Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants

Silvia García-Castillo1, M. Amelia Sánchez-Pina2 and Vicente Pallás1

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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Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus.


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Carnation mottle virus (CarMV) is the type member of the genus Carmovirus within the plant virus family Tombusviridae (Lommel et al., 2000). It is a 30 nm icosahedral plant virus with a single-stranded, positive-sense RNA genome (4·0 kb). Sequence analysis of the CarMV genome revealed the presence of five open reading frames (ORFs), of which the 5'-proximal ORF (p27) and its read-through product (p86) are thought, by homology with Turnip crinkle virus (TCV), to be essential for genome replication (White et al., 1995). The 3'-proximal ORF codes for the 38 kDa coat protein (CP) (Morris & Carrington, 1988; Russo et al., 1994). It has been suggested that the two overlapping internal ORFs (p7 and p9) are involved in cell-to-cell transport, as has been shown for homologous proteins of TCV (Hacker et al., 1992; Li et al., 1998). P7, the putative movement protein (MP) of CarMV, has RNA-binding properties (Marcos et al., 1999). The p7 RNA-binding domain has been mapped to a central part of the molecule, which is rich in basic amino acids and contains an {alpha}-helix at its C-terminal region (Vilar et al., 2001). It has been recently shown that the CarMV p9 is an intrinsic membrane protein inserted into the endoplasmic reticulum (ER) membrane with a Ncyt–Ccyt topology (Vilar et al., 2002).

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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Fig. 1. Accumulation kinetics of positive (a, b) and negative (c) RNA strands of CarMV. Total RNA extracts from inoculated C. quinoa leaves, at different hours (a) or days (b, c) p.i. were analysed by Northern blotting. The analysis of positive RNA strands was made with a digoxigenin-labelled riboprobe, obtained from clone pCarM Ec3, whereas the negative RNA strands were detected with the digoxigenin-labelled pCarM CP riboprobe (see text). V, Viral RNA purified from viral particles; T+ and T-, cold transcripts from genomic CarMV RNA, synthesized from clone pCarM CP to obtain positive and negative polarities, respectively. Since panel (c) had to be overexposed to detect negative strands, T- transcript was used in this gel at a 25-fold lower concentration than the T+ transcript. The ratio of T+/T- accumulation was, however, calculated from dot-blot analysis of serial dilutions of known concentrations (see text). M, total RNA extracts from mock-inoculated leaves; 0–36, h p.i.; 2–11, days p.i.; g, genomic CarMV RNA; sg1, subgenomic CarMV RNA of 1·7 kb that directs the translation of the p7 protein; sg2, subgenomic CarMV RNA of 1·5 kb that directs the translation of the CP.

 
The accumulation kinetics of plus-sense viral RNAs in C. quinoa plants showed that the two sgRNAs were detected at earlier infection stages than the gRNA. Furthermore, at these early infection stages (2–5 days p.i.), the two sgRNAs accumulated to much higher levels than the gRNA and interestingly, the amount of the sgRNA that directs the translation of the CP was 2-fold the amount of the sgRNA that directs the translation of the MP (Fig. 1a, b). Similar results were obtained in TCV-infected protoplasts (Wang & Simon, 1997) as well as in Tobacco necrosis virus A (TNV-A)-infected protoplasts, in which a differential expression of the sgRNAs was observed (Meulewaeter et al., 1992). The differential accumulation of the MP sgRNA and the CP sgRNA during early infection stages could be a consequence of the different affinity of the subgenomic promoters for the RNA polymerase, early in infection.

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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Fig. 2. Accumulation kinetics (a) and subcellular localization (b, c) of CarMV CP and p7. (a) Total protein extracts from inoculated leaves of C. quinoa at different days p.i. were analysed by Western blotting. Lanes 1–10, total protein extracts at different days p.i. Notice that a host protein that is recognized by the MP antiserum served as internal loading control. (b) Subcellular fractionation was carried out by differential centrifugation and subsequent Western blotting of inoculated leaves from C. quinoa plants at 2, 4 and 9 days p.i. P1, fraction enriched with nuclei and plastids; P30, membranous fraction; S30, cytosolic fraction; CW, cell wall fraction; i, inoculated leaf fractions. The total amount of tissue in the S30 fraction was 10-fold lower than in the other fractions. Lanes c, positive controls of CP or MP, obtained from purified virus particles (CP) and E. coli-expressed purified p7 (MP). m, mock-inoculated leaves.

 
Additionally, the subcellular localization of CP and MP was studied in CarMV-inoculated leaves of C. quinoa at 2, 4 and 9 days p.i., by differential centrifugation and subsequent Western blot analysis of the fractions obtained (Donald et al., 1993; Más et al., 2000) (Fig. 2b, c). The values obtained by densitometric quantification were corrected for the equivalent amounts of tissue present in each fraction. The results showed that CP was present mainly in the cytosolic fraction (Fig. 2b, lane S30), although it was also present in the membranous and the cell wall fractions [Fig. 2b, lanes P30 and CW (cell wall)] at 4 days p.i. The subcellular localization of p7 showed that, although it was mainly located in the cytosol, the amount of protein present in the CW fraction during later stages of infection increases to a greater extent than in the other fractions (Fig. 2c). The non-transient expression of p7, together with its progressive accumulation in the CW, is consistent with the recently proposed model for the intracellular movement of CarMV (Vilar et al., 2002). In this model, p7 binds viral RNA, which would induce a conformational change in this soluble protein, unveiling its C terminus, which would then interact with the cytoplasmically exposed C-terminal domain of p9. This RNA-mediated protein–protein interaction would confine the ternary complex to the membrane.

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 10–12 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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Fig. 3. Spatio-temporal analysis of the distribution of viral positive (a, b and e) and negative (c, d and f) RNA strands of CarMV by in situ hybridization on transverse sections of inoculated leaves from C. quinoa plants at 4 (a, c), 6 (b, d) and 11 (e, f) days p.i. The probes used for detection of the positive and negative RNA strands of CarMV were the same as those described for the Northern blot analysis (pCarMV Ec 3 and pCarMV CP). Blue colour indicates infected area. Notice that the negative RNA strands accumulated at lower levels than the positive RNA strands and that the infection area increased in the leaf sections with days p.i. The negative RNA strands were localized in the leading edge of the chlorotic lesions, several cells behind the infection front, whereas the centre of the chlorotic lesions presented no signal. PP, palisade parenchyma; SM, spongy mesophyll; P, phloem tissue; X, xylem tissue. Bars, 100 µm.

 


   ACKNOWLEDGEMENTS
 
We thank Dr M. Aranda (CEBAS-CSIC, Murcia) for critical reading of the manuscript and Mr P. Thomas for checking the English grammar. This research was supported by Grants BIO99-0854 and 1FD97-0520 from the Spanish granting agency DGYCIT and the EU. S. García-Castillo was the recipient of a fellowship from the Instituto de Fomento-Fundación Seneca from Comunidad de Murcia.


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Received 16 July 2002; accepted 21 October 2002.



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