Institut National de la Recherche Agronomique, UMR Biologie des Organismes et des Populations appliquée à la protection des plantes, Laboratoire de Zoologie1 and Station de Pathologie Végétale2, BP 35327, 35653 Le Rheu Cédex, France
Author for correspondence: Danièle Giblot Ducray-Bourdin. Fax +33 2 23 48 51 80. e-mail giblot{at}rennes.inra.fr
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Abstract |
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Introduction |
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The first barrier is the gut membrane, which the virus appears to traverse by an endocytosisexocytosis mechanism, presumably involving specific recognition between virus particles and aphid components. For PLRV, Garret et al. (1993) observed that in Myzus persicae (Sulz.) the site of this passage is the midgut. In contrast, this same aphid species acquires Soybean dwarf virus (SbDV; family Luteoviridae) through the hindgut (Gildow et al., 1994
). Rhopalosiphum padi (L.) and Sitobion avenae (Fab.) also acquire Cereal yellow dwarf virus-RPV (CYDV-RPV; genus Polerovirus, family Luteoviridae) and Barley yellow dwarf virus-PAV (BYDV-PAV; genus Luteovirus, family Luteoviridae) respectively, through their hindgut (Gildow, 1993
). However, in spite of the high degree of tissue specificity within and between vector species, the gut membrane does not seem to be very selective. In most cases, luteo- and poleroviruses were shown to be able to cross the gut membrane of both efficient and poor vector species (reviewed by Gildow, 1999
).
Two more selective barriers have been distinguished for BYDV and CYDV, the ASG basal lamina and basal plasmalemma. The mechanisms allowing virus particles to penetrate the ASG basal lamina are unknown. However, BYDV-PAV or CYDV-RPV particles were shown to attach specifically to ASG basal lamina and different types of interaction were described, depending on the aphid species (Peiffer et al., 1997 ). At the ASG basal plasmalemma, a receptor-mediated endocytosisexocytosis process similar to that at the midgut is apparently involved. Therefore, once the basal lamina has been crossed, virus transmission is probably still dependent on specific interactions that determine virus passage through the basal plasmalemma (Gildow & Rochow, 1980
; Gildow & Gray, 1993
). Although few studies have been done with PLRV, virus particles have also been observed specifically attached to ASG membrane, suggesting that virusvector interactions occur at this site for PLRV as well (Gildow, 1982
).
Luteo- and polerovirus particles contain two structural proteins, the coat protein (CP) and the minor capsid readthrough protein (RTP) (Bahner et al., 1990 ). Most results suggest that the CP alone allows transport through the gut membrane (van den Heuvel et al., 1993
; Chay et al., 1996
; Gildow, 1999
), whereas the role of the RTP remains much less clear. RTP has been shown to be necessary for BYDV transport through ASG membrane (Chay et al., 1996
) but more recent results suggested that it was also involved in the passage of Beet western yellows virus (BWYV; genus Polerovirus, family Luteoviridae) through the gut membrane (Brault et al., 2000
). For BWYV, the ability of RTP to mediate transmission has been associated with the conserved N-terminal half of the protein (Bruyère et al., 1997
). In contrast, PLRV isolates that had lost aphid transmissibility were shown to harbour amino acid changes in the non-conserved C-terminal domain of RTP (Jolly & Mayo, 1994
). Moreover, PLRV-like particles devoid of RTP were able to complete their route in M. persicae, from the gut lumen to the accessory salivary gland canal (Gildow, 1999
).
Several aphid species have been shown to transmit PLRV with various efficiencies, the more efficient one being M. persicae (Kennedy et al., 1962 ). Among other parameters, transmission depends largely on aphid species, clone, morph and instar (Björling & Ossiannilsson, 1958
; Upreti & Nagaich, 1971
; Hinz, 1966
; Robert & Maury, 1970
; Robert, 1971
) and virus isolate (Tamada et al., 1984
; Jolly & Mayo, 1994
). However, Bourdin et al. (1998)
showed that, among clones of the M. persicae complex which were efficient at transmitting the PLRV-HAT (Highly Aphid Transmissible) isolate PLRV-CU87, most transmitted the PLRV-PAT (Poorly Aphid Transmissible) isolate PLRV-14.2 with a low efficiency (025%), although two clones could transmit it at up to 80% efficiency. Interestingly, most of the poor vector clones belonged to the Myzus antirrhinii taxon within the M. persicae complex, showing that aphid genotype variation can affect virus transmission (Terradot et al., 1999
). Overall, these results provide evidence that both the properties of the virus and the vector and their interactions are involved in regulating the transmission process.
In this paper, we report that the efficiency with which PLRV particles cross M. persicae gut membrane determines the success of transmission. Comparison of CP and RTP sequences between PLRV-HAT and -PAT isolates revealed several amino acid changes that are associated with inefficient passage of virus particles across this membrane.
