scholarly journals Tobacco mosaic virus (TMV) Replicase and Movement Protein Function Synergistically in Facilitating TMV Spread by Lateral Diffusion in the Plasmodesmal Desmotubule of Nicotiana benthamiana

2008 ◽  
Vol 21 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Dana Guenoune-Gelbart ◽  
Michael Elbaum ◽  
Guy Sagi ◽  
Amit Levy ◽  
Bernard L. Epel
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Integral Membrane Protein ◽  
Movement Proteins ◽  
Green Fluorescent ◽  
Cell Spread ◽  
Er Membrane

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus (TMVMP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that TMVMP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of TMVMP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.

scholarly journals A Dysfunctional Movement Protein of Tobacco mosaic virus Interferes with Targeting of Wild-Type Movement Protein to Microtubules

2001 ◽  
Vol 14 (7) ◽  
pp. 895-904 ◽  
Author(s):  
Guy Kotlizky ◽  
Aviva Katz ◽  
Jessica van der Laak ◽  
Vitaly Boyko ◽  
Moshe Lapidot ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Free System ◽  
Intracellular Targeting ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Dependent Processes ◽  
Cell Spread

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.

scholarly journals Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

2020 ◽  
Vol 21 (4) ◽  
pp. 1414 ◽  
Author(s):  
Hui Li ◽  
Xiaobao Ying ◽  
Lina Shang ◽  
Bryce Redfern ◽  
Nicholas Kypraios ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Candidatus Liberibacter ◽  
Phloem Necrosis ◽  
Liberibacter Asiaticus ◽  
Green Fluorescent

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

scholarly journals Tobacco Mosaic Virus Movement Protein Interacts with Green Fluorescent Protein-Tagged Microtubule End-Binding Protein 1

2008 ◽  
Vol 147 (2) ◽  
pp. 611-623 ◽  
Author(s):  
Katrin Brandner ◽  
Adrian Sambade ◽  
Emmanuel Boutant ◽  
Pascal Didier ◽  
Yves Mély ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Binding Protein ◽  
Virus Movement ◽  
Green Fluorescent

Confined chromophores in tobacco mosaic virus to mimic green fluorescent protein

2015 ◽  
Vol 51 (82) ◽  
pp. 15122-15124 ◽  
Author(s):  
Quan Zhou ◽  
Fengchi Wu ◽  
Man Wu ◽  
Ye Tian ◽  
Zhongwei Niu
Keyword(s):  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Green Fluorescent

Grafting green fluorescent protein-like chromophores in the 4 nm channel of tobacco mosaic virus greatly enhances its fluorescence emission.

scholarly journals ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana

2004 ◽  
Vol 85 (10) ◽  
pp. 3123-3133 ◽  
Author(s):  
Tomas Canto ◽  
Stuart A. MacFarlane ◽  
Peter Palukaitis
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Host Responses ◽  
Reading Frame ◽  
Virus Accumulation ◽  
Green Fluorescent

Tobacco mosaic virus (TMV) contains a sixth open reading frame (ORF6) that potentially encodes a 4·8 kDa protein. Elimination of ORF6 from TMV attenuated host responses in Nicotiana benthamiana without alteration in virus accumulation. Furthermore, heterologous expression of TMV ORF6 from either potato virus X (PVX) or tobacco rattle virus (TRV) vectors enhanced the virulence of both viruses in N. benthamiana, also without effects on their accumulation. By contrast, the presence or absence of TMV ORF6 had no effect on host response or virus accumulation in N. tabacum plants infected with TMV or PVX. TMV ORF6 also had no effect on the synergism between TMV and PVX in N. tabacum. However, the presence of the TMV ORF6 did have an effect on the pathogenicity of a TRV vector in N. tabacum. In three different types of assay carried out in N. benthamiana plants, expression of TMV ORF6 failed to suppress gene silencing. Expression in N. benthamiana epidermal cells of the encoded 4·8 kDa protein fused to the green fluorescent protein at either end showed, in addition to widespread cytosolic fluorescence, plasmodesmatal targeting specific to both fusion constructs. The role of the ORF6 in host responses is discussed.

scholarly journals Genetic constructs creation using Golden Gate cloning method

2019 ◽  
Vol 25 ◽  
pp. 190-196 ◽  
Author(s):  
O. I. Varchenko ◽  
B. M. Krasyuk ◽  
A. A. Fedchunov ◽  
O. V. Zimina ◽  
M. F. Parii ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Regulatory Elements ◽  
Golden Gate ◽  
Genes Encoding ◽  
Green Fluorescent ◽  
Genetic Constructs

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

scholarly journals Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins

2005 ◽  
Vol 86 (4) ◽  
pp. 1223-1228 ◽  
Author(s):  
Tomas Canto ◽  
Peter Palukaitis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Subcellular Distribution ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Epidermal Cells ◽  
Green Fluorescent Proteins ◽  
Movement Proteins ◽  
Infected Cells ◽  
Green Fluorescent

The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubles.

scholarly journals Degradation of Tobacco Mosaic Virus Movement Protein by the 26S Proteasome

2000 ◽  
Vol 74 (7) ◽  
pp. 3330-3337 ◽  
Author(s):  
Christoph Reichel ◽  
Roger N. Beachy
Keyword(s):  
Molecular Weight ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
High Molecular Weight ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
26S Proteasome ◽  
Host Proteins ◽  
Subsequent Degradation ◽  
Green Fluorescent

ABSTRACT Cell-to-cell spread of tobacco mosaic virus is facilitated by the virus-encoded 30-kDa movement protein (MP). This process involves interaction of viral proteins with host components, including the cytoskeleton and the endoplasmic reticulum (ER). During virus infection, high-molecular-weight forms of MP were detected in tobacco BY-2 protoplasts. Inhibition of the 26S proteasome by MG115 andclasto-lactacystin-β-lactone enhanced the accumulation of high-molecular-weight forms of MP and led to increased stability of the MP. Such treatment also increased the apparent accumulation of polyubiquitinated host proteins. By fusion of MP with the jellyfish green fluorescent protein (GFP), we demonstrated that inhibition of the 26S proteasome led to accumulation of the MP-GFP fusion preferentially on the ER, particularly the perinuclear ER. We suggest that polyubiquitination of MP and subsequent degradation by the 26S proteasome may play a substantial role in regulation of virus spread by reducing the damage caused by the MP on the structure of cortical ER.

scholarly journals Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter

2004 ◽  
Vol 85 (6) ◽  
pp. 1727-1738 ◽  
Author(s):  
Michal Man ◽  
Bernard L. Epel
Keyword(s):  
Green Fluorescent Protein ◽  
Coat Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Regulatory Elements ◽  
Gfp Expression ◽  
Enhancer Element ◽  
Green Fluorescent ◽  
Rna Promoter

A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3′-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3′ terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem–loop structures, which are followed by an enhancer region.

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