. CDC48 functions are well characterized in mammalian and yeast systems but remain poorly investigated in plants. Yeast and mammalian CDC48s are essential, conserved chaperones involved in diverse cellular processes by controlling protein fate through extraction of substrates from membranes or complexes (Tsai et al., 2002; Meusser et al., 2005; Römisch, 2005; Rumpf and Jentsch, 2006; Schrader et al., 2009; Eisele et al., 2010; Meyer et al., 2012; Yamanaka et al., 2012). We show that virus infection leads to the induction of Arabidopsis (Arabidopsis thaliana) CDC48 isoforms and demonstrate a function of CDC48 in ER maintenance upon ER stress conditions. We further demonstrate that CDC48 interacts with MP and that CDC48 activity is required for MP degradation. Interaction of CDC48 with MP depends on the MP N terminus, which is required for degradation of the protein, for PD localization and microtubule accumulation of MP, and for function of MP in cell-to-cell transport of the viral RNA. Overexpressed CDC48 shifts MP subcellular localization from ER inclusions to microtubules, suggesting that CDC48 extracts the MP from ER-associated inclusions, where it accumulates in midstages of infection, to the cytosol, where it accumulates along microtubules during late infection stages. Moreover, overexpression of active, but not inactive, CDC48 inhibits virus movement. Our data demonstrate that a CDC48-dependent pathway leading to the clearance of ER-associated protein inclusions exists in plants, that plant viral MPs are substrates for this pathway, and that this pathway determines viral protein fate during infection. We suggest that CDC48-mediated extraction of MP from the ER is part of a plant defense response to remove MP from the ER, the compartment the virus uses for replication and movement.     

Biochemical Genetic Relationships Among Tunisian Hares (Lepus sp.), South African Cape Hares (L. capensis), and European Brown Hares (L. europaeus)

Tunisian hares (n = 45), currently assigned to Lepus capensis, were assayed for allelic variation at 40 allozyme loci, and allele frequencies at 32 loci were directly compared with earlier data of South African cape hares (L. capensis, n = 9) and European brown hares (L. europaeus, n = 244) to reveal genetic relationships among them. European mountain hares (L. timidus, n = 200) were used for outgroup comparison. In the Tunisian hares 27.5% of the loci were polymorphic with 2–4 alleles. Among all alleles at polymorphic loci, 15.1% occurred exclusively in Tunisian hares, 5.7% exclusively in cape hares, and 7.5% exclusively in brown hares at low frequencies. Not a single locus showed alternately fixed alleles between the samples of the L. capensis/L. europaeus complex. Levels of absolute and relative genetic differentiation among the samples of the L. capensis/ L. europaeus complex were low, relative to pairwise comparisons involving mountain hares. Diverse cluster analyses and multidimensional scaling of various pairwise genetic distance matrices concordantly grouped Tunisian hares with brown hares, and South African cape hares clustered only slightly farther apart, whereas mountain hares were distinctly separate. These results suggest regionally distinct phylogenetic units within an overall cohesive gene pool in the L. capensis/ L. europaeus complex, supporting Petter's view that all North African hares belong to L. capensis except for one local population of savanna hares, and that cape hares and brown hares are conspecific.     

Topographic organization of nerve fields

The vertebrate nervous system has topographic interconnections in many parts, known for example as retinotopy, somatotopy, etc. It is plausible that modifiable synapses play an important role in forming and refining these connections together with the sensory experiences. To elucidate the mechanism of topographic organization, we propose a simple model consisting of two nerve fields connected by modifiable excitatory synapses. The model also includes modifiable inhibitory synapses. The behavior of the model is described by a set of simultaneous non-linear integro-differential equations. By analyzing the equations, we obtain the equilibrium solution of topographic connections. It is also proved that a part of the presynaptic field which is frequently stimulated comes to be mapped on a large area of the postsynaptic field so that it has a good resolution.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Temporal and spatial fluctuations in bacterial abundances in four basins of a landfill leachate treatment (Etueffont, France)

The seasonal distribution of bacterial communities was assessed in a landfill leachate station located in Etueffont (Territoire de Belfort, France). Water samples were taken monthly from May 1998 to May 1999 from both the gross leachate and four lagooning basins. Bacterial numbers varied from 0.04x10(6) to 0.35x10(6) cells ml(-1) (m+/-sd=0.13x10(6)+/-0.12x10(6) cells ml(-1)). They decreased from the first to the last basin and were overall lower than those reported in literature for aquatic systems with comparable organic matter content. This may be ascribed to the leachate toxicity on bacteria.     

