3305
The Journal of Experimental Biology 212, 3305-3312
Published by The Company of Biologists 2009
doi:10.1242/jeb.028514
Aphid watery saliva counteracts sieve-tube occlusion: a universal phenomenon?
Torsten Will1, Sarah R. Kornemann1, Alexandra C. U. Furch1, W. Fred Tjallingii2 and Aart J. E. van Bel1,*
1
Plant Cell Biology Research Group, Department of General Botany, Justus-Liebig-University, Senckenbergstrasse 17-21, D-35390
Giessen, Germany and 2Department of Entomology, Agricultural University Wageningen, Binnenhaven 7, NL-6700 EH,
Wageningen, The Netherlands
*Author for correspondence ([email protected])
Accepted 14 July 2009
SUMMARY
Ca2+-binding proteins in the watery saliva of Megoura viciae counteract Ca2+-dependent occlusion of sieve plates in Vicia faba and
so prevent the shut-down of food supply in response to stylet penetration. The question arises whether this interaction between
aphid saliva and sieve-element proteins is a universal phenomenon as inferred by the coincidence between sieve-tube occlusion
and salivation. For this purpose, leaf tips were burnt in a number of plant species from four different families to induce remote
sieve-plate occlusion. Resultant sieve-plate occlusion in these plant species was counteracted by an abrupt switch of aphid
behaviour. Each of the seven aphid species tested interrupted its feeding behaviour and started secreting watery saliva. The
protein composition of watery saliva appeared strikingly different between aphid species with less than 50% overlap. Secretion of
watery saliva seems to be a universal means to suppress sieve-plate occlusion, although the protein composition of watery saliva
seems to diverge between species.
Key words: aphids, plant defence, sieve-tube occlusion, watery saliva.
INTRODUCTION
Sieve tubes – arrays of longitudinally arranged sieve elements (SEs)
– are the transport conduits for an assortment of metabolites such
as carbohydrates, amino acids, phytohormones and vitamins
(reviewed by van Bel, 2003). SEs are elongated cells that lack a
nucleus and vacuole and contain only an intact plasma membrane,
phloem plastids, a SE endoplasmic reticulum (Sjölund and Shih,
1983), a few mitochondria, and an extensive set of phloem-specific
proteins (reviewed by Hayashi et al., 2000). During SE development,
the terminal walls transform into sieve plates, perforated by sieve
pores (Evert, 1990) to connect adjacent SEs end to end.
Following production in the mesophyll and loading into sieve
tubes, plant nutrients are translocated through the transport phloem
towards the sinks, e.g. growing leaves, roots, stem or storage organs
(van Bel, 1996). Mass flow through sieve tubes is driven by a turgorpressure gradient between sources (high pressure) and sinks (low
pressure) (Münch, 1930; Gould et al., 2005).
Aphids (Homoptera: Aphididae: Aphidinae) puncture sieve tubes
with their stylets and passively ingest the nutrient-rich sieve-tube
content owing to the high endogenous pressure. During stylet
penetration, aphids are confronted with sieve-tube occlusion
mechanisms (Sjölund, 1997; Knoblauch and van Bel, 1998; Ehlers
et al., 2000). When damage occurs, occlusion of sieve pores
prevents the loss of sieve-tube sap (Evert, 1982; Schulz, 1998) and
the invasion of pathogens through the wound site (van Bel, 2003).
Sieve tubes are occluded by proteins and callose in response to
injury. The nature of protein-occlusion mechanisms depends upon
the plant family. Fabaceae possess parietal proteins, protein networks
and reversibly dispersive protein bodies, the so-called forisomes
(Knoblauch and van Bel, 1998; Ehlers et al., 2000; Knoblauch et
al., 2001). Cucurbitaceae possess parietal proteins and solubilized
phloem proteins (PP1) that form protein filaments after oxidation
(Leineweber et al., 2000). In electron-microscopic images of
Brassicaceae phloem, SEs show merely protein networks that span
the SE lumen and are attached to the cell periphery (Sjölund, 1997).
Sieve tubes of grasses appear virtually empty but may have an
occlusion mechanism based on solidification of soluble proteins
(Will and van Bel, 2006). To complement protein plugging, sieve
pores are occluded by callose deposition (McNairn and Currier,
1968), a universal mechanism in all families studied so far
[summarized by Behnke and Sjölund (Behnke and Sjölund, 1990)].
