1887

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Volume 91, Issue 6

Binding of monoclonal antibodies to the movement protein (MP) of : influence of subcellular MP localization and phosphorylation Free

Abstract

Monoclonal antibodies (mAbs) to recombinant movement protein (MP) of (TMV) were used to reveal the dependence of MP epitope accessibility to mAbs on subcellular MP localization and post-translational MP phosphorylation. Leaves of or were inoculated mechanically with TMV or agroinjected with an MP expression vector. At different time post-inoculation, ER membrane- and cell wall-enriched fractions (ER-MP and CW-MP, respectively) were isolated and analysed. The N-terminal region (residues 1–30) as well as regions 186–222 and 223–257 of MP from the CW and ER fractions were accessible for interaction with mAbs. By contrast, the MP regions including residues 76–89 and 98–129 were not accessible. The C-terminal TMV MP region (residues 258–268) was inaccessible to mAbs not only in CW-MP, but also in ER-MP fractions. Evidence is presented that phosphorylation of the majority of TMV MP C-terminal sites occurred on ER membranes at an early stage of virus infection, i.e. not after, but before reaching the cell wall. C-terminal phosphorylation of purified MP abolished recognition of C-proximal residues 258–268 by specific mAbs, which could be restored by MP dephosphorylation. Likewise, accessibility to mAbs of the C-terminal MP epitope in ER-MP and CW-MP leaf fractions was restored by dephosphorylation. Substitution of three or four C-terminal Ser/Thr residues with non-phosphorylatable Ala also resulted in abolition of interaction of mAbs with MP.

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2010-06-01
2024-04-26
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References

