1 Instituto de Biología Molecular y Celular de Plantas, UPV-CSIC, Avenida de los Naranjos s/n, 46022 Valencia, Spain
2 Departamento de Mejora y Patología Vegetal, CEBAS (CSIC), Campus Universitario de Espinardo, 30100 Murcia, Spain
Correspondence
Vicente Pallás
vpallas{at}ibmcp.upv.es
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ABSTRACT |
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MAIN TEXT |
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CarMV translates those ORFs that are not 5'-proximal from two subgenomic RNAs (sgRNAs) that are 3'-coterminal with the genomic RNA (gRNA). The smaller sgRNA (1·5 kb) directs the translation of the CP, whereas the larger one (1·7 kb) appears to be a bicistronic RNA that directs the translation of the two central small proteins by an unknown mechanism (Morris & Carrington, 1988; Russo et al., 1994
). The expression mechanism of carmoviruses is relatively well known (Morris & Carrington, 1988
; Russo et al., 1994
). However, there are few studies dealing with the in vivo accumulation kinetics of genomic and sgRNAs, and their translation products in this group of viruses, particularly in its type member, CarMV. In the present work, we analyse the spatio-temporal accumulation in Chenopodium quinoa plants of the RNAs and of the CP and MP (p7) proteins of CarMV. Total RNAs were extracted from inoculated leaves of C. quinoa at different times post-inoculation (p.i.) (Verwoed et al., 1989
) and analysed by Northern blot under denaturing conditions (Fig. 1
). The Northern blots were hybridized with two different DIG-labelled riboprobes: (i) clone pCarM Ec3 (Marcos et al., 1999
), containing the entire sequence of the CP gene and the 3' end of the CarMV genomic RNA, to detect the accumulation of CarMV genomic and sgRNAs (positive strands); (ii) clone pCarM CP, corresponding to the CP sequence without the 3' end of the CarMV gRNA, to detect the accumulation of the viral negative strands. The specificity of the positive and negative probes for recognizing their corresponding targets was evaluated by using viral RNA and transcripts of both polarities, whose concentration was previously quantified by spectroscopy (Fig. 1
, lanes V, T+ and T-, respectively). The sensitivity of both probes was similar, as checked by using serial dilutions of appropriate targets and analysing them by dot-blot hybridization (not shown) (Pallás et al., 1998
). The RNA bands detected in the Northern blot analysis were quantified by film densitometry.
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Comparative analysis revealed that the negative sgRNA strands were accumulated to a 100-fold lesser extent than the positive sgRNA strands (as calculated by dot blot hybridization; not shown), which continued to accumulate steadily throughout infection, whereas the negative sgRNA strands began to decrease during the later stages of infection (Fig. 1b, c). The fact that CarMV negative RNA strands do not encapsidate into viral particles could explain this different accumulation kinetics, since the negative strands would be more susceptible to cellular degradation processes. A similar accumulation of the negative gRNA strands has been described in TNV-A (Meulewaeter et al., 1992
) and Tobacco mosaic virus (TMV)-infected protoplasts (Ishikawa et al., 1991
). However, the tombusvirus Artichoke mottle crinkle virus (AMCV) showed no quantitative difference in accumulation kinetics either for the positive or for the negative strands (Tavazza et al., 1994
).
We then studied the expression pattern of two CarMV proteins (CP and p7) in C. quinoa-inoculated leaves by Western blot analysis (Fig. 2a), as previously described (Balsalobre et al., 1997
). The total proteins extracted were probed with two polyclonal antisera, one raised against CarMV p7 (Marcos et al., 1999
) and diluted 1 : 10 000, and the other raised against whole CarMV virions, kindly provided by Drs Darós and Flores (IBMCP, UPV-CSIC, Valencia, Spain) and diluted 1 : 5000. Both proteins were simultaneously detected at 2 days p.i., while the maximum accumulation level was reached at 10 days p.i. (Fig. 2
). A transient expression in vivo has been reported for most of the MPs studied, behaviour which has been explained by their involvement in initial infection stages; for example, the MPs of Alfalfa mosaic virus (AMV) (Berna et al., 1986
), TMV (Deom et al., 1990
), Red clover necrotic mosaic virus (RCNMV) (Osman & Buck, 1991
) and Cucumber mosaic virus (CMV) (Vaquero et al., 1996
). However, the CarMV p7 does not show a transient expression in vivo, since its levels of accumulation do not fall during late infection stages. This does not rule out the putative involvement of the CarMV p7 in cell-to-cell movement, since similar kinetics have already been described for other very well characterized MPs, such as the MP of Cauliflower mosaic virus (CaMV) (Perbal et al., 1993
) and the nepovirus Grapevine fanleaf virus (GFLV) (Ritzenthaler et al., 1995
).