Oxidative stress induction by Prunus necrotic ringspot virus infection in apricot seeds

Prunus necrotic ringspot rvirus (PNRSV) was able to invade the immature apricot seed including the embryo. The amount of virus was very high inside the embryo compared with that present in the cotyledons. PNRSV infection produced an oxidative stress in apricot seeds as indicated by the increase in lipid peroxidation, measured as thiobarbituric acid-reactive substances. This lipid peroxidation increase was parallelled with an imbalance in the seed antioxidant enzymes. A significant decrease in the ascorbate–GSH cycle enzymes as well as in peroxidase (POX) activity took place in infected seeds, suggesting a low capability to eliminate H₂O₂. No changes in superoxide dismutase (SOD) or catalase activity were observed. A significant decrease in polyphenoloxidase (PPO) activity was also observed. Native PAGE revealed the presence of three different SOD activity bands in apricot seeds: a Mn-containing SOD and two CuZn-containing SODs. Only an isozyme with catalase, glutathione reductase (GR) or PPO activity was detected in both healthy and infected apricot seeds. Regarding POX staining, three bands with POX activity were detected in native gels in both healthy and infected seeds. The gel results emphasise that the drop detected in POX, GR and PPO activities in PNRSV-infected apricot seeds by kinetic analyses was also evident from the results obtained by native PAGE. The oxidative stress and the imbalance in the antioxidant systems from PNRSV-infected apricot seeds resemble the hypersensitive response observed in some virus–host interactions. This defence mechanism would inactivate PNRSV during seed formation and/or the storage period or even during seed germination. Those results can explain the decrease in seed germination and the low transmission of PNRSV by seeds in apricot trees.     

HPLC‐DAD Analysis,Antioxidant and Protective Effects of Tunisian Rhanterium suaveolens against Acetamiprid Induced Oxidative Stress on Mice Erythrocytes

The present study was performed to assess the HPLC‐DAD analysis as well as antioxidant and protective effects of Tunisian Rhanterium suaveolens (Rs) against acetamiprid (ACT) induced oxidative stress on mice erythrocytes. The in vitro assays showed that the methanolic extract of Rs has an impressive antioxidant effect proved by testing the total antioxidant and scavenging activities using BCB, DPPH and ABTS assays, respectively. Moreover, qualitative and quantitative analysis using HPLC‐DAD revealed the richness of Rs in polyphenols where p‐Coumaric, Apigenin‐7‐glucoside and Ferulic acid were detected as the most abundant polyphenols. In the in vivo experiment, ACT, used as a toxicity model, was given to mice at a dose of 20 mg/kg. The latter was the origin of hemolytic anemia characterized by a significant decrease in red blood cells, hemoglobin and hematocrit levels and an increase in bilirubin, LDH, osmotic fragility, reticulocytes and white blood cells number. Characteristic erythrocyte morphological alterations were also determined as spherocytosis, schistocytosis and dacryocystitis. The oxidative status of ACT‐treated mice was also altered manifested by a significant increase in MDA and GSH levels and a decrease in SOD, CAT and GPx activities. When receiving the Rs methanolic extract at a dose of 300 mg/kg, all the parameters cited above were restored in mice. These remarkable corrections could only confirm the important antioxidant effect and the noticeable protective properties that possess Rs owing to its broad range of secondary bioactive metabolites.     

Profiles of the Essential Oils and Headspace Analysis of Volatiles from Mandragora autumnalis Growing Wild in Tunisia

Mandragora autumnalis Bertol . (Solanaceae family), synonym of M. officinalis Mill ., occurs in North Africa and grows natively in Northern and Central Tunisia, in humid to sub‐arid climates. The ripe fruits of mandrake are odiferous with a particular, indescribable, specific odor, shared, to a lesser extent, by the leaves and roots. We carried out an investigation of the essential oils (EOs) and of the aromatic volatiles emitted by fresh leaves, roots and ripe fruits of M. autumnalis growing wild in Central Tunisia. The EOs were obtained from freshly collected plant material by hydrodistillation, while the volatile emissions from the powdered M. autumnalis tissues were sampled by headspace solid phase microextraction (HS‐SPME); both types of samples were analyzed by gas chromatography‐mass spectrometry (GC/MS). Fifty‐one compounds representing 96.2–98.6 % of the total oil compositions were identified in the three tissues and belonged to different chemical classes specifically in 16 esters, 12 alcohols, 12 hydrocarbons, 6 ketones, 3 aldehydes and 3 acids. The main constituents were pentadecanoic acid (34.2 %) and hexadecanol (26.3 %). A total of 78 volatile compounds emanating from M. autumnalis tissues, representing 94.1–96.4 % of the total volatile compositions, were identified: 22 esters, 11 alcohols, 9 aldehydes, 14 ketones, 7 nitrogen, 10 hydrocarbons, 2 lactones, 1 sulfur and 2 ethers. Ethyl hexanoate (12.3 %) and 1,3‐butanediol (12.3 %) were at the highest relative percentages. This study characterizes and distinguishes M. autumnalis from Tunisia and attributes the compounds responsible for the intoxicating and particular odor of fruits. Chemosystematic of Mandragora autumnalis based on the identification of essential oils and headspace volatiles of each of its organ can be used to characterize this species according to its geographic distribution.