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

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.

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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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Replication of Tobacco Mosaic Virus on Endoplasmic Reticulum and Role of the Cytoskeleton and Virus Movement Protein in Intracellular Distribution of Viral RNA

The Journal of Cell Biology ◽

10.1083/jcb.147.5.945 ◽

1999 ◽

Vol 147 (5) ◽

pp. 945-958 ◽

Cited By ~ 169

Author(s):

Paloma Más ◽

Roger N. Beachy

Keyword(s):

In Situ Hybridization ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Viral Rna ◽

Pharmacological Agents ◽

Intracellular Targeting ◽

Cytoplasmic Filaments ◽

Infected Cells

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-3-0335 ◽

2008 ◽

Vol 21 (3) ◽

pp. 335-345 ◽

Cited By ~ 94

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.

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

Journal of Virology ◽

10.1128/jvi.74.7.3330-3337.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3330-3337 ◽

Cited By ~ 100

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.

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Intramolecular Complementing Mutations in Tobacco Mosaic Virus Movement Protein Confirm a Role for Microtubule Association in Viral RNA Transport

Journal of Virology ◽

10.1128/jvi.76.8.3974-3980.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3974-3980 ◽

Cited By ~ 39

Author(s):

Vitaly Boyko ◽

Jamie Alan Ashby ◽

Elena Suslova ◽

Jacqueline Ferralli ◽

Oliver Sterthaus ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Inclusion Bodies ◽

Fluorescent Protein ◽

Movement Protein ◽

Microscopic Analysis ◽

Viral Rna ◽

Cell Transport ◽

Substitution Mutations ◽

Green Fluorescent

ABSTRACT The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MPP81S-GFP and MPP81S;T104I;R167K-GFP differ in subcellular distribution. MPP81S-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MPP81S;T104I;R167K-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.

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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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