Journal of Experimental Botany, Vol. 60, No. 11, pp. 3269–3277, 2009
doi:10.1093/jxb/erp163 Advance Access publication 27 May, 2009
RESEARCH PAPER
Hypersensitive response to Aphis gossypii Glover in melon
genotypes carrying the Vat gene
Emilio Sarria Villada1, Elisa Garzo González2, Ana Isabel López-Sesé1, Alberto Fereres Castiel2 and
Marı́a Luisa Gómez-Guillamón1,*
1
2
Experimental Station ‘La Mayora’, Consejo Superior de Investigaciones Cientı́ficas (CSIC), E-29750 Algarrobo-Costa, Málaga, Spain
Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Cientı́ficas (CSIC), Serrano 115, E-28006 Madrid, Spain
Received 11 November 2008; Revised 30 March 2009; Accepted 29 April 2009
Abstract
Aphis gossypii Glover causes direct and indirect damage to Cucumis melo L. crops. To decrease the harmful effects
of this pest, one of the most economically and environmentally acceptable options is to use genetically resistant
melon varieties. To date, several sources of resistance carrying the Vat gene are used in melon breeding
programmes that aim to prevent A. gossypii colonization and the subsequent aphid virus transmission. The results
suggest that the resistance conferred by this gene is associated with a microscopic hypersensitive response
specific against A. gossypii. Soon after aphid infestation, phenol synthesis, deposits of callose and lignin in the cell
walls, damage to the plasmalemma, and a micro-oxidative burst were detected in genotypes carrying the Vat gene.
According to electrical penetration graph experiments, this response seems to occur after aphid stylets puncture
the plant cells and not during intercellular stylet penetration. This type of plant tissue reaction was not detected in
melon plants infested with Bemisia tabaci Gennadius nor Myzus persicae Sulzer.
Key words: Cotton aphid, Cucumis melo L., EPGs, resistance, susceptibility.
Introduction
Plants possess both passive and active mechanisms to
protect themselves against insect and pathogen attacks.
Passive mechanisms usually involve plant surface tissue
characteristics that prevent or impede the colonization of
insects. Among these characteristics, the most common are
high pubescence (Kishaba et al., 1992; Khan et al., 2000;
Gurr and McGrath, 2002), the presence of glandular
trichomes that store sticky or deterrent substances (Kellog
et al., 2002; Simmons et al., 2003; Wagner et al., 2004) and
the presence of deterrent compounds in the epicuticular wax
layer (Ni and Quisenberry, 1997; Shepherd et al., 1999;
Bahlmann et al., 2003). Active defence mechanisms are
commonly associated with resistance against pathogens
such as viruses, bacteria, fungi, and nematodes (Tanguy,
1971; Cohen et al., 1990; Southerton and Deverall, 1990;
Walters, 2003; Pegard et al., 2005; Menden et al., 2006;
Repka, 2006). They were also recently found to be
associated with plant resistance to insects (Walling, 2000)
but the information about active defence mechanisms of
plants to aphids is limited (Kaloshian, 2004; Smith and
Boyko, 2007). These responses include similarities with the
responses to pathogens and wounds and have been associated with jasmonic (Martinez de Ilarduya et al., 2003; ZhuSalzman et al., 2004; Gao et al., 2007), salicylic acid (Moran
and Thompson, 2001; Chaman et al., 2003; Martinez de
Ilarduya et al., 2003; De Vos et al., 2005), and ethylene
(Argandoña et al., 2001) signalling pathways.
Among the active mechanisms of plant defence, the
hypersensitive response is perhaps the best known. The
hypersensitive response involves rapid programmed cell
death elicited by plant recognition of an attack by
a pathogen or an insect (Morel and Dangl, 1997). Subsequently, an array of physiological and transcriptional
changes triggers the basal plant defence (Kaloshian, 2004)
that involves a rapid, complex reaction that includes,
among others, the production of reactive oxygen species
* To whom correspondence should be addressed: E-mail: [email protected]
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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3270 | Sarria et al.
(ROS), deposition of callose and lignin in cell walls,
accumulation of phytoalexins, and synthesis of pathogenesisrelated proteins (Chaman et al., 2003). This plant defence
mechanism has been described for sedentary herbivorous
insects such as galling insects (Ollerstam et al., 2002;
Fernandes et al., 2003; Ozaki and Sakamoto, 2006) but
until date, only two cases of hypersensitive response
associated with aphid resistance have been reported. Lyth
(1985) described macroscopic necrosis spots in apple leaves
after aphid infestation. Belefant-Miller et al. (1994) evaluated the plant response of barley to the Russian wheat
aphid and they observed collapsed autofluorescent cells
after aphid infestation in the resistant accession.