  1. Arce-Johnson, P., Kahn, T. W., Reimann-Philipp, U., Rivera-Bustamante, R. & Beachy, R. N.(1995). The amount of movement protein produced in transgenic plants influences the establishment, local movement, and systemic spread of infections by movement protein-deficient tobacco mosaic virus. Mol Plant Microbe Interact 8, 415–423.[CrossRef] [Google Scholar]
  2. Ashby, J., Boutant, E., Seemanpillai, M., Sambade, A., Ritzenthaler, Ch. & Heinlein, M.(2006). Tobacco mosaic virus protein functions as a structural microtubule-associated protein. J Virol 80, 8329–8344.[CrossRef] [Google Scholar]
  3. Berna, A.(1995). Involvement of residues within putative α-helix motifs in the behavior of the alfalfa and tobacco mosaic virus movement proteins. Phytopathology 85, 1441–1448.[CrossRef] [Google Scholar]
  4. Berna, A., Gafny, R., Wolf, S., Lucas, W. J., Holt, C. A. & Beachy, R. N.(1991). The TMV movement protein: role of the C-terminal 73 amino acids in subcellular localization and function. Virology 182, 682–689.[CrossRef] [Google Scholar]
  5. Brill, L. M., Nunn, R. S., Kahn, T. W., Yeager, M. & Beachy, R. N.(2000). Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein. Proc Natl Acad Sci U S A 97, 7112–7117.[CrossRef] [Google Scholar]
  6. Brill, L. M., Dechongkit, S., DelaBarre, D., Stroebel, J., Beachy, R. N. & Yeager, M.(2004). Dimerization of recombinant tobacco mosaic virus movement protein. J Virol 78, 3372–3377.[CrossRef] [Google Scholar]
  7. Citovsky, V.(1999). Tobacco mosaic virus: a pioneer of cell-to-cell movement. Philos Trans R Soc Lond B Biol Sci 354, 637–643.[CrossRef] [Google Scholar]
  8. Citovsky, V., Knorr, D., Schuster, G. & Zambryski, P.(1990). The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60, 637–647.[CrossRef] [Google Scholar]
  9. Citovsky, V., Wong, M. L., Shaw, A., Venkataram Prasad, B. V. & Zambryski, P.(1992). Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids. Plant Cell 4, 397–411.[CrossRef] [Google Scholar]
  10. Citovsky, V., McLean, B. G., Zupan, J. R. & Zambryski, P.(1993). Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase. Genes Dev 7, 904–910.[CrossRef] [Google Scholar]
  11. Cornell, R. B., Kalmar, G. B., Kay, R. J., Johnson, M. A., Sanghera, J. S. & Pelech, S. L.(1995). Function of the C-terminal domain of CTP : phosphocholine cytidylyltransferase. Effects of C-terminal deletions on enzyme activity, intracellular localization and phosphorylation potential. Biochem J 310, 699–708. [Google Scholar]
  12. Deom, C. M., Oliver, M. I. & Beachy, R. N.(1987). The 30KDa gene product of tobacco mosaic virus potentiates virus movement. Science 237, 389–394.[CrossRef] [Google Scholar]
  13. Dorokhov, Y. L., Alexandrova, N. M., Miroshnichenko, N. A. & Atabekov, J. G.(1983). Isolation and analysis of virus-specific ribonucleoprotein of tobacco mosaic virus infected tobacco. Virology 127, 237–252.[CrossRef] [Google Scholar]
  14. Dorokhov, Y. L., Alexandrova, N. M., Miroshnichenko, N. A. & Atabekov, J. G.(1984). The informosome-like virus-specific ribonucleoprotein (vRNP) may be involved in the transport of tobacco mosaic virus infection. Virology 137, 127–134.[CrossRef] [Google Scholar]
  15. Epel, B. L.(2009). Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host β-1,3-glucanases. Semin Cell Dev Biol 20, 1074–1081.[CrossRef] [Google Scholar]
  16. Gafny, R., Lapidot, M., Berna, A., Holt, C. A., Deom, C. M. & Beachy, R. N.(1992). Effects of terminal deletion mutations on function of the movement protein of tobacco mosaic virus. Virology 187, 499–507.[CrossRef] [Google Scholar]
  17. Gillespie, T., Boevink, P., Haupt, S., Roberts, A. G., Toth, R., Valentine, T., Chapman, S. & Oparka, K. J.(2002). Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus. Plant Cell 14, 1207–1222.[CrossRef] [Google Scholar]
  18. Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levi, A. & Epel, B.(2008). Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of N. benthamiana. Mol Plant Microbe Interact 21, 335–345.[CrossRef] [Google Scholar]
  19. Haley, A., Hunter, T., Kiberstis, P. & Zimmern, D.(1995). Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco protoplasts. Plant J 8, 715–724.[CrossRef] [Google Scholar]
  20. Heinlein, M. & Epel, L.(2004). Macromolecular transport and signaling through plasmodesmata. Int Rev Cytol 235, 93–164. [Google Scholar]
  21. Heinlein, M., Epel, B. L., Padgett, H. S. & Beachy, R. N.(1995). Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270, 1983–1985.[CrossRef] [Google Scholar]
  22. Heinlein, M., Padgett, H. S., Gens, J. S., Pickard, B. G., Casper, S. J., Epel, B. L. & Beachy, R. N.(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.[CrossRef] [Google Scholar]
  23. Kahn, T. W., Lapidot, M., Heinlein, M., Reichel, C., Cooper, B., Gafny, R. & Beachy, R. N.(1998). Domains of the TMV movement protein involved in subcellular localization. Plant J 15, 15–25.[CrossRef] [Google Scholar]
  24. Karpova, O. V., Ivanov, K. I., Rodionova, N. P., Dorokhov, Yu. L. & Atabekov, I. G.(1997). Nontranslatability and dissimilar behavior in plants and protoplasts of viral RNA and movement protein complexes formed in vitro. Virology 230, 11–21.[CrossRef] [Google Scholar]
  25. Karpova, O. V., Rodionova, N. P., Ivanov, K. I., Kozlovsky, S. V., Dorokhov, Y. L. & Atabekov, J. G.(1999). Phosphorylation of tobacco mosaic virus movement protein abolishes its translation repressing ability. Virology 261, 20–24.[CrossRef] [Google Scholar]
  26. Kawakami, S., Watanabe, Y. & Beachy, R. N.(2004). Tobacco mosaic virus infection spreads cell to cell as intact replication complexes. Proc Natl Acad Sci U S A 101, 6291–6296.[CrossRef] [Google Scholar]
