Plant Sciences Research Section, Imperial College London, Wye Campus, Ashford TN25 5AH, UK
Correspondence
Glen Powell
g.powell{at}imperial.ac.uk
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ABSTRACT |
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MAIN TEXT |
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Uptake of virions occurs when the maxillary stylet tips puncture the plasma membrane of an epidermal cell (Lopez-Abella et al., 1988; Powell, 1991
). These brief (315 s) intracellular punctures can be monitored using the DC electrical penetration graph (EPG) technique (Tjallingii, 1988
) and appear as distinct potential drops (pds) in the recorded signal (Tjallingii, 1985
). Three successive intracellular activities occur during each short pd, characterized as subphases II-1, II-2 and II-3, respectively (Powell et al., 1995
; Martin et al., 1997
; Fig. 1
a). The third intracellular activity (II-3) is associated with efficient uptake of non-persistently transmitted viruses and therefore represents active ingestion of cytosolic fluid by the aphid (Powell et al., 1995
; Martin et al., 1997
; Powell & Hardie, 2000
).
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While the egestion and salivation scenarios provide intriguing alternatives for the inoculation mechanism, direct experimental evidence for either case is lacking. None of the three intracellular subphases has been experimentally associated with either egestion or salivation. Observations of aphids penetrating Parafilm and ejecting ink particles have been extrapolated to plants in support of the ingestionegestion hypothesis (Harris & Bath, 1973; Harris, 1977
), but there is no direct evidence that aphids egest during plant penetration. The ingestionsalivation hypothesis is also unproven: it has been proposed that subphase II-1 represents a distinctive phase of watery salivation by aphids (Cherqui & Tjallingii, 2000
), but there is no definitive evidence that aphids inject saliva into cells during brief punctures. The aim of the present study was to exploit the unique properties of Pea enation mosaic virus (PEMV) to investigate the occurrence of intracellular salivation and its importance for the inoculation of non-persistently transmitted viruses.
PEMV represents an obligate symbiosis between two distinct viruses (de Zoeten & Skaf, 2001): an enamovirus (PEMV-1, a member of the family Luteoviridae) and an umbravirus (PEMV-2). PEMV-1 confers transmission by aphids in a circulative (non-propagative) manner and so, in common with luteoviruses, acquired PEMV accumulates in the accessory salivary glands and is inoculated via salivation (de Zoeten & Skaf, 2001
; Reavy & Mayo, 2002
; Gray & Gildow, 2003
). PEMV-2 plays no known role in determining interactions with aphid vectors, but influences the site of successful virus inoculation within plants. By conferring cell-to-cell movement, PEMV-2 enables escape from the phloem limitation that is typical of luteoviruses, so that PEMV can be inoculated during the superficial, brief epidermal probes that also characterize optimal inoculation of non-persistently transmitted viruses (Nault & Gyrisco, 1966
). This unique combination of features makes inoculation of PEMV an ideal marker for aphid salivation during stylet penetration of the epidermis. The occurrence of PEMV inoculation during intracellular (EPG-recorded) activities was therefore investigated in order to determine whether and when intracellular salivation occurs.