Aphis gossypii feeding on melon causes direct damage to
the plant by removing photoassimilates and indirect damage by transmitting pathogenic viruses. One of the most
effective strategies to lessen these harmful effects is to use
genetically resistant commercial melons. To date, three
main sources of resistance to aphids are mostly used in
melon breeding: PI 161375, PI 414723, and TGR-1551. All
three lines carry the dominant gene, Vat (Virus aphid
transmission) that confers resistance to A. gossypii and to
A. gossypii virus transmission (Pitrat and Lecoq, 1980;
Klingler et al., 2001; Sarria et al., 2008).
The Vat gene was recently cloned and functional studies
in transgenic melon, cotton, and tomato confirmed the
resistance character this gene controls (Dogimont et al.,
2007). This Vat gene putatively encodes a cytoplasmic
protein that has a nucleotide binding site and leucine-rich
repeat domains (NBS-LRR) (Brotman et al., 2002), belonging to the R gene family, which is widely associated with
active defence mechanisms against pathogens and insects
(Kaloshian, 2004). Consequently, it is logical that, in melon
genotypes that carry the Vat gene, an active defence
mechanism against A. gossypii would be present. However,
the mechanism of resistance conferred by this gene remains
unclear. Shinoda (1993) first described early deposits of
callose in leaf tissues of resistant melon plants in response
to A. gossypii infestation. No other work related to the
active defence mechanism associated with the Vat gene has
been reported. Reports of sap composition (Chen et al.,
1996, 1997, 1999) do not reveal any obvious translocable
factor that could affect aphid behaviour. The grafting
experiments carried out by Kennedy and Kishaba (1977)
and Sarria et al. (2006) indicate that the plant response to
A. gossypii infestation occurs closely around the probing
site.
In this work, several different melon genotypes with
specific Vat-conferred resistance to A. gossypii were used to
investigate the responses of epidermal and mesophyll cells
to A. gossypii probing activities. This response was compared with those observed after Myzus persicae Sulzer and
Bemisia tabaci Gennadius infestations. In addition, the
electrical penetration graph (EPG) technique (Tjallingii,
1978) was used to study the aphid probing and feeding
activities that triggered the observed response. A possible association between this plant response and the resistance to
virus transmission is also discussed.
Materials and methods
Insects and plant material
Aphis gossypii and B. tabaci (biotype Q) were collected from
infested melon crops in a greenhouse (Almerı́a, Spain). One
only nymph from a female aphid was used to create the clone
and maintain it under artificial conditions. They were reared
on plants of the susceptible Spanish melon cultivar ‘ANC57’. Myzus persicae was collected from sweet pepper plants
growing in an open field in Alcalá de Henares (Spain) and
colonies were reared on plants of the pepper cultivar
‘Italiano’. Aphid colonies were maintained in a growth
chamber at 25 C (light) and 20 C (dark) with a photoperiod
of 16:8 h (light:dark), while whitefly colonies were maintained in a greenhouse at an average temperature of 28 C.
Melon genotypes used in the experiments were PI 414723,
PI 161375, and TGR-1551, which are melon lines whose
resistance to A. gossypii is controlled by the Vat gene (Pitrat
and Lecoq, 1980; Klingler et al., 2001, Sarria et al., 2008);
‘AR 5’, an aphid-resistant cantaloupe cultivar in which the
Vat gene was introgressed from PI 414723 (McCreight et al.,
1984); and ‘Bola de Oro’, ‘PMR-5’, and ‘PMR 6’, which are
aphid-susceptible cultivars used as controls in the experiments. ‘PMR 5’ and ‘AR 5’ are near-isogenic melon lines.
The melon genotypes PI 414723, PI 161375, and TGR1551 are also tolerant to B. tabaci while ‘AR 5’, ‘PMR 5’,
‘PMR 6’, and ‘Bola de Oro’ are susceptible to this whitefly
species (Soria et al., 1999; Sauvion et al., 2005).
Melon plants were grown in plastic pots (12 cm diameter)
filled with soil-substrate composed of peat (60%), litonite
(10%), and compost (30%) and maintained in the same
environmental conditions as aphid colonies. Plants 4-weeksold, i.e. having five or six true leaves, were used in the
experiments.
Infestation procedure
Plants were infested with ten young wingless adults of
A. gossypii that were previously starved for 2 h. Aphids were
placed inside a clip cage (25 mm diameter) on the adaxial
surface of the leaf in the experiments; to observe aphid
stylet tracks, insects were placed on the abaxial leaf surface.
