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Plant Physiol, August 2001, Vol. 126, pp. 1349-1350
THE HOT AND THE CLASSIC
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INTRODUCTION |
Plasmodesmata are
cytoplasmic bridges between plant cells. Due to their small dimensions,
they were traditionally regarded as allowing only the passage of small
molecules and metabolites. It was surprising, therefore, that large
structures such as plant viruses were also found to move from cell to
cell via plasmodesmata. The last decade has witnessed great advances in
our understanding of how this happens. In tobacco mosaic virus (TMV)
infection, for example, a 30-kD viral movement protein (TMV MP) is
essential for the cell-to-cell spread of infection and, in part,
determines the host range of the virus. In the initially infected cell,
TMV MP is produced by the transcription of an RNA derived from the invading virus. TMV MP then associates with viral RNA (vRNA) molecules and mediates their transport into neighboring cells. This nucleoprotein complex is directed toward plasmodesmata via interactions with the host
cell's cytoskeleton. Recent evidence suggests that the targeting of
TMV MP/vRNA complexes to plasmodesmata may involve binding to cell wall
pectin methylesterase (PME). This month's The Hot and the
Classic summarizes some of the more important papers concerning
TMV MP, as determined by citation analysis, that have been published in
recent years.
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TMV MP Modifies Size Exclusion Limit (SEL) of
Plasmodesmata |
Wolf et al. (1989) developed a method for delivering
plasma membrane-impermeant fluorescent probes to the cytosol of
spongy mesophyll cells of tobacco (Nicotiana tabacum)
leaves. This technique was used to study plasmodesmatal size exclusion
limits in transgenic plants that express the TMV MP gene. Movement of
fluorescently tagged dextran with an average molecular mass of 9.4 kD
and an approximate Stokes radius of 2.4 nm occurred between cells of the transgenic plants, whereas the SEL for the control plants was 700 to 800 D. Thus, expression of the TMV MP gene in tobacco plants
increases the plasmodesmatal SEL 10-fold between adjacent mesophyll cells.
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TMV MP Specifically Localizes to Secondary
Plasmodesmata |
In young tobacco leaves, almost all plasmodesmata are primary in
nature. Secondary plasmodesmata are formed in a basipetal pattern as
the leaves undergo expansion (Ding et al., 1992). This pattern is
observed regardless of whether the plant is transgenically altered to
express TMV MP. In those plants in which TMV MP is transgenically
expressed, it accumulates predominantly in the secondary plasmodesmata
and is associated with a filamentous material. Dye-coupling experiments
revealed a close temporal correlation between the modification of
plasmodesmatal SEL and the localization of TMV MP in secondary
plasmodesmata. This association is not seen in the
bundle-sheath/vascular parenchyma boundary, suggesting that
another mechanism is needed for phloem invasion by the virus.
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The TMV MP-Nucleic Acid Complex Is a Thin Filament |
Citovsky et al. (1992) used electron microscopy to visualize the
complexes that TMV MP forms with single-stranded DNA and RNA. These
complexes are long, unfolded, and very thin (1.5-2.0 nm in diameter).
The complexes are compatible in size with the TMV MP-induced increase
in plasmodesmatal permeability, making them likely candidates for the
structures involved in the cell-to-cell movement of TMV. Mutational
analysis using single and double deletion mutants of TMV MP revealed
three regions potentially important for protein function.
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Dynamics of TMV MP Function Visualized |
Waigmann et al. (1994) developed a microinjection assay for
monitoring the dynamics of TMV MP function directly in wild-type plants. Their results indicate that TMV MP influences plasmodesmal SEL
several cells distant from the injection site, indicating either that
TMV MP itself crosses plasmodesmata or that TMV MP induces a diffusible
signal capable of dilating plasmodesmatal pores. The region of TMV MP
responsible for increasing plasmodesmatal SEL was mapped to the
carboxyl-terminal end of the protein.
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Microtubules Target TMV MP to Plasmodesmata |
Heinlein et al. (1995) fused TMV MP with green
fluorescent protein (GFP). A modified virus that contained this MP:GFP
fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after
infection. Double-labeling fluorescence microscopy suggests that TMV MP
interacts and coaligns with microtubules (MTs). The distribution of TMV
MP is disrupted by treatments that disrupt MTs, but not by cytochalasin
B, which disrupts filamentous F-actin. McLean et al. (1995) achieved
similar results by means of fluorescent immunolocalization, except that
they noted a subtle interaction of TMV MP with actin filaments. In
vitro binding assays also suggested that TMV MP binds to actin.
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TMV MP Is Most Active in the Leading Edge of the
Infection |
Plasmodesmatal permeability in tobacco epidermal
cells was examined in expanding infection sites of TMV expressing
MP-GFP (Oparka et al., 1997). The infection sites were circular in
profile and within 3 d following inoculation had developed a
brightly fluorescent leading edge. Co-localization of MP-GFP with
callose demonstrated that nearly all epidermal cell plasmodesmata
were targeted with MP-GFP. MP-GFP was located in the center of the plasmodesmal pore. Increase in plasmodesmatal SEL, as determined by the
passage of microinjected 10-kD Texas Red dextran, was restricted predominantly to cells within the highly fluorescent leading edge and
was virtually absent from cells in the center of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labeled with MP-GFP. Injections outside the fluorescent infection site failed to show movement of dextran, whereas
dextran injected into cells at the leading edge moved inwards toward
the center of the lesion but not outwards into cells lacking GFP.
