Institut de Biologie Moléculaire des Plantes du CNRS et de l'Université Louis Pasteur, 12 rue du Gé néral Zimmer, Strasbourg 67084 cedex , France1
Author for correspondence: Véronique Ziegler- Graff.Fax +33 388 61 44 42.e-mail veronique.ziegler-graff{at}ibmp-ulp.u- strasbg.fr
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Abstract |
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Introduction |
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The fact that BW6.4 efficiently replicates and assembles into virions in Chenopodium quinoa mesophyll protoplasts (Reutenauer et al., 1993 ; Brault et al., 1995
) suggested that the diminished accumulation of BW6.4 in whole plants reflects a requirement for the RTD for vascular transport of the virus (Brault et al., 1995
). It is difficult, however, to eliminate the possibility that the low virus titre in whole plants is instead due to less efficient replication of the RTD-null mutant in the specialized nucleate cells of the phloem compartment where virus replication normally occurs. In this paper, we have addressed this question by comparing the distribution and level of multiplication of BW6.4 and wild-type BWYV in phloem cells of systemically infected leaves of agro-infected plants. Our findings indicate that deletion of the RTD greatly diminishes both the number and the size of infection foci in the phloem tissue but does not markedly reduce the virus levels attained within those cells which become infected.
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Methods |
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Tissue printing.
Petioles of systemically infected leaves of N. clevelandii were hand-cut with a razor blade and the freshly cut surface was gently pressed to a nitrocellulose membrane for several seconds. Serial prints were obtained by repeated application of this procedure at 12 mm intervals along the petiole. The membrane was incubated overnight at 4 °C in PBS (8 mM Na 2HPO4, 1·5 mM KH2PO4 , 150 mM NaCl, 2·5 mM KCl, pH 7·4) containing 0·5% Tween 20 and 5% dried milk (blocking solution). The membrane was next washed three times with PBS plus 0·5% Tween 20 and then incubated for 4 h at room temperature in blocking solution containing a 1:5000 dilution of a rabbit polyclonal antibody raised against BWYV coat protein (Bruyère et al., 1997 ). The tissue prints were washed and then incubated for 2 h in blocking solution containing alkaline phosphatase (AP) coupled to goat anti-rabbit secondary antibody (Bio-Rad). After three washes in PBS, bound antibody was rendered visible by incubating the membrane with 5-bromo-4-chloro-3- indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) in 0·1 M diethanolamine, pH 9·6. Preliminary tests were carried out with serial dilutions of the anti-coat protein antibody and the AP- coupled secondary antibody to show that the chromogenic response was proportional to the amount of coat protein antibody immobilized on the membrane in our experimental conditions.
Immuno-histology.
Upper non-inoculated leaves were collected from N. clevelandii 24 weeks post-agro-inoculation (p.i.). Excised tissue fragments were immersed immediately in fixing solution (4% formaldehyde, 50% ethanol, 5% acetic acid), vacuum infiltrated and left overnight at 4 °C. Samples were then dehydrated by immersion in a graded series of ethanol baths, transferred to Histo-clear II (National Diagnostics) and finally embedded in paraffin (Paraplast+; Oxford Labware). Serial sections (10 µm thick) were cut with a microtome, deparaffined with toluene, rehydrated in a graded series of ethanol baths, equilibrated in PBS and then immersed in PBS containing 0·05% Triton X-100. The sections were next immersed in 0·5% BSA in PBS followed by treatment with PBS containing 2% non- immune goat serum and 0·5% BSA. The sections were incubated overnight at 4 °C with anti-coat protein antibody and then with AP-coupled secondary antibody as above. Bound antibody was rendered visible by incubating with the AP substrate Fast Red TR/Naphthol AS-MX (Sigma).
Electron microscopy.
Transmission electron microscopy was carried out essentially as described (Ritzenthaler et al., 1995 ) on thin sections of glutaraldehyde-fixed leaf tissue from healthy or systemically BW6.4- and BW0-infected leaves of N. clevelandii.
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Results and Discussion |
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No immuno-labelled veins were detected in sections of either the BW6.4- or BW0-infected tissue sampled 1 week p.i. (Table 2). At 2 and 4 weeks p.i., immuno- labelled phloem cells associated with minor veins were observed for both the BW6.4-infected and the BW0-infected samples. The intensity of staining of the cells in the BW6.4-infected (Fig. 2g
i
) and the BW0-infected (Fig. 2k
m
, o
q
) samples was comparable but the number of veins labelled was markedly different. Thus at 2 weeks p.i., only 5% of the veins observed in section in the BW6.4-infected tissue were immuno-labelled whereas 80% of the veins in the BW0-infected tissue contained at least one immuno-labelled cell (Table 2
). In the few BW6.4-infected veins that were observed, immuno-labelling was always confined to a single cell (Fig. 2g
i
). In the BW0-infected veins, labelling was occasionally observed in single cells at 2 weeks p.i. (Fig. 2k, l
), but it was much more common to observe clusters of two or more adjacent immuno-labelled phloem cells when the vein was observed in transverse section (Fig. 2m
), or when the same vein was traced through a series of sections (not shown).