Induction of callose synthesis (Kauss et al., 1983), coagulation
of soluble proteins (Will and van Bel, 2006) and dispersion of
forisomes (Knoblauch et al., 2001) appear to be Ca2+ dependent.
Occlusion is triggered by Ca2+ influx induced by damage
(Knoblauch and van Bel, 1998). Aphids seem to have developed
strategies to prevent sieve-tube occlusion during stylet penetration,
one of which is the secretion of Ca2+-binding watery saliva
(Tjallingii, 2006; Will and van Bel, 2006; Will et al., 2007).
As shown by the electrical penetration graph (EPG) technique
[for an overview, see Walker (Walker, 2000)], watery saliva is
massively secreted into the SE lumen at the beginning of sieve-tube
penetration (Prado and Tjallingii, 1994). Some watery-saliva
proteins of the aphid species Megoura viciae (Buckton) have Ca2+binding properties (Will et al., 2007). This was demonstrated by
confronting forisomes with salivary proteins in vitro. Forisome
dispersal (which occludes sieve plates) can be triggered in vitro by
supply of Ca2+, and dispersal can subsequently be reversed by adding
Ca2+ chelators (Knoblauch et al., 2001) or concentrated aphid saliva
(Will et al., 2007).
While feeding on SEs, aphids switch from phloem sap ingestion
(EPG waveform E2) to secretion of watery saliva (EPG waveform
E1) upon leaf-tip burning (Will et al., 2007). Leaf-tip burning was
observed to induce an electropotential wave, accompanied by Ca2+
influx into the sieve-tube lumen resulting in sieve-tube occlusion
(Fromm and Bauer, 1994; Furch et al., 2007). As demonstrated by
Gould and colleagues (Gould et al., 2004), sieve-tube occlusion is
accompanied by a decrease of sieve-tube pressure (Gould et al.,
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3306 T. Will and others
Table 1. Aphid species reared for experimental use in electrical penetration graph (EPG) and protein studies
Aphid species
Host plant
Acyrtosiphon pisum (Harris) (green variant)
Acyrtosiphon pisum (Harris) (red variant)
Megoura viciae (Buckton)
Aphis fabae (Scopoli)
Myzus persicae (Sulzer)
Macrosiphum euphorbiae (Thomas)
Rhopalosiphum padi (Linnaeus)
Schizaphis graminum (Rondani)
Vicia faba cv. Witkiem major
Vicia faba cv. Witkiem major
Vicia faba cv. Witkiem major
Vicia faba cv. Witkiem major
Brassica napus cv. unspecified
Cucurbita maxima cv. Gele Reuzen
Hordeum vulgare cv. unspecified
Hordeum vulgare cv. unspecified
Host specificity
EPG data
(Table 1; Figs 2, 3)
Protein composition
of watery saliva (Fig. 5)
O
O
M
P
P
P
O
O
+
–
+
+
+
+
+
+
+
+
+
+
–
+
–
+
M, monophagous; O, oligophagous; P, polyphagous.
2004). This pressure decrease inside the sieve-tube lumen most likely
triggers the switch in aphid behaviour (Will et al., 2008).
We investigated whether watery saliva acts as a universal tool in
aphid–plant interactions through suppression of sieve-tube occlusion.
We used the behavioural switch from phloem-sap ingestion to
secretion of watery saliva (Will et al., 2007) as an indicator for
suppression of sieve-tube occlusion by watery saliva in several
aphid–plant combinations. Watery saliva from different aphid species
was collected to compare their protein composition and investigate
potential overlap with regard to Ca2+-binding proteins.
MATERIALS AND METHODS
Aphid and plant breeding
Aphid species were reared on their host plants (Table 1) in Perspex
cages with large gauze-covered windows in a controlled room
environment with a 17 h:7 h light (L):dark (D) regime at a
temperature of 25°C. The same aphid/host plant combinations for
rearing were also used for experiments.
Plants (Table 1) were grown in a greenhouse at 20°C with natural
lighting plus additional lamp light (SONT Agro 400 W; Phillips,
Eindhoven, The Netherlands) with a 14 h:10 h L:D period and were
used in a vegetative phase.