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Finally, the spatial distribution of positive and negative RNA strands of CarMV was studied at different days p.i. by in situ hybridization using the probes described for the Northern blot analysis (Fig. 3). Sampled tissue was harvested from mock- and CarMV-inoculated C. quinoa leaves at 4, 6 and 11 days p.i. Fixation, paraffin embedding and hybridization were carried out as previously described (García-Castillo et al., 2001
). Consistent with the results obtained in the Northern blot analysis, the in situ hybridization studies showed that the positive RNA strands accumulated to a greater extent than the negative RNA strands in all the infection stages and cellular types analysed. The results obtained from viral positive RNA strands reflect those previously obtained for the spatial distribution of the CP (García-Castillo et al., 2001
), where each leaf cellular type (epidermis, palisade parenchyma, mesophyl and vascular bundles) within the symptomatic areas were infected (Fig. 3a, b, e
). However, unlike positive RNA strands (Fig. 3a, b and e
), the negative RNA strands were not observed in the centre of the viral chlorotic lesions (Fig. 3c, d, f
). The signal from the negative RNA strands was localized at the edges of the chlorotic lesions (Fig. 3c, d, f
). Moreover, the signal at the leading edges on both sides of the lesion was connected throughout the central, non-infected area by infected spongy parenchyma and lower epidermis cells, suggesting a progression of the virus from the upper to the lower epidermis (Fig. 3c, d, f
). Therefore, virus replication seemed to be spatially regulated, since it was localized behind the infection front or at the leading edges of the chlorotic lesions. This result also suggests that replication is a requirement for cell-to-cell movement, as has similarly been described for the potyvirus Pea seed-borne mosaic virus (PSbMV), where replication was restricted to 1012 cells behind the infection front in infected immature pea cotyledons (Wang & Maule, 1995
). Studies on the subcellular fate of plant viral MPs fused to GFP (Huang & Zhang, 1999
; Széczi et al., 1999
) and involving immunogold labelling at the electron microscope level (Roberts et al., 1998
) showed that MPs are located within the cells ahead of, at or behind the infection front. These results and ours strongly suggest the existence of synchronization between replication and cell-to-cell movement.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Berna, A., Briand, J. P., Stussi-Garaud, C. & Godefroy-Colburn, T. (1986). Kinetics of accumulation of the three non-structural proteins of alfalfa mosaic virus in tobacco plants. J Gen Virol 67, 11351147.
Deom, C. M., Schubert, K. R., Wolf, S., Holt, C. A., Lucas, W. J. & Beachy, R. N. (1990). Molecular characterization and biological function of the movement protein of tobacco mosaic virus in transgenic plants. Proc Natl Acad Sci U S A 87, 32843288.[Abstract]
Donald, R. G. K., Zhou, H. & Jackson, A. O. (1993). Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley. Virology 195, 659668.[CrossRef][Medline]
García-Castillo, S., Marcos, J. F., Pallás, V. & Sánchez-Pina, M. A. (2001). Influence of the plant growing conditions on the translocation routes and systemic infection of Carnation mottle virus in Chenopodium quinoa plants. Physiol Mol Plant Pathol 58, 229238.[CrossRef]
Hacker, D. L., Petty, I., Wei, N. & Morris, T. J. (1992). Turnip crinkle virus genes required for RNA replication and virus movement. Virology 186, 18.[Medline]
Huang, M. & Zhang, L. (1999). Association of the movement protein of alfalfa mosaic virus with the endoplasmic reticulum and its trafficking in epidermal cells of onion bulb scales. Mol Plant Microbe Interact 12, 680690.