To assess the plant response to insect infestation, experiments with groups of ten wingless M. persicae and 30
B. tabaci individuals per plant were conducted following the
same methodology as described for A. gossypii.
For each set of experiments, at least three plants per
genotype and one leaf disc per plant were evaluated; insectfree clip cages on melon leaves were used as controls.
Sampling procedure and microscopy
Aphid stylet sheaths are composed of proteins and were
stained with fuchsine as described by Brennan et al. (2001).
Leaf areas where aphids had been feeding for 24 h were
thinly sectioned using a hand-microtome. Samples were
fixed in ethanol (70%) and then immersed in an acid fuchsin
solution [10 parts 70% ethanol and 1 part 0.2% acid fuchsin
Hypersensitive response to Aphis gossypii in melon | 3271
in 95% ethanol:glacial acetic acid (1:1, v/v)]. Stylet sheaths
on these samples appeared bright orange under an epifluorescence microscope Leica DM LB2 (Ex 515–560, DM 580).
To show the autofluorescence in cells around the stylet
sheaths, these samples were also observed under UV light
using a different microscope filter (Ex 340–380, DM 400).
The dynamics of autofluorescence displayed in cells was
evaluated following the Belefant-Miller et al. (1994) procedure using leaf discs.
To observe callose and lignin depositions after aphid
infestation, leaf discs were first fixed and decoloured in
absolute ethanol. Callose deposition was revealed following
the methodology employed by Cohen et al. (1990), in which
decoloured leaf discs were immersed in an aniline blue
solution (0.01% aniline blue in 7 mM K2HPO4, pH 8.9) for
24 h. Callose deposition was observed, coloured in bluewhite, using an epifluorescence microscope Leica DM LB2,
(Ex 340–380, DM 400). Lignin was observed by staining
decoloured leaf discs with a phloroglucinol solution (0.02%
phloroglucinol in HCl:ethanol 1:1 v/v) as described by
Davidson et al. (1995). Observations of lignin deposition,
coloured in red, were made using a light microscope (Leica
DM LB2).
Evans Blue stain diffuses into dead cells (Gaff and Okong
‘O-Ogola, 1971) and thus it was used to observe plasmalemma damage. Leaf discs were completely submerged in
a 0.1% (w/v) aqueous solution of Evans Blue, and then
subjected to two 5 min cycles under vacuum followed by
a single 20 min cycle under vacuum. Samples were then
washed by vacuum infiltration of phosphate-buffered saline
plus 0.05% (v/v) Tween three times for 15 min each (Wright
et al., 2000). Cells with plasmalemma damage appeared blue
under a light microscope.
The increase in peroxidase activity was evaluated as
described by Martinez de Ilarduya et al. (2003). Leaf discs
were vacuum-infiltrated with 1 mg ml1 3,3#-diaminobenzidine
(DAB), pH 3.8, for 20 min and incubated in the DAB solution for an additional 6 h period at room temperature (25 C).
Finally, leaf discs were immersed in 95% ethanol to clear the
tissue; DAB polymerizes instantly and locally at sites of
peroxidase activity into a reddish-brown polymer that is
stable in most solvents (Thordal-Christensen et al., 1997).
Cell collapse was assessed through observation of the leaf
surface under the scanning electron microscope (SEM) since
electrolyte leakage points to the existence of damage in
cellular membranes (Goodman, 1968). Leaf discs of the
infested area were fixed in glutaraldehyde (4% v/v, in
phosphate buffer 0.2 M, pH 7) at 4 C for 24 h and
thoroughly rinsed in fresh phosphate buffer. Subsequently,
they were dehydrated by immersion in an acetone solution
series (25, 50, 75, and 100% v/v), 15 min each. Samples were
mounted on SEM tubes, coated with a thin layer of gold and
observed using a JEOL JSM-840 SEM operating at 15 KeV.
Monitoring of aphid probing
Monitoring of A. gossypii and M. persicae probing and
feeding behaviour was conducted using a methodology
similar to the one described by Garzo et al. (2002). A gold
wire 1 cm long (20 lm diameter) was attached to the
dorsum of a young adult aptera by immobilizing it with
a vacuum-operated plate and touching the aphid with
a small droplet of conducting glue (water-based silver glue).
The other end of the gold wire was attached to a 3 cm long
copper wire (0.2 mm diameter) and connected to the input
of the first stage amplifier with a 1 GX input resistance and
503 gain (Tjallingii, 1985, 1988). The plant electrode 10 cmlong (2 mm diameter) copper rod, was inserted in the soil of
the potted plant and connected to the plant voltage output
of the EPG device (Giga-4 manufactured by Wageningen
University). PROBE 3.0 for Windows software (Laboratory
of Entomology, Wageningen University) was used for data
acquisition and real-time display of EPG waveforms.