Sieve-tube occlusion visualised by confocal laser scanning
microscopy
In order to document sieve-tube occlusion in response to leaf-tip
burning, Brassica napus and Hordeum vulgare plants were prepared
as described previously (Knoblauch and van Bel, 1998). A few
cortical cell layers were locally removed down to the phloem from
the lower side of the main vein of a mature leaf that remained
attached to the plant during the entire experiment. The layers were
removed by manual paradermal slicing with a new razor blade, while
avoiding damage to the phloem. Immediately after cutting a shallow
window (~10 mm long, 2 mm wide and 6 cm away from the leaf
tip), we bathed the free-lying phloem tissue in a buffer solution
(Hafke et al., 2005) and the plant was allowed to recover for
30–60 min. The leaf was fixed in an upside-down position with
double-sided adhesive tape onto a small Perspex table. The state of
the tissue was checked under the microscope. If the phloem
appeared to be undamaged (indicated by thin sieve plates), a loading
area (5 mm⫻5 mm) was made by gently rubbing the lower side of
the leaf vein, located 2–3 cm upstream from the observation window,
with fine sandpaper (Fig. 1A).
A stock solution of the phloem-mobile fluorochrome 5(6)
carboxyfluorescein diacetate (CFDA)-mixed isomers (Invitrogen,
Karlsruhe, Germany) was prepared by solubilisation of 1mg CFDA
in 1ml DMSO. A working solution of 1μl stock solution in 2ml buffer
solution (Hafke et al., 2005) was applied at the loading site. CFDA
permeates the plasma membrane in the non-fluorescent acetate form
and is cleaved by cytosolic enzymes producing membraneimpermeant fluorescent carboxyfluorescein (CF) (handbook from
Molecular Probes, Eugene, OR, USA). CF trapped inside SEs is
transported by mass flow in the sieve tubes as observed by confocal
laser scanning microscopy (CLSM; Leica TCS 4D; Leica
Microsystems, Heidelberg, Germany) (Knoblauch and van Bel,
1998). After CF had reached the observation window, the leaf tip
was burnt for 3s (Fig.1A,B; maximum area 1cm2) to induce blockage
of SEs. To visualize stoppage of mass flow in SEs, CF was
photobleached at the observation window for approximately 3min.
If phloem transport was blocked, the photobleached SEs would remain
non-fluorescent because of the lack of fluorochrome supply (Fig.1C).
After burning and subsequent photobleaching, a time course of
the events in the SEs was recorded. The experiment was repeated
four times for each plant species with new individuals. Digital image
processing was done with Corel PHOTO-PAINT®10 to optimize
brightness, contrast and colouring.
Aphid behaviour monitored by EPGs
Aphid behaviour was monitored using the EPG technique modified
by Tjallingii (Tjallingii, 1988). A gold wire electrode (1 cm length
and 20 μm diameter) was attached to the dorsum of an adult apterous
aphid using electrically conductive silver glue (Electrolube,
Swadlincote, Derbyshire, UK). A vacuum device was used to
immobilize aphids during electrode attachment (van Helden and
Tjallingii, 2000). The aphid electrode was connected to a DC EPG
Giga-8 (Tjallingii, 1978; Tjallingii, 1988) and the EPG output was
recorded with PROBE 3.5 (hardware and software from EPGSystems, Wageningen, The Netherlands, www.epgsystems.eu). A
second electrode (plant electrode) was inserted into the soil of potted
plants. In this electrical circuit, the ‘living’ components, represented
by the aphid and plant, act as a variable resistor, and plant cell
membrane potentials present additional voltage sources (reviewed
by Walker, 2000). The whole setup was placed in a Faraday cage
to shield it from electromagnetic influence.
Randomly selected adult and apterous aphids were placed on their
particular host plant species (Table 1) on the midrib of the lower
side of a mature leaf 6±0.5 cm away from the leaf tip. When aphids
showed ingestion of phloem sap for longer than 30 min, recognized
by an E2 waveform in the EPG (Tjallingii, 1988), the leaf tip (of
maximum area 1 cm2) was carefully burned for 3 s to trigger a
phloem-mediated electrical long-distance signal (Furch et al., 2007).