In order to maximize virus acquisition and subsequent experimental transmission, PEMV vectors (clone JF01/29 of the pea aphid, Acyrthosiphon pisum) were born and reared on virus-infected plants (tick bean, Vicia faba var. minor). Apterous aphids approaching the final moult to the adult stage (i.e. late IV instars) were collected from these plants and allowed access to healthy test plants (newly germinated tick bean seedlings at the hook stage growing in perlite/compost mix in individual 7·5 cm pots). Although PEMV is a circulatively transmitted (and therefore saliva-inoculated) virus, it was important to eliminate the possibility that additional inoculation occurred via carry-over of virus in or on the stylets. This was achieved by confining aphids (under an inverted glass tube) to a first test plant overnight (2026 h), allowing extended access to a virus-free food source. The following day, only aphids that had moulted to the adult stage (and therefore shed and replaced their stylets) were selected as an additional precaution against inoculation of stylet-retained virus. These aphids were then starved in plastic Petri dishes for 24 h before being used in experiments, in order to encourage the probing behaviour that characterizes efficient inoculation of non-persistently transmitted viruses (Powell, 1993) and PEMV (Nault & Gyrisco, 1966
). After the starvation period, each aphid was attached to a fine (20 µm diameter) gold wire for EPG recording and allowed access to seven consecutive EPG test plants for a single probe on each. EPG signals were recorded directly on to a PC hard disk (STYLET 3.7 software) and simultaneously displayed on a second PC running the Picoscope for Windows package (Pico Technology). The majority of probes terminated naturally within 20 s, but those that lasted up to 30 s were terminated artificially by lifting the gold wire. In addition, a subset of randomly selected probes (n=175) was terminated earlier than this, by lifting the wire during intracellular subphase II-1, II-2 or II-3. All test plants (eight plants per aphid: one overnight test plus seven EPG tests) were transferred to a nicotine-fumigated glasshouse and symptoms of PEMV infection were scored visually 1417 days after inoculation. A total of 80 aphids was used in the experiment, accounting for 640 test plants during the course of the study. Over the same period, a further 400 plants were not subjected to experimental aphid exposure but were grown in the same glasshouse environment; none of these test plants developed symptoms of infection. The efficiency of PEMV transmission to the first (overnight) test plant was very high (98·8 %), but 10 insects did not inoculate any of the EPG test plants in the subsequent recordings and were excluded from the statistical analysis. The remaining aphids (n=70) inoculated PEMV to 32·0 % of 490 EPG test plants overall and showed no significant decline in inoculation efficiency from the first (31·4 %) to the last (28·6 %) EPG test plant. Data for all seven EPG tests were therefore pooled for subsequent analysis (n=490).
The occurrence of virus inoculation was absolutely dependent on cell puncture by the maxillary stylets. All 157 occurrences of PEMV inoculation were associated with a clearly recorded pd (Table 1), confirming that the virus was delivered into cells during intracellular salivation. Analysis of those cell punctures that were interrupted artificially showed that only subphase II-1 was necessary for inoculation. Aphids allowed further intracellular activities, and interrupted during subphases II-2 or II-3, did not inoculate virus at a significantly higher efficiency than those allowed subphase II-1 only (Fig. 1b
;
2=2·21; d.f.=2; P>0·05). The increase in inoculation efficiency between cell punctures interrupted during II-1 and those interrupted during II-2 (Fig. 1b
), although not significant (
2=2·14; d.f.=1; P>0·05), may be related to the duration of II-1 activity; II-1 was inevitably cut short for insects that were interrupted during this subphase. A further analysis was therefore carried out (across all recorded pds) to assess whether the duration of each subphase was related to the occurrence of inoculation. Cell punctures effecting successful inoculation showed significantly longer duration of II-1 than punctures that did not inoculate (t=4·63; d.f.=355; P<0·001; Fig. 2
), a result that lends further support to the link between this first intracellular activity and virus release. By contrast, the durations of subphases II-2 and II-3 showed no relationship with virus inoculation (Fig. 2
).
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A great deal of speculation has been based on reported egestion by aphids (Harris & Bath, 1973) but, following the ingestionsalivation hypothesis (Martin et al., 1997
) and the present supportive findings, it now seems appropriate to question whether egestion occurs during epidermal penetration. While the first and third intracellular subphases appear to represent salivation and ingestion, respectively, the behaviour represented by the intermediate period (II-2) remains unclear. Harris & Harris (2001)
have speculated that II-2 represents egestion and have presented a scheme whereby this subphase is responsible for inoculation of non-persistently transmitted viruses, but there is no evidence that II-2 is linked with ejection of stylet contents. The data reported by Martin et al. (1997)
showed clearly that the activity associated with virus inoculation was subphase II-1, not II-2.
The common duct at the maxillary stylet tips represents less than 1 % of the total aphid stylet length (Forbes, 1969) and the vast majority of acquired virions are retained at more proximal sites within the food canal (Wang et al., 1996
). However, very few (probably less than 100) virus particles are involved in the non-persistent transmission process (Pirone & Thornbury, 1988
) and the presence of virions at more proximal locations does not provide evidence that they function in transmission (Pirone & Perry, 2002
). The common duct represents the likely functional retention site and, although many virions flow past this position during ingestion, there may be no credible mechanism by which they can be inoculated.
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ACKNOWLEDGEMENTS |
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Received 23 September 2004;
accepted 26 October 2004.
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