INRA, 28 rue de Herrlisheim, 68021 Colmar Cedex, France1
Department of Plant Pathology, Pennsylvania State University, University Park, PA 16802, USA2
IBMP, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France3
Instituto Biológico-CEIB, PO Box 70, 13001-970 Campinas, SP, Brazil4
De Ruiter Seeds, PO Box 1050, 2660 BB Bergschenhoek, Netherlands5
Author for correspondence: Véronique Brault. Fax +33 3 89 22 49 33. e-mail brault{at}colmar.inra.fr
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Abstract |
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Introduction |
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Earlier studies on Luteoviridae, especially on the barley yellow dwarf complex, composed mainly of Barley yellow dwarf virus-PAV and -MAV (BYDV-PAV and -MAV), and Cereal yellow dwarf virus-RPV (CYDV-RPV), revealed that digestive and salivary epithelia have differential specificity to luteovirus particles (Rochow, 1969 ; Gildow, 1999
). Particles of many luteoviruses, with some exceptions (Gildow, 1999
), can be transported across the gut wall of different aphid species, whether efficient vectors or non-vectors. Therefore, the ability of luteoviruses to cross the gut wall is a trait generally unrelated to vector-specificity. However, the gut wall may affect transmission efficiency (Rouzé-Jouan et al., 2001
). The salivary gland barrier, on the other hand, appears to be virus species-specific and accounts for most of the transmission specificity between vector and virus species. Moreover, the salivary gland barrier has been shown recently to function at two different levels: the basal lamina, which can be penetrated by some luteoviruses with certain non-efficient vector species, and the basal plasmalemma, whose permeability ultimately dictates vector specificity (Peiffer et al., 1997
). In addition to these two specificity barriers, efficient retention and transmission of luteoviruses require the virions to associate with Buchnera GroEL, produced by endosymbiotic bacteria (Buchnera spp.) of aphids and released into their haemolymph (van den Heuvel et al., 1994
; 1997
; van den Heuvel, 1999
).
Transmission electron microscopy (TEM) is a powerful approach to analyse virusvector relationships (Ammar, 1991 ). Ultrastructural studies aimed at the localization of luteovirus particles in organs and cells of vector and non-vector aphids (Gildow, 1999
) revealed the site of passage (acquisition) of particles across the digestive tract. Virions of BYDV-PAV and -MAV (Luteovirus) and CYDV-RPV (Polerovirus) are acquired across the hindgut of their vectors, which are several species of cereal aphids (Gildow, 1999
). However, Potato leafroll virus (PLRV; Polerovirus) is known to be acquired in the midgut of Myzus persicae (Garret et al., 1993
; Gildow et al., 2000b
), whereas Soybean dwarf virus (SbDV; unassigned member of the Luteoviridae) is acquired through the hindgut of the same species (Gildow et al., 2000a
). Furthermore, Pea enation mosaic virus-1 (PEMV-1; Enamovirus) is acquired through the midgut of Acyrthosiphon pisum (Harris & Bath, 1972
). The site of passage of BWYV in M. persicae has not yet been determined.