In EPG recordings, the time point of burning was marked by
generating three –50 mV pulses by pressing the EPG amplifier
calibration button. EPG recordings of control insects (no leaf-tip
burning) were made under identical experimental conditions,
including the three –50mV pulses. Burning experiments and controls
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Aphids counteract plant defence
3307
Fig. 1. Transient stoppage of mass flow in
sieve tubes of Brassica napus induced by
leaf-tip burning. (A) Phloem transport of
carboxyfluorescein (CF) was observed by
confocal laser scanning microscopy
(CLSM) at a tissue window 6 cm from the
burning site. 5(6)Carboxyfluorescein
diacetate (CFDA) was applied onto a
loading area located 2–3 cm upstream
from the observation window. (B,E) The
membrane-impermeable CF was
transported by mass flow through sieve
tubes (marked with an asterisk). (C,F) CF
was photobleached at the observation
area immediately after leaf-tip burning,
resulting in a decrease of fluorescence.
(C,F–I) Upon burning, reduced
fluorescence remained stable for more
than 30 min, which indicates stoppage of
mass flow due to sieve-plate occlusion.
(D,J) An increase in fluorescence between
30 and 60 min implies recovery of CF
supply from the upstream loading area
due to release of sieve-tube blockage and
hence resumption of translocation. Time
points of observation are given in the
lower right corner of each image (leaf tip
was burnt at t=0). Scale bars in E–J,
10 μm.
were repeated 12 times in most cases (except for Rhopalosiphum
padi, burning N=13; Macrosiphum euphorbiae, control N=7) and
recording time was set to 1 h.
EPG waveforms were analyzed by waveform pattern and auto
power spectra (APS) (example waveforms and APS are presented
in Fig. 2) according to Prado and Tjallingii (Prado and Tjallingii,
1994), using the PROBE analysis module. APS present the
waveform frequency (Hz) vs the relative magnitude with a maximum
of 1, which provides a major characteristic of waveform identity.
Recorded EPGs were analyzed with regard to switches in
behaviour expressed as transitions from one waveform to another,
e.g. from E2 (ingestion of sieve-tube sap) to E1 (secretion of watery
saliva into sieve-tube lumen) as observed for M. viciae (Will et al.,
2007). EPGs from each aphid were scored as a ‘reaction’ if they
changed from E2 to E1 and as a ‘non-reaction’ if they remained in
E2 for 10 min after leaf-tip burning, or for controls, within 10 min
of the three –50 mV pulses.
Statistical analysis
Statistical analysis was done with Fisher’s exact test using SigmaStat
3.0 software (SPSS, Chicago, IL, USA). Each treatment was
compared against its control group. The level for significance was
set to P=0.05.
Aphid saliva collection and saliva concentration
Watery saliva secreted by the aphid species M. viciae, Acyrtosiphon
pisum (red and green variant), M. euphorbiae, Aphis fabae and
Schizaphis graminum was collected and concentrated according to
a previous method (Will et al., 2007). Saliva from 100 aphids in
1 μl of saliva/diet-concentrate was used as a benchmark for
concentrating the diet/saliva mixture of each aphid species. The final
sample was partitioned in aliquots of 10 μl and kept frozen at –80°C.
1-D SDS-PAGE
1-D SDS-PAGE of the saliva concentrate (sample of 1000 aphids
per lane) was carried out according to Laemmli (Laemmli, 1970)
using a 4% stacking gel and a 10% separation gel in a MiniProtean
3 Electrophoresis System (Bio-Rad Laboratories, Hercules, CA,
USA). Precision Plus Protein All Blue standards (Bio-Rad) was used
as a protein size marker. Fourfold concentrated reducing sample
buffer (Roti-Load 1; Carl-Roth, Karlsruhe, Germany) was added to
each saliva sample in the proportion 1:3. Protein gels were silver
stained (Switzer and Merril, 1979; Schoenle et al., 1984). Similarities
of protein band patterns were calculated using Quantity One® 1-D
Analysis Software (Bio-Rad) including all detected bands and setting
the band match tolerance to 1%. Protein band intensity was not
weighted because of the varying amounts of protein in the respective
samples.
RESULTS
Time course of events in sieve tubes after leaf-tip burning
CFDA was applied to the loading site on B. napus leaves and
transported downstream in the CF form through the sieve tubes.
