scholarly journals Effects of Green Fluorescent Protein or β-Glucuronidase Tagging on the Accumulation and Pathogenicity of a Resistance-Breaking Lettuce mosaic virus Isolate in Susceptible and Resistant Lettuce Cultivars

2000 ◽  
Vol 13 (3) ◽  
pp. 316-324 ◽  
Author(s):  
Sylvie German-Retana ◽  
Thierry Candresse ◽  
Emmanuel Alias ◽  
René-Pierre Delbos ◽  
Olivier Le Gall
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Wild Type Virus ◽  
Lettuce Mosaic Virus ◽  
Recombinant Viruses ◽  
Virus Accumulation ◽  
Resistance Breaking ◽  
Green Fluorescent

The RNA genome of a resistance-breaking isolate of Lettuce mosaic virus (LMV-E) was engineered to express the jellyfish green fluorescent protein (GFP) or β-glucuronidase (GUS) fused to the helper-component proteinase (HC-Pro) to study LMV invasion and spread in susceptible and resistant lettuce cultivars. Virus accumulation and movement were monitored by either histochemical GUS assays or detection of GFP fluorescence under UV light. The GFP- and GUS-tagged viruses spread systemically in the susceptible lettuce cultivars Trocadero and Vanguard, where they induced attenuated symptoms, compared with the wild-type virus. Accumulation of the GFP-tagged virus was reduced but less affected than in the case of the GUS-tagged virus. Systemic movement of both recombinant viruses was very severely affected in Vanguard 75, a lettuce cultivar nearly isogenic to Vanguard but carrying the resistance gene mo12 . Accumulation of the recombinant viruses in systemically infected leaves was either undetectable (GUS-tag) or erratic, strongly delayed, and inhibited by as much as 90% (GFP-tag). As a consequence, and contrary to the parental virus, the recombinant viruses were not able to overcome the protection afforded by the mo12 gene. Taken together, these results indicate that GUS or GFP tagging of the HC-Pro of LMV has significant negative effects on the biology of the virus, abolishing its resistance-breaking properties and reducing its pathogenicity in susceptible cultivars.

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

2002 ◽  
Vol 92 (2) ◽  
pp. 169-176 ◽  
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.

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 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 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 Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.

1996 ◽  
Vol 44 (6) ◽  
pp. 581-589 ◽  
Author(s):  
E Z Monosov ◽  
T J Wenzel ◽  
G H Lüers ◽  
J A Heyman ◽  
S Subramani
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Ultrastructural Localization ◽  
Yeast Cells ◽  
Fluorescent Properties ◽  
Green Fluorescent

We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

scholarly journals Studies on the Movement of Cowpea Mosaic Virus Using the Jellyfish Green Fluorescent Protein

Virology ◽  
1998 ◽  
Vol 242 (1) ◽  
pp. 22-27 ◽  
Author(s):  
Jan Verver ◽  
Joan Wellink ◽  
Jan Van Lent ◽  
Kodetham Gopinath ◽  
Ab Van Kammen
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Cowpea Mosaic Virus ◽  
Green Fluorescent

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.

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