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Methods |
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Nonviruliferous aphids were reared under controlled conditions (16 h light/8 h dark; 20 °C) on Chinese cabbage (Brassica campestris Linné var. pekinensis) to keep them free of PLRV (Chuquillanqui & Jones, 1980 ). Clone Mp3, used in this study, has been recently characterized as belonging to the Myzus antirrhinii taxon within the M. persicae complex (Terradot et al., 1999
). Although it is efficient at transmitting several PLRV isolates, it is a very poor vector of PLRV-14.2 (less than 10% transmission) (Bourdin et al., 1998
).
Virus purification.
Virus isolates were purified following the method described by Tamada & Harrison (1980) and modified as followed. About 300 g frozen infected P. floridana leaves was homogenized with 3 vols 0·1 M sodium citrate, pH 6, containing 0·5%
-mercaptoethanol and 5% Celluclast (Novo Nordisk). The homogenate was incubated at room temperature for 23 h, filtered through muslin and the pH adjusted to 7. The virus preparation was emulsified with 1/3 vol. chloroform and 1/3 vol. butanol and centrifuged for 15 min at 5000 r.p.m. (rotor JA14, Beckman). Polyethylene glycol (PEG) 6000 and NaCl were added to the aqueous phase to 8% (w/v) and 0·2 M, respectively. After overnight incubation at 4 °C, the suspension was centrifuged for 15 min at 10000 r.p.m. (rotor JA14, Beckman) and the pellets were resuspended in phosphate buffer (0·01 M, pH 7) containing 1% Triton X-100. Further purification was performed by ultracentrifugation through a 30% sucrose cushion followed by sucrose density-gradient centrifugation. The peak-containing fractions of the gradients were pooled and the virus was pelleted by ultracentrifugation. Virus concentration was calculated by measuring the optical density of the virus preparation at 260 nm and using
260=8·6 (Takanami & Kubo, 1979
). Yields were 300400 µg/kg leaf.
Virus transmission experiments.
Three virus acquisition procedures were designed. Virus-free young apterous adults were employed in all cases.
Plant-to-plant transmission experiments.
Batches of aphids were allowed to feed on PLRV-14.2- or -CU87-infected cuttings of P. floridana for a 3 day acquisition access period (AAP) under controlled conditions (16 h light/8 h dark, 20 °C).
Membrane feeding.
Aphids were fed for a 24 h AAP, through a stretched Parafilm membrane, on purified virus suspensions of PLRV-14.2 or -CU87 containing 100 µg/ml of virus.
Microinjection of purified virus.
Purified PLRV-14.2 or -CU87 (1020 nl containing 100 µg/ml of virus) was microinjected into the haemocoel of immobilized aphids using a pantograph micromanipulator (Micro Instruments Ltd) and an Inject-Matic apparatus regulated by an electronic programme (A. Gabay, Geneva, Switzerland).
In each procedure, following the AAP, three aphids were then trans ferred to each of 20 healthy P. floridana seedlings for a 3 day inoculation access period (IAP). Three replicates were performed and in the first test, purified virus from the same preparation was used in both the membrane feeding and microinjection procedures. At the end of the IAP, aphids were killed with an insecticide spray (Pirimicarb).
Virus infection was assessed through symptom expression 23 weeks after inoculation and confirmed using DAS-ELISA (Clark & Adams, 1977 ) 23 weeks later. Test plants were considered infected with PLRV when the DAS-ELISA absorbance values were greater than twice the average values of healthy P. floridana.
Back inoculations were performed from plants that had been inoculated by microinjected aphids. Two plants infected with each isolate and originating from two different replicates were chosen. Twenty test plants were inoculated with three aphids following a 3 day AAP on each source plant. Transmission efficiency was assessed as described above.
For each procedure, mean transmission rates obtained with PLRV-14.2 and -CU87 were transformed using angular transformation and analysed by one- or two-way ANOVA (analysis of variance), using the GLM (General Linear Model) procedure of the SAS (Statistical Analysis Software) package before being compared using Duncans multiple range test (SAS Institute Inc., 1995 ).
Sequencing of CP and RTP genes.
The ORFs corresponding to the CP and the RTP were sequenced for both PLRV-14.2 and -CU87. All the primers were designed based on the sequence of the Canadian isolate, PLRVC (Keese et al., 1990 ). Total RNAs from infected plant tissues were extracted using the RNeasy plant Mini Kit (Qiagen), following the manufacturers instructions. For cDNA synthesis, 10 µl of total RNAs was used. Reverse transcription was primed with an oligonucleotide complementary to nucleotides 58635882 (PLRV1), and a PCR product of about 2·5 kb was synthesized with oligonucleotides PLRV1 and PLRV2 (complementary to nucleotides 33823401). PCR products were purified (Concert rapid PCR purification system; Gibco-BRL) and approximately 100 ng of DNA was used as matrix for sequencing reactions (ABI Prism Big Dye dRhodamine terminator cycle sequencing ready reaction kit; Perkin-Elmer). For each isolate, purified PCR products obtained in at least three independent PCR reactions were sequenced as overlapping fragments, using 10 internal forward primers, with an ABI 310 automated DNA sequencer (Perkin-Elmer). Sequences were assembled and analysed using the program BioEdit (Hall, 1999
), available at http://www.mbio.ncsu.edu/RNaseP/info/programs/BIOEDIT/bioedit.html.