Extensive TEM observations led Gildow (1987 , 1999
) to propose a model of receptor-mediated endocytosis and exocytosis to explain the transport of luteovirus particles through the different barriers in the aphid. These processes rely on the presence of putative receptors in the different cell membranes. The narrow specificity of luteovirus transmission is thought to be regulated by interactions of viral capsid components with specific receptors, whose nature and localization remain to be determined (van den Heuvel et al., 1999
). The capsid of Luteoviridae members is composed of two proteins, the major coat protein (CP), encoded by ORF 3, and the minor readthrough (RT) protein, encoded by ORF 3 and the downstream ORF 5, which is expressed by a stop codon readthrough mechanism. The RT component is known to play a role in virus accumulation in planta (Brault et al., 1995
; Chay et al., 1996
; Bruyère et al., 1997
). Moreover, the RT protein is important for virusvector interactions, as demonstrated by experiments with BWYV or BYDV-PAV mutants carrying no RT protein or a mutated version thereof (Brault et al., 1995
; Chay et al., 1996
; Bruyère et al., 1997
; Brault et al., 2000
). Thus, a BWYV mutant carrying a deletion of ORF 5 (BWYV-6.4) was found to be non-transmissible when acquired either by feeding or by microinjection of purified virions into the haemocoel (Brault et al., 1995
). Moreover, this mutant was unable to bind to Buchnera GroEL in vitro and was rapidly degraded in the haemolymph when microinjected into aphids (van den Heuvel et al., 1997
). These findings suggest that the RT protein is required for stability of virions in the haemolymph and/or their transport to the ASG (Brault et al., 1995
). In the case of BYDV-PAV, even though the RT protein was required for efficient transmission, it was not necessary for virus uptake in the haemolymph. The RT protein, however, was required for transport of virions through the ASG cells (Chay et al., 1996
). More recently, however, the role of the RT protein in transmission has been called into question by experiments with virus-like particles (VLP) obtained through baculovirus expressing the CP gene of PLRV fused to a histidine tag (Gildow et al., 2000b
). Such VLPs, containing no RT protein, were shown to circulate in the vector M. persicae in a way similar to wild-type virions; moreover, these VLPs could readily be observed in the salivary canal. These observations suggested that RT protein may not be required for passage of virions through both gut and salivary cells, or for their diffusion in the haemocoel. Aiming to better understand the role of RT protein in the transmission process, TEM observations were undertaken to localize particles of the mutant BWYV-6.4 in both the gut cells and the ASG of M. persicae. Nucleic acid sequence-based amplification (NASBA; Kieviets et al., 1991
) was used to assess whether this mutant was present in the haemolymph.
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Methods |
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Microinjection of purified virus at approximately 100 µg/ml for wild-type BWYV (BWYV-WT) and BWYV-6.4 was performed as previously described (Bruyère et al., 1997 ). After a 72 h IAP on M. perfoliata, microinjected aphids were prepared for ultrastructural TEM examination of the ASG. The plants were tested for virus infection 4 weeks later by ELISA.
Sample preparation.
For ultrastructural TEM examination, aphids were fixed and embedded as previously described (Gildow & Gray, 1993 ) with some modifications. Aphids were bisected in the middle of the thorax into an anterior part containing the foregut and the salivary glands, and a posterior part containing the midgut joined to the hindgut. This operation was carried out in the primary fixative (5% glutaraldehyde, 2% formaldehyde, 0·01% CaCl2, 0·05% NaN3 in 0·02 M sodium cacodylate, pH 7·4). After an overnight fixation at 4 °C, the tissues were post-fixed in 1% osmium tetroxide, transferred to 2% aqueous uranyl acetate, and finally dehydrated in an acetone series before being embedded in EponAraldite plastic. Ultrathin sections (6080 nm) were stained by two successive incubations in 2% uranyl acetateisobutanol (9:1) for 45 min and in 0·04% lead citrate for 15 min.
In order to facilitate visualization of virions at the basal pole of cells, some aphids were microinjected with a non-diluted BWYV polyclonal antiserum (Bio-Rad/Sanofi) or BWYV anti-P19 (Reutenauer et al., 1993 ) polyclonal antiserum, after a 72 h AAP on purified virus, as previously described (Gildow, 1982
, 1993
). These aphids were fixed and embedded 3 h after microinjection. All observations were made with a Philips EM 208 transmission electron microscope operating at 80 kV.
Virus purification and detection.
BWYV-WT and BWYV-6.4 were purified from agro-infected Nicotiana clevelandii (Brault et al., 1995 ) according to the method of van den Heuvel et al. (1991)
. Purified virions were stored at -80 °C in citrate buffer (0·1 M sodium citrate, pH 6·0) containing 25% sucrose. Virus infection was assayed by double antibody sandwich ELISA (Clark & Adams, 1977
) using an anti-BWYV polyclonal antiserum (Bio-Rad/Sanofi).
Virus stability.
To assess stability of the particles in the aphid diet, RNA was extracted from purified virus in the presence of Tris-saturated phenol and 100 mM TrisHCl pH 8·0, 50 mM NaCl, 10 mM EDTA, 1% SDS and 2 mg/ml proteinase K (Euromedex). After incubation at 60 °C for 10 min, samples were extracted with phenolchloroform and ethanol-precipitated. Viral RNA was detected by Northern blot analysis (Veidt et al., 1992 ).
Viral RNA was also extracted from whole aphids or haemolymph samples as described by Chay et al. (1996) . Haemolymph was collected by removing a mesothoracic leg with fine forceps and applying gentle pressure on the abdomen to force haemolymph droplets from the wound. The samples were collected using a fine glass capillary.