Following phloem transport of CF in B. napus leaves, the leaf tip
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3308 T. Will and others
Fig. 2. Electrical penetration graph (EPG) recording for an ingesting aphid (Macrosiphum euphorbiae) in response to heat shock-triggered sieve-tube
occlusion in Cucurbita maxima plants. The aphid was localized 4.5 cm away from the burning site. Burning (red arrow) took place 320 s after start of the
recording. (A) A 1 h overview showing behavioural changes of the ingesting aphid in response to leaf-tip burning. Horizontal arrows mark the start and end of
a specific behaviour. (B–D) Details (10 s each) for three different time intervals, marked by vertical dashed arrows in A. Graphs on the left are waveform
recordings, graphs on the right are auto power spectra (APS) that present the waveform frequency (Hz) vs the relative magnitude with a maximum of 1,
which provide a major characteristic of waveform identity. (B) 140–130 s prior to heat shock, the aphid shows an E2 profile which indicates ingestion. (C) 10 s
after heat shock the aphid changes its behaviour from ingestion (E2) to a short pure E1 period that is followed by a period of mixed behaviour (E2/E1;
ingestion and salivation), which lasts approximately 3 min (visible in A). Then, 148 s after heat shock, the aphid begins to perform steady secretion of watery
saliva (E1) into the sieve element (SE) lumen (A). From then on, the aphid again displays mixed behaviour (E2/E2). (D) The original behaviour (E2) is
resumed approximately 25 min after the burning-induced behaviour switch.
was burnt (Fig. 1A,B) in order to induce sieve-plate occlusion.
Subsequently, CF was photobleached at the observation window
(Fig. 1C). The rationale for these treatments is that fluorescence
will not recover at the site of observation until the sieve plates are
unblocked (Fig. 1D). Fluorescence inside the observed SEs
(marked with an asterisk in Fig. 1) was reduced by photobleaching
(Fig. 1E,F). A continued bleached appearance (Fig. 1F–I)
demonstrates the absence of fluorochrome supply and is evidence
of stoppage of mass flow over a period of 30 min (Fig. 1C).
Between 30 and 60 min after burning (Fig. 1J) fluorescence
recovered, indicating that mass flow had resumed (Fig. 1D).
Similar results were obtained for all replicates (N=4) and for both
plant species (data for H. vulgare not shown).
EPG and leaf-tip burning
As with V. faba (Will et al., 2007), leaf-tip burning was adopted
to observe whether food deprivation caused by sieve-tube
occlusion triggers behavioural responses of other aphid species.
Each aphid species (Table 1) switched its behaviour from ingestion
(E2: Fig. 2B; Fig. 3) to secretion of watery saliva (E1: Fig. 2C;
Fig. 3), as shown in detail for the aphid species M. euphorbiae
(Fig. 2A–D) and in 1 h overviews for other tested aphid species
(Fig. 3). The behavioural switch was observed for all tested
aphid–plant combinations (Table 2) shortly after leaf-tip burning
(Figs 2 and 3) (M. persicae: 6 s; M. euphorbiae: 10 s; A. fabae:
13 s; S. graminum: 17 s; R. padi: 18 s; A. pisum: 23 s). The response
is significant with P-values that range from <0.001 (M. viciae,
A. fabae, M. euphorbiae, R. padi and S. graminum) and 0.012
(M. persicae) to 0.036 (A. pisum, green variant) (Table 2).
Macrosiphum euphorbiae resumes ingestion, preceded by mixed
E1/E2 behaviour, approximately 25 min after leaf-tip burning
(Fig. 2A,D).
Apart from the typical E2–E1 switch, individuals showed different
behaviour profiles within a species. An example of analysis of the
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Aphids counteract plant defence
3309
Fig. 3. EPG for different species of
ingesting aphids on their host
plants in response to heat-shock
triggered sieve-tube occlusion.
Overviews of 1 h duration are
presented for different aphid
species–host plant combinations.
