Mechanical Wounding Caused by Inoculation Influences the Photosynthetic Response of Nicotiana benthamiana Plants to Plum Pox Potyvirus

Plants of Nicotiana benthamiana (Gray) (60 d old) were mechanically inoculated by a spreading of the fourth and fifth leaves with inoculum with or without plum pox potyvirus (PPV). Changes in growth parameters and selected photosynthetic characteristics were followed in control and inoculated plants in the locally affected leaves (LA) during 11 d after inoculation (DAI), in systemically affected leaves immature at time of inoculation (SAI) during 14–25 DAI, and in systemically affected leaves developed after the inoculation (SAD) during 28–39 DAI. The pure mechanical damage caused by inoculation induced a decrease in the net photosynthetic rate (P _N) in LA and SAD leaves, and an increase in the steady-state value of the non-photochemical chlorophyll (Chl) fluorescence quenching q_N. The q_N increase appeared in certain time intervals in all measured leaves on plants, so it could be regarded as indication of a systemic reaction of plant to the local mechanical injury. The viral infection developed in LA leaves and spread to SAI and SAD leaves was documented by the ELISA-DASI method. The plant height and area of SAI and SAD leaves were lower in infected plants. The combined effect of mechanical damage and viral infection caused a decrease in P _N only in LA and SAD leaves. In SAD leaves, an increased relative height of the J step (V_J) in the O-J-I-P Chl fluorescence transient together with a lower B/A band ratio of thermoluminescence glow curves reflected a damage to the acceptor side of photosystem 2 (PS2) caused by the viral infection, and a faster kinetics of the induction of the photochemical quenching coefficient q_P of Chl fluorescence indicated a faster Q_A ^– re-oxidation in the remaining undamaged centres of PS2.     

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

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Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.     

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.     

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of β‐aspartic acid (Asp)‐Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP‐1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant‐expressed CGP fed in combination with plant‐made CGPase was hydrolysed in the intestine, and high levels of ß‐Asp‐Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.     

Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts

Aims

The local concentration of extracellular Ca²⁺ ([Ca²⁺]_o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca²⁺]_o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca²⁺]_o to osteoblastic proliferation.

Methods

Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.

Results

Our data showed that elevating [Ca²⁺]_o evoked a sustained increase of [Ca²⁺]_c in a dose-dependent manner. This [Ca²⁺]_c increase was blocked by TMB-8 (Ca²⁺ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (a PLC inhibitor) strongly reduced the [Ca²⁺]_o-induced [Ca²⁺]_c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca²⁺]_o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca²⁺]_o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, respectively, but not affected by Cav channels antagonists.

Conclusions

Elevating [Ca²⁺]_o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca²⁺ concentration.     

Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway

The small GTPase protein Sar1 is known to be involved in both the initiation of COPII-coated vesicle formation and scission of the nascent vesicle from the endoplasmic reticulum. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here, we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low-curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three-dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.     

Virion Incorporation of the Herpes Simplex Virus Type 1 Tegument Protein VP22 Occurs via Glycoprotein E-Specific Recruitment to the Late Secretory Pathway