Detection of viral RNA by AmpliDet RNA.
To detect BWYV RNA in haemolymph samples of M. persicae, AmpliDet RNA (Leone et al., 1998 ) was used. This method combines the NASBA technique (Kievits et al., 1991
) with molecular beacon probes (Tyagi & Kramer, 1996
; Eun & Wong, 2000
).
Two different primer sets (Table 1) were designed to amplify viral RNA, based on the nucleotide sequence of the CP (P1-CP, P2-CP) and the readthrough domain (RTD expressed from ORF 5) (P1-RTD, P2-RTD). The P1 primers contain both a target complementary sequence and a 5'-T7 polymerase recognition site sequence. To detect the amplicons, two molecular beacons, MB-CP and MB-RTD, complementary to the target sequences were used (Table 1
). Each beacon carried a target-specific sequence and 5' and 3' arm sequences of 6 nucleotides, tagged either with a fluorophore (6-carboxyfluorescein; FAM) or a quencher (DABCYL). The arm sequences are complementary to each other and form a double-stranded structure if no target is present. In this conformational state fluorescence is quenched. Upon hybridization of the molecular beacon to its target, the double-stranded structure is broken and the fluorophore is no longer quenched. The sensitivity of the molecular beacons was estimated as 50 and 5 fg of purified virus, for MB-CP and MB-RTD, respectively. The fluorescence of FAM was measured at 538 nm using a Fluoroscan FL instrument.
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Results |
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Aphid acquisition of BWYV-WT occurred following membrane feeding on a purified virus suspension. Efficiency of virus acquisition was tested by transferring some of the membrane-fed aphids onto test plants for transmission tests. Subsequent ELISA measurements revealed that all 10 test plants fed on by aphids that had been previously acquisition-fed with BWYV-WT were infected, indicating that a high number of aphids had acquired and transmitted the virus. The transmission test also demonstrated that the purified BWYV-WT used in our experiments retained transmissibility and infectivity following purification and storage at -80 °C. None of the 10 plants fed on by buffer-fed aphids (aphid control) became infected, confirming that the aphid culture was non-viruliferous.
In the anterior midgut (stomach), BWYV-WT particles were frequently observed in the lumen mixed with other gut contents, but particles were never observed attached to the apical membranes of stomach cells. Particles were never observed to occur within the stomach cell cytoplasm in structures similar to those described below for the posterior midgut. Therefore, there is no evidence to support the idea that the stomach is involved in BWYV acquisition by M. persicae.
BWYV-WT particles were consistently observed in the lumen of the posterior midgut and the hindgut (Table 2) following membrane feeding. This observation demonstrates that aphids were ingesting the virus in detectable amounts. However, BWYV-WT particles were observed only within cells of the midgut and not in the hindgut cells, indicating that BWYV-WT was acquired specifically through the midgut. In the posterior midgut (intestine), virions freely suspended in the lumen were only occasionally observed, but particles were frequently observed immediately adjacent to or in contact with the midgut cell membranes lining the lumen. Virus particles appeared to accumulate in close proximity to intestinal cell membranes. Individual particles associated with the midgut apical plasmalemma accumulated at the tips of microvilli of cells (Fig. 1a
), or within apical plasmalemma invaginations forming narrow channels into midgut cells (Fig. 1b
).
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As a control, membrane-fed aphids from the same experiment were injected with an antiserum directed against a non-structural protein of BWYV (P19, putative movement protein). In these aphids, isolated particles were observed in the basal lamina (three of eight aphids examined; Table 2) but virus aggregates were never observed, providing further evidence that the virus-like particles observed after injection of anti-BWYV antiserum were indeed BWYV particles aggregated after exocytosis from midgut cells.
Virions were occasionally observed suspended in the hindgut lumen, but virus was not observed attached to the hindgut apical cell membrane. Particles were never observed within the cytoplasm of hindgut cells in membrane structures, or embedded in the basal lamina surrounding the hindgut as described above (Table 2). These observations provided no evidence that the hindgut is involved in BWYV acquisition. Virions observed in the hindgut lumen are presumed to be passing out of the aphid through the alimentary canal.