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Immuno-labelling of veins after agro-infection with RTD mutants
N.clevelandii plants were also agro-infected with BW6.106 and BW6.40 (Fig. 1), mutants with an in-frame deletion in either the conserved N-terminal half (BW6.106) or the non- conserved C-terminal half (BW6.40) of the RTD. We have shown previously that neither of these mutations inhibited synthesis of RT protein but that, in the case of BW6.106, the mutated RT protein was not incorporated into virions and the mutant virus accumulated poorly in agro-infected plants (Bruyère et al ., 1997
). Virions of mutant BW6.40, on the other hand, incorporated RT protein and accumulated to near wild-type levels following agro-infection (Bruyère et al., 1997
). Examination of leaf thin-sections taken 4 weeks p.i. revealed a pattern of immuno-labelled cells in the BW6.40- infected leaves similar to that observed in BW0-infected leaves, i.e. staining typically appeared in numerous contiguous cells (Fig. 2n
). In the BW6.106-infected systemic leaves, on the other hand, the distribution of immuno-labelled phloem cells was similar to that observed in the BW6.4-infected leaves, i.e. stained cells were infrequent and isolated (Fig. 2j
). Thus, even a relatively small deletion in the RTD can interfere with virus movement provided it falls within the conserved portion of the RTD. At present, we cannot conclude whether the inefficient spread of BW6.106 is due to the deletion of an important sequence motif or to the fact that the RT protein is not incorporated into virions.
Elimination of the RTD does not alter the infected cell types
The major cell types in veins of N. clevelandii are companion cells (C), phloem parenchyma cells (PP), sieve elements (SE), xylem parenchyma cells and xylem tracheary elements, all within a sheath of bundle cells (BS) (Ding et al., 1988 ). BWYV infection is generally confined to the C and PP cells. Immuno- labelling of small cells lining the sieve tubes was observed for both the BW6.4- and the BW0-infected samples (e.g. Fig. 2g
,h
, k
, l
, q
). Their small size and their proximity to the SE suggest that these cells are C cells. We also observed, for both types of inoculum, immuno-labelling of large rounded cells which are probably PP cells (Fig. 2i
, o
). Finally, some of the smaller immuno-labelled cells which were not immediately adjacent to an SE in the section cannot be placed with confidence in either category.
Electron microscopy can be used to better discriminate between C cells and PP cells because C cells typically have much smaller vacuoles than PP cells and contain a higher density of ribosomes. Thin sections of systemic leaves of N. clevelandii agro-infected with BW6.4 and BW0 were examined and cells in small veins that contained pseudocrystalline arrays of virus-like particles (VLP) were located. In agreement with earlier observations (Esau & Hoefert, 1972 ; D'Arcy & de Zoeten, 1979
; Shepardson et al., 1980
), arrays of VLP were visualized in both C cells and PP cells in the BW0-infected tissue (data not shown). The same cell types also contained VLP arrays in the BW6.4-infected tissue (Fig. 3
a, b
), but no such arrays were observed in comparable sections from healthy tissue (data not shown). Although not enough infected cells were examined by electron microscopy to allow statistical analysis, we conclude that the RTD is not absolutely required for infection of either cell type. Furthermore, the presence of extensive arrays of VLP in both the BW6.4- and the BW0- infected cells is consistent with the above observations of immuno- labelled cells by light microscopy, which indicate that, once a C or PP cell is infected, progeny virions accumulate abundantly even when the RTD is absent. In several sections from BW6.4-infected leaves, VLPs were observed in the plasmodesmata joining a C cell and an SE (Fig. 3c
, e
) or a PP cell and an SE (Fig. 3d
, f
). Such images provide additional evidence that the RTD is not strictly essential for movement of virions through plasmodesmata.
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We suggest that wild-type BWYV can move from an infected C or PP cell to a neighbouring cell either directly via connecting plasmodesmata, or indirectly, by transiting through an SE with connections to both cells. The abundance of isolated infected cells in the BW6.4-infected systemic leaves could reflect a strong requirement for the RTD for cell-to-cell movement of the former type. An alternative explanation would be that inefficient vascular movement of BW6.4 has sufficiently diminished the rate of virus movement into and out of the SE that transit via the vasculature to a neighbouring phloem cell is unlikely to occur during the experimental observation period. Evidently, it will be necessary to learn more about the relative importance in a wild-type infection of the cell-to-cell and vascular movement pathways in order to distinguish between these possibilities.
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Received 20 April 1999;
accepted 2 July 1999.