Horizontal arrows indicate the
start and end of a particular
behaviour. Response to leaf-tip
burning (red arrow) occurs within
only a few seconds [in these
measurements: M. persicae: 6 s;
A. fabae: 13 s; S. graminum: 17 s;
Rhopalosiphum padi: 18 s; A.
pisum (green variant): 23 s]. All
aphid species show a change
from ingestion (E2) to secretion of
watery saliva (E1). Subsequent to
the immediate response, which is
universal, further behaviour is
quite diverse. A mixed
ingestion/salivation behaviour
(mixed E1/E2) is observed for R.
padi and S. graminum. Mixed
behaviour is followed by ingestion
(E2) for R. padi. SE penetration
can also be interrupted, as
demonstrated by pathway
activities (path), observed for A.
pisum, A. fabae, M. persicae and
S. graminum. The short
occurrence of F in the EPG of S.
graminum reflects penetration
problems inside the apoplast.
behaviour of 12 M. viciae individuals in reaction to SE occlusion
demonstrates the variability of the responses (Fig. 4). Ten of 12
aphids reacted to leaf-tip burning by secretion of watery saliva
followed by a mixed E1/E2 behaviour. The return from mixed E1/E2
behaviour to ingestion occurred most frequently (5/10; Fig. 4).
Another frequent switch (4/10) was that from mixed behaviour back
to secretion of watery saliva (E1), followed by a broad variety of
behavioural profiles (Fig. 4). Other aphid species showed a similar
diversity of behaviour.
Comparison of watery saliva from six aphid species by 1-D
SDS-PAGE
Protein band patterns of the different aphid species/biotypes (Fig. 5)
show only partial overlap (Fig. 5, red bands) when matched to M.
viciae as the reference species (Fig. 5, green bands). Even if aphids
are reared and fed on the same host plant [M. viciae, A. pisum (both
variants) and A. fabae on V. faba], the similarity is low. A
calculation for similarity shows values of 19.2–43.1% [43.1% S.
graminum; 34.1% A. pisum (green variant); 32.9% A. pisum (red
THE JOURNAL OF EXPERIMENTAL BIOLOGY
3310 T. Will and others
Fig. 4. Behaviour succession tree – individual behaviour after
distant leaf-tip burning exemplified by M. viciae. Sequence of
behaviour during a 1 h recording period of 12 aphids following
leaf-tip burning (time course from top to bottom). Green numbers
in brackets represent the number of individuals that exhibit a
particular behaviour. The thickness of the arrows corresponds
with the behavioural frequency. Most frequently, E1 (secretion of
watery saliva) is followed by a mixed E1/E2 behaviour and E2
(ingestion). Another frequently noted behaviour succession is
mixed E1/E2 behaviour, E1, mixed E1/E2 behaviour again and
then E2. While seven aphids stay inside the penetrated SEs and
return to ingestion before the end of the 1 h observation period,
only 3 of 10 aphids stop SE penetration, indicated by pathway
activities (path), of which two return to ingestion. Four groups can
be distinguished: aphids that (i) show no reaction, (ii) penetrate a
new SE, (iii) stay inside the original SE or (iv) stop feeding during
the 1 h recording period.
variant); 19.2% A. fabae; 30.1% M. euphorbiae] when compared
with M. viciae. The protein pattern varies not only between species
but also between variants of A. pisum (green and red). Watery saliva
proteins of most species are distributed over the whole protein
marker range (Fig. 5).
Protein concentration varies strongly between species (Fig.5),
although each sample was collected from 1000 aphids. Roughly, the
amount of secreted saliva seems to be positively correlated with the
size of the aphid species. The total protein amount in watery-saliva
samples can affect the number of detectable protein bands (Will et
al., 2007). Thus, a comparison of samples with differing protein
concentrations is not conclusive for determining differences in protein
bands (e.g. Fig.5, red and green biotype of A. pisum). Nevertheless,
samples with comparable protein concentrations (e.g. Fig.5, M.
