Intracellular distribution of cowpea mosaic virus movement protein as visualised by green fluorescent protein fusions

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

PLANT PHYSIOLOGY ◽

10.1104/pp.108.117481 ◽

2008 ◽

Vol 147 (2) ◽

pp. 611-623 ◽

Cited By ~ 56

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

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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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Formation of Surface Tubules and Fluorescent Foci in Arabidopsis thaliana Protoplasts Expressing a Fusion between the Green Fluorescent Protein and the Cauliflower Mosaic Virus Movement Protein

Virology ◽

10.1006/viro.2000.0292 ◽

2000 ◽

Vol 271 (1) ◽

pp. 58-64 ◽

Cited By ~ 44

Author(s):

Zhong Huang ◽

Yu Han ◽

Stephen H. Howell

Keyword(s):

Arabidopsis Thaliana ◽

Green Fluorescent Protein ◽

Mosaic Virus ◽

Fluorescent Protein ◽

Movement Protein ◽

Cauliflower Mosaic Virus ◽

Virus Movement ◽

Green Fluorescent

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

Virology ◽

10.1006/viro.1997.8982 ◽

1998 ◽

Vol 242 (1) ◽

pp. 22-27 ◽

Cited By ~ 22

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

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Are Tubules Generated by the 3a Protein Necessary for Cucumber Mosaic Virus Movement?

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.11.985 ◽

1999 ◽

Vol 12 (11) ◽

pp. 985-993 ◽

Cited By ~ 42

Author(s):

Tomas Canto ◽

Peter Palukaitis

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Nicotiana Benthamiana ◽

Fluorescent Protein ◽

Movement Protein ◽

Virus Movement ◽

Wild Type ◽

Gene Encoding ◽

Green Fluorescent ◽

Infected Tobacco

The 3a movement protein of cucumber mosaic virus (CMV), fused to the jellyfish green fluorescent protein (3a-GFP) generated surface punctate aggregates as well as tubules protruding from infected tobacco and Nicotiana benthamiana protoplasts. Fluorescent tubules also appeared on the surface of protoplasts prepared from transgenic tobacco plants expressing 3a-GFP, indicating that the 3a protein is the only viral component required for the formation of the tubules. CMV with a mutation in the gene encoding the 3a protein, M8 CMV, could infect tobacco systemically, but tubules were not detected protruding from infected protoplasts when the mutated 3a protein was fused to the GFP [(M8)3a-GFP]. This indicates that the ability of the 3a protein to generate tubules in the surface of protoplasts is not a function required for the spread of CMV in tobacco. On the other hand, the (M8)3a-GFP did not traffic through plasmodesmata interconnecting tobacco epidermal cells, in contrast to the wild-type 3a-GFP. This suggests that there may be a correlation between the ability of the 3a protein to assemble tubules in protoplasts and its ability to promote movement within certain tissues.

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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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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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