Acquisition of the non-transmissible BWYV-6.4 RT-minus mutant
The fate of the non-transmissible mutant BWYV-6.4, which lacks the RT protein, was first analysed in membrane-fed M. persicae. To test for transmissibility of BWYV-6.4, 10 Montia perfoliata seedlings were given a 3 day inoculation feeding with aphids previously membrane-fed for 72 h on purified BWYV-6.4 virions. None of these plants became infected after 4 weeks, confirming the reported inability of M. persicae to transmit this mutant (Brault et al., 1995 ).
When aphids were acquisition-fed for 72 h on BWYV-6.4, virions were present as free particles suspended in the lumen of the midgut and the hindgut in 10 of 26 aphids observed (38% vs 100% for BWYV-WT) and 5 of 15 (33% vs 82% for BWYV-WT), respectively (Table 2). This observation confirms that aphids were ingesting virus during feeding and that detectable amounts of the virus remained intact during the 72 h membrane feeding. The virus particles were dispersed in low concentrations in the lumen (Fig. 4a
), but crystal-like arrays of particles were sometimes also found in the midgut (Fig. 4b
) and the hindgut lumen (Fig. 4c
). No virus particles were visualized in the cytoplasm of intestine or hindgut cells in any of the aphids examined (Table 2
). This indicated that BWYV-6.4 was not being acquired into cells in amounts detectable by TEM.
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Association of BWYV-WT and BWYV-6.4 with the aphid accessory salivary glands
Localization of BWYV-WT in the ASG was analysed following purified virus acquisition either by membrane feeding or by microinjection into the haemolymph (Table 3). The ability of M. persicae to transmit BWYV-WT following microinjection of purified virus was tested by allowing injected aphids a 3 day inoculation feeding on Montia perfoliata seedlings, followed by ELISA testing for infection. All 10 test plants fed on by the BWYV-WT-injected aphids were infected, demonstrating that the aphids were viruliferous and that the purified virus was infectious. None of 5 test plants became infected when fed on by non-injected aphids. The ASG of M. persicae consists of four secretory cells connected at their apical poles to the salivary ducts through a branched network of microvilli-lined secretory canals (Gildow, 1987
). The entire ASG is surrounded by a complex matrix consisting of collagen and laminin glycoproteins assembled into a network containing several other components, and is capable of preventing luteovirus transmission (Peiffer et al., 1997
). When purified BWYV-WT was membrane-fed or injected into aphids, virions were consistently observed embedded within the basal lamina (Fig. 5a
) surrounding the ASG in all 9 aphids examined (Table 3
). Therefore, the ASG basal lamina does not present a barrier to BWYV-WT transmission in M. persicae. BWYV-WT particles were seen in invaginations of the basal plasmalemma (Fig. 5b
), and occasionally in coated or tubular vesicles within the cytoplasm (Table 3
), indicative of virus uptake into the ASG. Particles were present in the salivary canal in several aphids.
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Structural stability of BWYV-6.4 virions during acquisition
Rapid degradation of BWYV-6.4 particles in the aphid diet could account for the low number of particles visualized in the aphid gut and ASG. That this was not the case was shown by analysis of RNA extracted from the diet containing either BWYV-WT or BWYV-6.4, 0 and 72 h after the beginning of acquisition. Both BWYV-WT and BWYV-6.4 genomic RNA were detected in the samples (Fig. 6). The bands were less intense when RNA were extracted 72 h after acquisition, indicating that some particle breakdown and RNA degradation occurs in the diet. This degradation, however, occurred to similar extents for BWYV-WT and BWYV-6.4, indicating that there is no particular instability of BWYV-6.4 virions. Stability of BWYV-6.4 particles was also confirmed by TEM visualization of equivalent amounts of each type of particle (BWYV-WT or BWYV-6.4) in samples of the diet following the 72 h acquisition feeding (data not shown). Furthermore, observations of BWYV-WT and BWYV-6.4 particles from artificial diet immuno-captured onto anti-BWYV-coated grids revealed that the virions remained structurally intact for up to 7 days at room temperature. Thus, loss of structural integrity does not seem to explain the reduced ability to visualize BWYV-6.4 in membrane-fed aphids or the inability of aphids to transmit this mutant.