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Results |
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When back inoculations were performed from infected plants that had been inoculated by microinjected aphids, PLRV-14.2 was still poorly transmitted whereas PLRV-CU87 was transmitted up to 100% (Table 2). These observations confirmed that the virus material used in microinjection had not become contaminated with a HAT isolate.
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Discussion |
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Plant-to-plant transmission experiments first showed that, although Mp3 aphids were very poor vectors of PLRV-14.2 and PLRV-V, they efficiently transmitted PLRV-CU87 and other PLRV isolates (Table 1; Bourdin et al., 1998
and unpublished data). This finding establishes that Mp3 aphids are able to feed properly on PLRV-infected P. floridana and that their intrinsic behavioural properties cannot account for the observed poor transmissibility. Nor is low transmissibility of PLRV-14.2 linked to lower virus availability from infected source plants since, when PLRV-14.2 was provided at the same concentration as PLRV-CU87 using the membrane-feeding protocol, it was still poorly transmitted (Table 1
). Therefore, although we cannot rule out the possibility that the distribution of PLRV-14.2 particles in phloem tissues of infected P. floridana is uneven (van den Heuvel et al., 1995
), such a distribution, if it exists, is not responsible for the differences in acquisition by aphids in our experiments.
After microinjection, Mp3 aphids transmitted PLRV-14.2 with 50 to 73% efficiency, showing that the ASG membrane was easily crossed by this isolate and arguing that virus particles passed through the gut membrane with a very low efficiency when naturally ingested (Table 1). This is in contrast with most previous results which indicated that the ASG basal lamina and/or basal plasmalemma are responsible for vector specificity of several viruses in the Luteoviridae family (reviewed by Gildow, 1999
). For example, when Rochow (1969)
microinjected purified BYDV isolates to non-vector aphid species, virus particles were not transmitted, suggesting that the gut membrane played no role in the observed specificity. Rochow et al. (1975)
later confirmed that the ASG regulated BYDV transmission specificity. In only one case has the gut membrane previously been shown to be responsible for transmission specificity: CYDV-RPV particles could not reach the haemolymph of the non-vector Metopolophium dirhodum (Wlk.) and were never observed attached to the gut apical plasmalemma of this aphid species (Gildow, 1993
). This led the author to conclude that M. dirhodum lacked the receptors to recognize CYDV-RPV (Gildow, 1999
). However, our results suggest that, for PLRV at least, such specificity is not controlled in an all-or-nothing fashion. Although Mp3 aphids were shown to be non-vectors of PLRV-14.2 in most cases, they did transmit PLRV-14.2 very poorly in some experiments. Moreover, they could transmit other isolates very efficiently. This suggests that these aphids possess at their gut membrane the receptor(s) needed for efficient transcytosis and that PLRV-14.2 particle transport through the midgut is more probably impeded by low affinity between virus particles and their receptor(s) in the aphids rather than by an absence of the appropriate receptor(s).
Sequence comparisons have revealed a number of alterations in the PLRV-14.2 CP and RTP with regard to the other isolates (Fig. 1). One or more of these changes presumably account for the poor transmissibility of PLRV-14.2. Two of the changes found in the RTP seem of particular interest. The first change (QN to RS) at amino acids 271272 is very close to the strictly conserved ED sequence found at position 267268. When the ED motif was replaced by alanine residues in a BWYV infectious cDNA clone, the resulting progeny was unable to cross M. persicae gut membrane (Brault et al., 2000
). The second change was the substitution of amino acids KA at position 554555 by amino acids ET and was located in the Myzus homology domain i.e. a sequence that is highly conserved among poleroviruses transmitted by M. persicae (Mayo & Ziegler-Graff, 1996
). More recent studies, however, have cast doubt on the importance of the Myzus homology domain on transmission of BWYV (Bruyère et al., 1997
).
Evidently, more information is needed to determine the effect of the substitutions found in PLRV-14.2 CP and RTP on transmission efficiency. However, the similar transmission rates of PLRV-14.2 and -CU87 after microinjection suggest that, although the observed amino acid changes can affect the passage of PLRV-14.2 through the gut membrane, they had little or no effect on transport through ASG basal lamina and basal plasmalemma. Moreover, it is of interest, that none of the changes previously reported to affect PLRV-V transmission (Jolly & Mayo, 1994 ) were found in PLRV-14.2. This suggests that different sequence modifications can have similar effects on transmission and supports the hypothesis of structural redundancy within the RTP (Brault et al., 2000
).
The respective roles of virus structural proteins in virus recognition within the aphid are at present unsettled because of the conflicting results obtained by different authors (Brault et al., 1995 ; Chay et al., 1996
; Bruyère et al., 1997
; Gildow, 1999
). The PLRV-14.2/Mp3 model will undoubtedly help to clarify these questions.
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Acknowledgments |
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Footnotes |
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References |
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Received 2 June 2000;
accepted 5 September 2000.