  27. Kiselyova, O. I., Yaminsky, I. V., Karger, E. M., Frolova, O. Y., Dorokhov, Y. L. & Atabekov, J. G.(2001). Visualization by atomic force microscopy of tobacco mosaic virus movement protein-RNA complexes formed in vitro. J Gen Virol 82, 1503–1508. [Google Scholar]
  28. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef] [Google Scholar]
  29. Lee, J.-Y. & Lucas, W. J.(2001). Phosphorylation of viral movement proteins – regulation of cell-to-cell trafficking. Trends Microbiol 9, 5–8.[CrossRef] [Google Scholar]
  30. Lehto, K., Bubric, P. & Dawson, W. O.(1990). Time course of TMV 30K protein accumulation in intact leaves. Virology 174, 290–293.[CrossRef] [Google Scholar]
  31. Lucas, W. J.(2006). Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes. Virology 344, 169–184.[CrossRef] [Google Scholar]
  32. Mas, P. & Beachy, R. N.(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.[CrossRef] [Google Scholar]
  33. McLean, B. G., Zupan, J. & Zambryski, P. C.(1995). Tobacco mosaic virus movement protein associates with cytoskeleton in tobacco cells. Plant Cell 7, 2101–2114.[CrossRef] [Google Scholar]
  34. Melcher, U.(2000). The ‘30K’-superfamily of viral movement proteins. J Gen Virol 81, 257–266. [Google Scholar]
  35. Moore, P. J., Fenczik, C. A., Deom, C. M. & Beachy, R. N.(1992). Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus. Protoplasma 170, 115–127.[CrossRef] [Google Scholar]
  36. Oparka, K. J., Prior, D. A., Santa Cruz, S., Padgett, H. S. & Beachy, R. N.(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] [Google Scholar]
  37. Padgett, H. S., Epel, B. L., Kahn, T. W., Heinlein, M., Watanabe, Y. & Beachy, R. N.(1996). Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection. Plant J 10, 1079–1088.[CrossRef] [Google Scholar]
  38. Reichel, C. & Beachy, R.(1998). Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum. Proc Natl Acad Sci U S A 95, 11169–11174.[CrossRef] [Google Scholar]
  39. Reichel, C. & Beachy, R.(2000). Degradation of tobacco mosaic virus movement protein by the 26S proteasome. J Virol 74, 3330–3337.[CrossRef] [Google Scholar]
  40. Rhee, Y., Tzfira, T., Chen, M. H., Waigmann, E. & Citovsky, V.(2000). Cell-to-cell movement of tobacco mosaic virus: enigmas and explanations. Mol Plant Pathol 1, 33–39.[CrossRef] [Google Scholar]
  41. Sambade, A., Brandner, K., Hofmann, Ch., Seemanpillai, M., Mutterer, J. & Heinlein, M.(2008). Transport of TMV movement protein particles associated with the targeting of RNA to plasmodesmata. Traffic 9, 2073–2088.[CrossRef] [Google Scholar]
  42. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  43. Scofield, S. R., Tobias, C. M., Rathjen, J. P., Chang, J. H., Lavelie, D. T., Michelmore, R. W. & Staskawicz, B. J.(1996). Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato. Science 274, 2063–2066.[CrossRef] [Google Scholar]
  44. Sukhacheva, E. A., Tul'kina, L. G., Karger, E. M., Sheveleva, A. A., Stratonova, N. V. & Dorokhov, Yu. L.(2005). Mapping of epitopes of the recombinant movement protein of the tobacco mosaic virus with the use of monoclonal antibodies. Russ J Bioorganic Chem 31, 433–438.[CrossRef] [Google Scholar]
  45. Szécsi, J., Ding, X. S., Lim, C. O., Bendahmane, M., Cho, M. J., Nelson, R. S. & Beachy, R. N.(1999). Development of tobacco mosaic virus infection sites in Nicotiana benthamiana. Mol Plant Microbe Interact 12, 143–152.[CrossRef] [Google Scholar]
  46. Tomenius, K., Clapham, D. & Meshi, T.(1987). Localization of immunogold cytochemistry of the virus-coded 30k protein in plasmodesmata of leaves infected with tobacco mosaic virus. Virology 160, 363–371.[CrossRef] [Google Scholar]
  47. Trutnyeva, K., Bachmaier, R. & Waigmann, E.(2005). Mimicking carboxyterminal phosphorylation differentially effects subcellular distribution and cell-to-cell movement of Tobacco mosaic virus movement protein. Virology 332, 563–577.[CrossRef] [Google Scholar]
  48. Tyulkina, L. G., Skurat, E. V., Zvereva, A. S., Dorokhov, Yu. L. & Atabekov, J. G.(2006). Movement protein stimulates Tobacco mosaic virus reproduction in infected cells. Dokl Biochem Biophys 409, 253–256.[CrossRef] [Google Scholar]
  49. Waigmann, E., Lucas, W. J., 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 U S A 91, 1433–1437.[CrossRef] [Google Scholar]
  50. Waigmann, E., Chen, M.-H., Bachmaier, R., Ghoshroy, S. & Citovsky, V.(2000). Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein. EMBO J 19, 4875–4884.[CrossRef] [Google Scholar]
  51. Watanabe, Y., Ooshika, I., Meshi, T. & Okada, Y.(1984). Subcellular localization of the 30K protein in TMV-inoculated tobacco protoplasts. Virology 133, 18–24.[CrossRef] [Google Scholar]
  52. Wolf, S., Deom, C. M., Beachy, R. N. & Lucas, W. L.(1989). Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit. Science 246, 377–379.[CrossRef] [Google Scholar]
  53. Wright, K. M., Wood, N. T., Roberts, A. G., Chapman, S., Boevink, P., Mackenzie, M. & Oparka, K. J.(2007). Targeting of TMV movement protein to plasmodesmata requires the actin/ER network: evidence from FRAP. Traffic 8, 21–31.[CrossRef] [Google Scholar]
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Binding of monoclonal antibodies to the movement protein (MP) of Tobacco mosaic virus: influence of subcellular MP localization and phosphorylation
J. Gen. Virol. 91, 1621 (2010); https://doi.org/10.1099/vir.0.018002-0
/content/journal/jgv/10.1099/vir.0.018002-0
/content/journal/jgv/10.1099/vir.0.018002-0
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