The mechanism by which herpesviruses acquire their tegument is not yet clear. One model is that outer tegument proteins are recruited by the cytoplasmic tails of viral glycoproteins. In the case of herpes simplex virus tegument protein VP22, interactions with the glycoproteins gE and gD have been shown. We have previously shown that the C-terminal half of VP22 contains the necessary signal for assembly into the virus. Here, we show that during infection VP22 interacts with gE and gM, as well as its tegument partner VP16. However, by using a range of techniques we were unable to demonstrate VP22 binding to gD. By using pulldown assays, we show that while the cytoplasmic tails of both gE and gM interact with VP22, only gE interacts efficiently with the C-terminal packaging domain of VP22. Furthermore, gE but not gM can recruit VP22 to the Golgi/trans-Golgi network region of the cell in the absence of other virus proteins. To examine the role of the gE-VP22 interaction in infection, we constructed a recombinant virus expressing a mutant VP22 protein with a 14-residue deletion that is unable to bind gE (ΔgEbind). Coimmunoprecipitation assays confirmed that this variant of VP22 was unable to complex with gE. Moreover, VP22 was no longer recruited to its characteristic cytoplasmic trafficking complexes but exhibited a diffuse localization. Importantly, packaging of this variant into virions was abrogated. The mutant virus exhibited poor growth in epithelial cells, similar to the defect we have observed for a VP22 knockout virus. These results suggest that deletion of just 14 residues from the VP22 protein is sufficient to inhibit binding to gE and hence recruitment to the viral envelope and assembly into the virus, resulting in a growth phenotype equivalent to that produced by deleting the entire reading frame.The herpesvirus tegument is the virion compartment located between the DNA-containing capsid and the virus envelope (6). Although it is well defined that the viral capsid assembles in the nucleus (37, 38) and the viral envelope is acquired from cellular membranes (3, 24), the mechanism of tegument protein acquisition is still to be established. At least 20 virus-encoded components are recruited into the herpes simplex virus type 1 (HSV-1) tegument (32), and there is increasing evidence to suggest that subsets of these proteins may be added as assembly progresses along the maturation pathway (28). To ensure efficient incorporation, it is likely that individual tegument proteins are specifically targeted to their cellular site of recruitment. Such targeting could involve interaction with a viral partner, a cellular partner, or both. A clearer understanding of how individual tegument proteins are acquired by newly assembling virions will help to define the herpesvirus assembly pathway.A number of protein-protein interactions between individual tegument proteins (13, 40, 42), and between tegument proteins and glycoproteins (19, 20, 22, 32), have been described that may provide useful insight into the assembly process. In particular, the interaction of tegument proteins with the cytoplasmic tails of virus glycoproteins provides an attractive mechanism for the virion recruitment of at least the outer components of the tegument. In the case of VP22, the homologues from pseudorabies virus (PRV) and HSV-1 have been shown to interact with the cytoplasmic tail of gE (19, 20, 32). However, the role of this interaction in virus infection has not yet been clearly defined and the fact that additional glycoprotein interactions have been described, with gM in the case of PRV and gD in the case of HSV-1, may point to potential redundancy in the mechanism of VP22 packaging (4, 19, 20). In addition, we and others have previously shown that HSV-1 VP22 interacts directly with a second tegument protein, namely, VP16 (13, 33), an interaction that could provide an alternative route for VP22 to enter the virion. In a previous study, we concluded that the region of VP22 containing its VP16 interaction domain was required but not sufficient for optimal VP22 packaging into the assembling virion, with an additional C-terminal determinant also involved (23). We also demonstrated that the same region of VP22 that was required for virion packaging was essential to target the protein to its characteristic cytoplasmic trafficking complexes, suggesting that these specific sites may be the location in the cell for VP22 assembly into the virion (23). Since that study, O''Regan and coworkers have reported that the C-terminal half of HSV-1 VP22 also contains the binding site for gE (32), providing a possible candidate for an additional VP22 binding partner. Furthermore, as HSV-1 VP22 has been shown to bind to gD and PRV VP22 interacts with gM, it is possible that the C terminus of VP22 contains a gD and/or a gM binding site (4, 20).In the present study, we aimed to clarify the molecular mechanism by which VP22 is recruited into the virus particle. We show that HSV-1 VP22 binds efficiently to VP16, gE, and gM in the infected cell, but we cannot detect an interaction with gD. We show that the packaging domain of VP22 binds to the cytoplasmic tail of gE but not gM and that the same region of VP22 is recruited to the secretory pathway by gE in the absence of other virus proteins. Finally, we show that a mutant VP22 protein lacking a 14-residue peptide from its packaging domain is unable to interact with gE during infection, exhibits a different subcellular localization, and fails to assemble into the virus particle. This is the first characterization of a single protein-protein interaction essential for the packaging of an HSV-1 tegument protein.     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

Cytomegalovirus miRNAs Target Secretory Pathway Genes to Facilitate Formation of the Virion Assembly Compartment and Reduce Cytokine Secretion

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Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world''s most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials ⁹, vaccines ^{14, 15}, electronic materials, chemical tools, and molecular electronic containers^{4, 5, 10, 11, 16, 18, 12}.In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups ⁸. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications ⁸. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed ¹³. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus³, Carnation mottle virus¹², Cowpea chlorotic mottle virus⁶, Tobacco mosaic virus¹⁷, Turnip yellow mosaic virus¹, and MRFV ¹³.     

A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing. Two approaches were used. First, the extent of phytoene desaturase (PDS) gene silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS gene in N. benthamiana. The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between gene silencing efficiency and percentage homology of the heterologous gene sequence with the endogenous gene sequence. It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous gene sequences is not absolutely required for gene silencing.     

Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

Background

Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.

Results

All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.

Conclusions

Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case.