Ishikawa, M., Meshi, T., Ohno, T. & Okada, Y. (1991). Specific cessation of minus-strand RNA accumulation at an early stage of tobacco mosaic virus infection. J Virol 65, 861868.[Medline]
Li, W., Qu, F. & Morris, T. J. (1998). Cell-to-cell movement of turnip crinkle virus is controlled by two small open reading frames that function in trans. Virology 244, 405416.[CrossRef][Medline]
Lommel, S., Matelli, G. P. & Russo, M. (2000). Family Tombusviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 791825. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Marcos, J. F., Vilar, M., Perez-Payá, E. & Pallás, V. (1999). In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the putative movement protein from carnation mottle carmovirus (CarMV). Virology 255, 354365.[CrossRef][Medline]
Más, P., Sánchez-Pina, M. A., Balsalobre, J. M. & Pallás, V. (2000). Subcellular localisation of Cherry leaf roll virus coat protein and genomic RNAs in tobacco leaves. Plant Sci 153, 113124.[CrossRef][Medline]
Meulewaeter, F., Cornelissen, M. & van Emmelo, J. (1992). Subgenomic RNAs mediate expression of cistrons located internally on the genomic RNA of tobacco necrosis virus strain A. J Virol 66, 64196428.[Abstract]
Morris, T. J. & Carrington, J. C. (1988). Carnation mottle virus and viruses with similar properties. In The Plant Viruses, vol. 3, Polyhedral Virions with Monopartite RNA Genomes, pp. 73112. Edited by R. Koenig. New York: Plenum Press.
Osman, T. A. M. & Buck, K. M. (1991). Detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants. J Gen Virol 72, 28532856.[Abstract]
Pallás, V., Más, P. & Sánchez-Navarro, J. A. (1998). Detection of plant RNA viruses by non-isotopic dot-blot hybridisation. Methods Mol Biol 81, 461468.[Medline]
Perbal, M. C., Thomas, C. L. & Maule, A. J. (1993). Cauliflower mosaic virus gene I product (P1) forms tubular structures which extend from the surface of infected protoplasts. Virology 195, 281285.[CrossRef][Medline]
Ritzenthaler, C., Pinck, M. & Pinck, L. (1995). Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo. J Gen Virol 76, 907915.[Abstract]
Roberts, I. M., Wang, D., Findlay, K. & Maule, A. J. (1998). Ultrastructural and temporal observations of the cylindrical inclusions (Cls) show that the Cl protein acts transiently in aiding virus movement. Virology 245, 173181.[CrossRef][Medline]
Russo, M., Burgyan, J. & Martelli, G. (1994). Molecular biology of Tombusviridae. Adv Virus Res 44, 381428.[Medline]
Széczi, 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, 143152.
Tavazza, M., Lucioli, A., Calogero, A., Pay, A. & Tavazza, R. (1994). Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus. J Gen Virol 75, 15151524.[Abstract]
Vaquero, C., Sanz, A. I., Serra, M. T. & García-Luque, I. (1996). Accumulation kinetics of CMV RNA 3-encoded proteins and subcellular localization of the 3a protein in infected and transgenic tobacco plants. Arch Virol 141, 987999.[Medline]
Verwoed, T. C., Dekker, B. M. M. & Hoekema, A. (1989). A small scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17, 2362.[Medline]
Vilar, M., Esteve, V., Pallás, V., Marcos, J. F. & Pérez-Payá, E. (2001). Structural properties of Carnation mottle virus p7 movement protein and its RNA-binding domain. J Biol Chem 276, 1812218129.
Vilar, M., Saurí, A., Monné, M., Marcos, J. F., von Heijne, G., Pérez-Payá, E. & Mingarro, I. (2002). Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane. J Biol Chem 277, 2344723452.
Wang, D. & Maule, A. J. (1995). Inhibition of host gene expression associated with plant virus replication. Science 267, 229231.
Wang, J. & Simon, A. (1997). Analysis of the two subgenomic RNA promoters for turnip crinkle virus in vivo and in vitro. Virology 232, 174186.[CrossRef][Medline]
White, K. A., Skuzeski, J. M., Li, W., Wei, N. & Morris, T. J. (1995). Immunodetection, expression strategy and complementation of turnip crinkle virus p28 and p88 replication components. Virology 211, 525534.[CrossRef][Medline]
Received 16 July 2002;
accepted 21 October 2002.
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