viciae, A. fabae and M. euphorbiae) show a low similarity as well,
which suggests that dissimilarity is not due to a difference in the
amount of protein at least in the comparison among the samples of
M. viciae, S. graminum, A. fabae and M. euphorbiae in Fig.5.
DISCUSSION
Sieve-tube occlusion mechanisms are presumably involved in plant
defence against phloem-feeding insects by blockage of nutrient
supply (Caillaud and Niemeyer, 1996). Aphids appear to be able to
counteract sieve-tube occlusion by secretion of watery saliva
(Tjallingii, 2006; Will and van Bel, 2006; Will et al., 2007). This
notion is based on the following observations: (i) aphids start
secreting watery saliva (E1) at the beginning of each SE penetration
(Prado and Tjallingii, 1994), (ii) M. viciae switches to secretion of
watery saliva in response to sieve-tube occlusion triggered by distant
leaf-tip burning (Will et al., 2007), (iii) watery saliva concentrate
of M. viciae inhibits Ca2+-induced dispersion of forisomes (legume
proteins involved in sieve-plate plugging) (Will et al., 2007), and
(iv) several watery saliva proteins of M. viciae are able to bind Ca2+
in biochemical assays (Will et al., 2007). To answer the question
of whether suppression of sieve-tube occlusion by aphid watery
saliva is a general phenomenon, the leaf-burning test (Will et al.,
2007) was extended to a variety of aphid–host plant combinations
(Table 1). Aphid behavioural responses were monitored with EPG
recordings (Figs 2 and 3; Table 2) and protein composition of watery
saliva was compared among different aphid species/biotypes (Fig.5).
Callose-mediated sieve-tube occlusion induced by remote leaftip burning has been visualized for broad bean, tomato (Furch et
al., 2007) and cucurbits (A.C.U.F., unpublished). Prior to callose
deposition, sieve tubes are plugged by proteins such as giant protein
Table 2. Aphid behaviour change induced by leaf-tip burning experiments
Plant
Aphid species
Vicia faba
Megoura viciae
Acyrtosiphon pisum (green variant)
Aphis fabae
Myzus persicae
Macrosiphum euphorbiae
Rhopalosiphum padi
Schizaphis graminum
Brassica napus
Cucurbita maxima
Hordeum vulgare
Leaf-tip burning
(reaction/non-reaction)
Control
(reaction/non-reaction)
Fisher’s exact test, P-value
(significance level P=0.05)
10/2
10/2
10/0
9/3
12/0
13/0
12/0
0/12
4/8
3/9
2/10
0/7
0/12
1/11
<0.001
0.036
<0.001
0.012
<0.001
<0.001
<0.001
Behaviour of different aphid species was monitored with the electrical penetration graph technique (i) after sieve-tube occlusion in response to leaf-tip burning
and (ii) in control groups on different plants. Data from each aphid were scored as ‘reaction’ if there was a change from E2 to E1 and scored as ‘nonreaction’ if E2 behaviour remained for 10 min after leaf-tip burning. ‘Reaction’ of aphids from the control group describes a change of behaviour by chance
during the 10 min observation period. Statistical analysis with Fisher’s exact test shows highly significant differences between burning experiments and
controls which are independent from aphid species and host plant.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
Aphids counteract plant defence
Fig. 5. Comparison of watery saliva from six aphid species by 1-D SDSPAGE. Lane 1 contains protein standard (molecular masses in kDa given
on the left side of the gel). Lanes 2–7 [Megoura viciae on V. faba (N=10);
S. graminum on H. vulgare (N=1); A. pisum (green variant) on V. faba
(N=3); A. pisum (red variant) on V. faba (N=1); A. fabae on V. faba (N=1);
M. euphorbiae on C. maxima (N=2)] contain watery saliva concentrate
obtained from 1000 aphids each, separated on a 10% gel. Lanes 3–7 were
matched with a tolerance of 1% to lane 2 (M. viciae, marked green) by
Quantity One® software (Bio-Rad). Matched protein bands were marked
red and numbered by protein band numbers of M. viciae, while nonmatching protein bands were labelled yellow.
bodies (forisomes) in V. faba (Knoblauch et al., 2001) or probably
structural phloem proteins in cucurbits (Leineweber et al., 2000).
Sieve-plate plugging by forisomes was reversed by the time
(15–20 min after burning) that callose deposition reached its
maximum level (Furch et al., 2007). Thus, sieve-plate occlusion
seems to be a two-step event in which protein plugging precedes
callose deposition (Will and van Bel, 2006).
Both Rosopsida and Liliopsida employ callose deposition on sieve
plates for occlusion (Eleftheriou, 1990; Evert, 1990). In addition,
Rosopsida also employs structural proteins to occlude SEs (Evert,
1990), whilst we (Will and van Bel, 2006) speculated about the
existence of soluble proteins in phloem sap of Liliopsida which
coagulate in response to wounding. All in all, the rapidity of the
observed sieve-tube occlusion in B. napus (Rosopsida) and H.
vulgare (Liliopsida) (Fig. 1) and the speed of aphid reaction to leaftip burning (Figs 2 and 3) suggest sieve-plate plugging by proteins
prior to callose deposition, because the latter is a slower response
that requires several minutes for synthesis (Furch et al., 2007).