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Discussion |
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The BWYV-6.4 mutant has been shown to be non-transmissible by membrane-fed or microinjected aphids (Brault et al., 1995 ). Our results show that in membrane-fed aphids BWYV-6.4 virions were only occasionally observed in the gut lumen. Instability of viral particles in the aphids diet was not responsible for the infrequent presence of BWYV-6.4, as shown by TEM observations of intact virus in the diet and the detection of viral genomic RNA extracted from the diet following membrane feeding. The low amount of BWYV-6.4 particles visualized in the gut lumen could result from a lack of adsorption on the apical plasmalemma leading to a rapid elimination of the particles in the honeydew. However, the crystals of BWYV-6.4 observed in the gut lumen could also reduce the number of free particles seen in the lumen. Apart from the intestine lumen, BWYV-6.4 virions were also observed between the basal plasmalemma and basal lamina, in the basal lamina or between two basal laminae of adjacent intestine cells, but only in aphids microinjected with anti-BWYV antibodies. BWYV-6.4 particles, however, were never detected in the cytoplasm of intestine cells. We assume that intracellular BWYV-6.4 virions were present but simply too rare to be detected by our observations. Even if large amounts of BWYV-6.4 particles are aggregated in the crystals found in the lumen, we can assume that this arrangement of particles cannot be endocytosed in intestinal cells. The release of BWYV-6.4 virions in the haemocoel was also demonstrated by our detection of viral RNA in the haemolymph using the AmpliDet RNA technique. This mutant, which is unable to bind Buchnera GroEL (van den Heuvel et al., 1997
), is probably degraded in the haemolymph before attaining the ASG. Mutant virions were not detected near or within ASG cells, even when purified BWYV-6.4 was microinjected directly into the haemocoel. These results confirm those reported by Chay et al. (1996)
, who showed that a BYDV RT protein-deficient mutant was detected by reverse transcriptionPCR in the haemolymph of the vector, but that particles were never observed in the ASG basal lamina. On the other hand, contrasting results have been obtained with PLRV VLPs produced in a baculovirus system (Gildow et al., 2000a
). In this work, RT protein-deficient VLPs were found in the vector at the same locations as observed for the wild-type virions. It cannot be ruled out, however, that the histidine-tag fused at the N terminus of the CP, potentially exposed on the surface of the VLPs, could stabilize the capsid of these VLPs by interacting with Buchnera GroEL and, thus, functionally substitute for the RT protein.
Based on our results, the RT protein of BWYV does not seem to be strictly required for the transport of virions through intestinal cells, although the presence of RT protein greatly enhances the efficiency of this process. Based upon these findings, at least two models can be proposed to describe virus uptake by intestinal cells. In the first model, putative receptors on the midgut apical plasmalemma could recognize a protruding or easily accessible domain of the viral capsid consisting of both CP and RT motifs. Deletion of the RT protein, as in BWYV-6.4, would not abolish virus recognition by the receptor, but this process would occur with lower efficiency. The second model postulates that luteovirus endocytosis in aphids may involve multiple receptors. This situation could occur at the inner surface of the gut, where virions must first be captured from the food bolus before being endocytosed. Involvement of multiple receptor binding events has already been described for entry into human cells by adenoviruses (Nemerow, 2000 ), which have been shown to associate first with an attachment receptor by means of a fibre protruding from the viral capsid. Internalization of the virus is triggered by interaction of the second receptor (integrin) with the base of the fibre. Applied to the BWYVaphid system, such a model would hypothesize that the first type of receptor, thought to be frequent along the gut epithelium, would recognize motifs of the RT protein. The second type of receptor, which would be less common and would recognize motifs of the CP, would intervene in the slower process of endocytosis. The uneven distribution of receptors and their different specificities could account for the differential efficiency of transcellular transport between wild-type virions and the RT protein-deficient mutant.
In summary, we have detected specific association of BWYV with the aphid midgut tissue during the virus acquisition process and presented evidence indicating that the RT protein of BWYV and presumably other members of the Luteoviridae is not absolutely required for recognition and acquisition into the aphid vector haemocoel. However, efficiency of acquisition was greatly reduced for the RT-minus mutant BWYV-6.4, suggesting an important role for the readthrough domain in the virus recognition and transcellular transport process. BWYV-6.4 was never detected at the ASG and was not transmitted to plants, indicating that it was either acquired at concentrations too low for such phenomena to be detected by the methods employed here or the mutant virus was inactivated in the haemocoel due to its inability to associate with protective GroEL homologues.
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Acknowledgments |
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References |
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Received 20 February 2001;
accepted 10 May 2001.