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

Limitations on the use of fused green fluorescent protein to investigate structure–function relationships for the cauliflower mosaic virus movement protein

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1851-1855 ◽  
Author(s):  
Carole L. Thomas ◽  
Andrew J. Maule
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Cauliflower Mosaic Virus ◽  
Virus Movement ◽  
Wild Type ◽  
Tubule Formation ◽  
Mouth Disease Virus ◽  
Green Fluorescent

To investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP–MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.

scholarly journals Effects of Movement Protein Mutations on the Formation of Tubules in Plant Protoplasts Expressing a Fusion Between the Green Fluorescent Protein and Cauliflower mosaic virus Movement Protein

2001 ◽  
Vol 14 (8) ◽  
pp. 1026-1031 ◽  
Author(s):  
Zhong Huang ◽  
Yu Han ◽  
Stephen H. Howell
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Cauliflower Mosaic Virus ◽  
Virus Movement ◽  
Plant Protoplasts ◽  
Tubule Formation ◽  
Leaf Mesophyll ◽  
Green Fluorescent

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MPER2A, a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MPN6 and GFP-MPN13, two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MPWT and GFP-MPER2A form tubules containing both MP fusions, indicating that although the GFP-MPER2A cannot induce tubule formation, GFP-MPER2A can coassemble or colocalize with CFP-MPWT in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

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 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 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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