Activation of occlusion seems to depend on increasing Ca2+
concentration in SEs (Kauss et al., 1983; Knoblauch and van Bel,
1998; Knoblauch et al., 2001). Induction of sieve-plate occlusion
by electropotential waves (Fromm and Bauer, 1994; Furch et al.,
2007) and turgor shocks (Knoblauch et al., 2001; Hafke et al., 2007)
triggers a release of Ca2+ into the SE lumen which then induces
occlusion. Furch and colleagues (Furch et al., 2007) demonstrated
3311
that sieve-tube occlusion is reversible probably through active
removal of Ca2+ ions.
The crucial role of Ca2+ in sieve-tube occlusion makes it an
outstanding target for insects and pathogens in order to interfere
with occlusion mechanisms. Therefore, the switch from ingestion
to secretion of watery saliva, provoked by leaf-tip burning (Figs 2
and 3; Table 2), has been interpreted as an attempt to suppress sieveplate occlusion (Will et al., 2007). In addition to the Ca2+-binding
proteins that were described for M. viciae, one was recently
identified in the saliva of A. pisum (Carolan et al., 2009). Aphids
may use proteins in watery saliva as a tool for Ca2+ binding (Will
and van Bel, 2006; Will et al., 2007). Thus, secretion of watery
saliva into the SE lumen can be regarded as a method to counteract
sieve-plate occlusion, although it does not seem to succeed in all
cases (Figs 3 and 4).
This study indicates that the involvement of watery salivation in
the suppression of plant defence is independent of aphid species
[N=7; monophagous (1), oligophagous (3) and polyphagous (3)],
host plant species (N=4) and host family (Fabaceae, Brassicaceae,
Cucurbitaceae and Poaceae) (Figs 2 and 3; Table 2).
Detailed behavioural analysis of the aphid species M. viciae
(Fig. 4) shows that 10 of 12 aphids reacted to leaf-tip burning and
that seven of these returned to ingestion without interruption of sievetube penetration. The mixed E1/E2 behaviour appears to express
testing of the penetrated sieve tube for its further suitability as a
source of nutrition. A change of turgor pressure in sieve tubes caused
by occlusion (Gould et al., 2004) represents a relevant signal for
aphids to initiate the behaviour switch from ingestion to salivation,
observed in vitro for M. persicae (Will et al., 2008).
Aphid behaviour that has been observed by EPG thus far [e.g.
Brevicoryne brassicae, M. viciae and M. euphorbiae (Tjallingii,
1985); R. padi (Prado and Tjallingii, 1994); Aphis gossypii and M.
persicae (Martin et al., 1997); A. pisum (Tjallingii and Gabrys,
1999)] suggests that aphids possess only a small and conserved
repertoire of behaviour which hardly varies among aphid species
and host plants. The key for adaptation to the respective host plant(s)
may lie in the watery saliva, which is astonishingly variable in
quantity and protein composition, even if aphid species feed on the
same host plant (Fig. 5). The variable composition may reflect an
evolutionary adaptation to a variety of defence mechanisms of host
plant(s) (e.g. phenols, hydrogen peroxide) other than Ca2+-induced
occlusion. Several watery-saliva proteins which are not involved in
Ca2+ binding may function in the detoxification of such compounds
(Miles and Oertli, 1993; Urbanska et al., 1998).
LIST OF ABBREVIATIONS
APS
CF
CFDA
CLSM
DC
EPG
SE
auto power spectrum
carboxyfluorescein
5(6)carboxyfluorescein diacetate
confocal laser scanning microscopy
direct current
electrical penetration graph
sieve element
We thank Gregory P. Walker (Department of Entomology, University of California,
Riverside, USA) for critical reading of the manuscript. We are grateful to Edgar
Schliephake (Julius Kühn-Institut, Institut für Resistenzforschung und
Stresstoleranz, Quedlinburg, Germany) for supplying a starter colony of M. persicae.
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