Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector

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).

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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

Journal of General Virology ◽

10.1099/vir.0.79838-0 ◽

2004 ◽

Vol 85 (6) ◽

pp. 1727-1738 ◽

Cited By ~ 13

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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Novel N Gene-Associated, Temperature-Independent Resistance to the Movement of Tobacco Mosaic Virus Vectors Neutralized by a Cucumber Mosaic Virus RNA1 Transgene

Journal of Virology ◽

10.1128/jvi.76.24.12908-12916.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12908-12916 ◽

Cited By ~ 23

Author(s):

Tomas Canto ◽

Peter Palukaitis

Keyword(s):

Salicylic Acid ◽

Green Fluorescent Protein ◽

Tobacco Mosaic Virus ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Fluorescent Protein ◽

N Gene ◽

Nicotiana Glutinosa ◽

Tobacco Plants ◽

Green Fluorescent

ABSTRACT The N gene conditions for resistance to Tobacco mosaic virus (TMV) but only below 28°C. However, a TMV-based vector expressing green fluorescent protein (TMV-GFP) showed only limited movement at 33°C in tobacco plants harboring the N gene and other genes cointrogressed from Nicotiana glutinosa. TMV-GFP moved efficiently in tobacco plants that either lacked these genes or that contained the N gene but were transgenic for RNA1 of Cucumber mosaic virus. These findings identified novel temperature-independent resistance to the movement of TMV-GFP which could be neutralized by a different viral transgene. Using the N gene and nahG gene-transgenic tobacco, we show that this novel resistance is manifested specifically by the N gene itself and operates via a pathway independent of salicylic acid.

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

Journal of Virology ◽

10.1128/jvi.74.23.11339-11346.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11339-11346 ◽

Cited By ~ 58

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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms21041414 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1414 ◽

Cited By ~ 5

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.

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

Microbiology ◽

10.1099/0022-1317-81-7-1851 ◽

2000 ◽

Vol 81 (7) ◽

pp. 1851-1855 ◽

Cited By ~ 41

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.

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The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce

Phytopathology ◽

10.1094/phyto.2002.92.2.169 ◽

2002 ◽

Vol 92 (2) ◽

pp. 169-176 ◽

Cited By ~ 15

Author(s):

T. Candresse ◽

O. Le Gall ◽

B. Maisonneuve ◽

S. German-Retana ◽

E. Redondo

Keyword(s):

Green Fluorescent Protein ◽

Mosaic Virus ◽

Fluorescent Protein ◽

Lettuce Mosaic Virus ◽

Recombinant Viruses ◽

Systemic Movement ◽

Resistance Breaking ◽

Recessive Genes ◽

Lettuce Breeding ◽

Green Fluorescent

Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo11 and mo12, are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo12 confers resistance with most isolates, the genes mo11 or mo12 confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo11, for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo12 lettuce lines because its systemic movement was restricted in mo12 lines but not in susceptible or in mo11 lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs.

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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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.8.1026 ◽

2001 ◽

Vol 14 (8) ◽

pp. 1026-1031 ◽

Cited By ~ 9

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.

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