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TMV MP Localized to Endoplasmic Reticulum
(ER) |
Heinlein et al. (1998) used TMV expressing MP:GFP in combination
with antibody staining to identify the host cell components in which MP
and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected tobacco protoplasts. MP:GFP and
replicase colocalized to the ER and were present in large, irregularly
shaped, ER-derived structures that may represent "viral factories."
The ER-derived structures required an intact cytoskeleton, and MTs
appeared to redistribute MP:GFP from these sites during late stages of
infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in
protoplasts, the MP:GFP was targeted to distinct, punctate sites near
the plasma membrane. Treating protoplasts with cytochalasin D and
brefeldin A at the time of inoculation prevented the accumulation of
MP:GFP at these sites. It is proposed that the punctate sites anchor
the cortical ER to plasma membrane and are related to sites at which
plasmodesmata form. The authors present a model that postulates a role
for the ER and cytoskeleton in targeting TMV MP and viral
ribonucleoprotein from sites of virus synthesis to the plasmodesmata.
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Association of vRNA with Plasma Membrane Protrusions
Requires TMV MP |
The distribution of TMV vRNA in infected tobacco protoplasts was
visualized by in situ hybridization (Mas and Beachy, 1999). Immunostaining of the ER lumenal-binding protein concurrent with in
situ hybridization revealed that vRNA colocalized with the ER. At
midstages of infection, vRNA accumulated in large, irregular bodies
associated with cytoplasmic filaments, whereas at late stages vRNA was
dispersed throughout the cytoplasm and was associated with hair-like
protrusions from the plasma membrane containing ER. TMV 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
MTs, whereas disruption of the cytoskeleton with pharmacological
agents produced severe changes in vRNA localization. Viral mutants
lacking functional TMV MP revealed that TMV MP was not required for
association of vRNA with 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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TMV MP Binds to PME |
A 38-kD cell wall protein that binds to TMV MP was identified as
PME by Dorokhov et al. (1999) and Chen et al. (2000). Chen et al.
(2000) report that deletion of the PME-binding region results in the
inactivation of TMV cell-to-cell movement. These data suggest that PME
may be a host cell receptor involved in cell-to-cell movement of TMV or
that that TMV MP may modify the activity of PME during the formation of
secondary plasmodesmata.
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LITERATURE CITED |
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Chen MH, Sheng JS, Hind G, Handa AK, Citovsky V
(2000)
Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement.
EMBO J
19: 913-920[CrossRef][ISI][Medline]
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Citovsky V, Wong ML, Shaw AL, Prasad BVV, Zambryski P
(1992)
Visualization and characterization of tobacco mosaic-virus movement protein-binding to single-stranded nucleic-acids.
Plant Cell
4: 397-411[Abstract/Free Full Text]
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Ding B, Haudenshield JS, Hull RJ, Wolf S, Beachy RN, Lucas WJ
(1992)
Secondary plasmodesmata are specific sites of localization of the tobacco mosaic-virus movement protein in transgenic tobacco plants.
Plant Cell
4: 915-928[Abstract/Free Full Text]
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Dorokhov YL, Makinen K, Frolova OY, Merits A, Saarinen J, Kalkkinen N, Atabekov JG, Saarma M
(1999)
A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein.
FEBS Lett
461: 223-228[CrossRef][ISI][Medline]
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Heinlein M, Epel BL, Padgett HS, Beachy RN
(1995)
Interaction of tobamovirus movement proteins with the plant cytoskeleton.
Science
270: 1983-1985[Abstract/Free Full Text]
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Heinlein M, Padgett HS, Gens JS, Pickard BG, Casper SJ, Epel BL, Beachy RN
(1998)
Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection.
Plant Cell
10: 1107-1120[Abstract/Free Full Text]
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Mas P, Beachy RN
(1999)
Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA.
J Cell Biol
147: 945-958[Abstract/Free Full Text]
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McLean BG, Zupan J, Zambryski PC
(1995)
Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.
Plant Cell
7: 2101-2114[Abstract]
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Oparka KJ, Prior DAM, SantaCruz S, Padgett HS, Beachy RN
(1997)
Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV).
Plant J
12: 781-789[CrossRef][ISI][Medline]
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Waigmann E, Lucas WJ, Citovsky V, Zambryski P
(1994)
Direct functional assay for tobacco mosaic-virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.
Proc Natl Acad Sci USA
91: 1433-1437[Abstract/Free Full Text]
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Wolf S, Deom CM, Beachy RN, Lucas WJ
(1989)
Movement protein of tobacco mosaic virus modifies plasmodesmatal exclusion limit.
Science
246: 377-379[Abstract/Free Full Text]
Peter V. Minorsky
Department of Biology
Vassar College
Poughkeepsie, NY 12604
© 2001 American Society of Plant Physiologists
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TOP
INTRODUCTION
TMV MP Modifies Size...