Aphids were placed on a restricted leaf area (5 mm
diameter) and artificially interrupted and removed from
plants at the start of the first probe. The following
differential treatments were used: first probe interrupted
during stylet pathway (C) and before any potential drops
(intracellular stylet punctures) were recorded; first probe
interrupted immediately after a single potential drop was
recorded; first probe interrupted immediately after five or
more potential drops were recorded. One hour after aphids
were removed from the plants, leaf discs (5 mm diameter)
were immersed in absolute ethanol and stained to observe
callose deposition, as explained above.
Results
Characterization of the response to A. gossypii
infestation
Twenty-four hours after A. gossypii infestation, several
events in epidermal and mesophyll tissues were observed.
Fuchsin stained the aphid stylet sheaths, which revealed the
aphid stylet tracks; they were observed as a bright orange
line through the intercellular spaces of the epidermal and
mesophyll tissues of all melon accessions (Fig. 1A). When
these same samples were observed under UV light (Ex 340–
380, DM 400), the cells around the stylet tracks showed
a clear autofluorescence only in tissues of the resistant
melon accessions (Fig. 1B).
According to these preliminary observations and since
autofluorescence in cells has been described as a signal of
the plant resistance (Cohen et al., 1990; Belefant-Miller
et al, 1994), several events related to a possible melon plant
response 24 h after A. gossypii infestation were examined at
the microscopic level in aphid-resistant and -susceptible
genotypes.
Blue-white spots revealing strong callose depositions in
many epidermal and mesophyll cell walls were observed in
all the genotypes carrying the Vat gene (‘AR 5’, PI 161375,
PI 414723, and TGR-1551) (Fig. 1C). No such reaction was
observed in plants of any of the susceptible genotypes (‘Bola
de Oro’, ‘PMR 5’, and ‘PMR 6’).
Deposits of lignin were observed in red in epidermal and
mesophyll cells of the aphid-resistant genotypes (Fig. 1D).
3272 | Sarria et al.
Fig. 1. Microphotographs of leaf samples of an aphid-resistant melon, TGR-1551, infested with A. gossypii Glover. Bright orange stylet
tracks (white arrowheads), stained with acid fuchsin and using the correct microscope filter (Ex 515–560, DM 580), were observed in thin
leaf sections (A). When the same leaf section was observed under another microscope filter (Ex 340–380, DM 400), cells around stylet
tracks showed autofluorescence (B, black arrow). Aniline blue and phloroglucinol stains revealed callose (C) and lignin depositions (D),
respectively, in epidermal and mesophyll cells. Increases in peroxidase activity, using DAB (E), and plasmalemma damage, using Evans
Blue (F), were observed 10 min after A. gossypii infestation. Cell collapse was observed 12 h after infestation (G). Legend, e, epidermis;
m, mesophyll; s, stoma; t, trichome; v, vascular tissues; white arrowhead, stylet sheath; black arrow, plant cell response; bar¼50 lm.
No lignin deposit was observed in cells of any of the aphidsusceptible lines.
The above events were observed 24 h after aphid infestation and may be associated with a general plant defence
response. To determine the dynamics of these events at
different periods after A. gossypii infestation, new experiments were designed using TGR-1551 and ‘Bola de Oro’
genotypes and the near isogenic melon lines ‘AR 5’ and
‘PMR 5’. In these experiments, leaf samples were taken at
10, 20, 40, 90 min, 4 h 30 min, 6, 12, 24, and 72 h after
aphid infestation.
Following A. gossypii probing activities, callose deposition was observed in epidermal cells of the resistant
genotypes, TGR-1551 and ‘AR 5’, 20 min after A. gossypii
infestation, and autofluorescence of cells appeared 40 min
after, whereas lignin deposition was observed 4.5 h after
aphid infestation. No sign of response to aphid infestation
was evident in plants of the susceptible genotypes (‘Bola de
Oro’ and ‘PMR 5’), with the exception of slight callose
deposits next to sieve elements, which were only observed
72 h after aphid infestation.
These results pointed to the possible existence of an active
plant response to A. gossypii and to the occurrence of
a programmed cell death in aphid-resistant genotypes.
Thus, additional events associated with a hypersensitive
response were evaluated in all genotypes: plasmalemma
integrity and peroxidase activity.
An increase in peroxidase activity (Fig. 1E) and plasmalemma damage (Fig. 1F), related to programmed cell death,
were clearly observed just 10 min after aphid infestation
(shorter periods were not considered) only in genotypes
carrying the Vat gene. To complete the characterization of
this response, cell collapse after aphid infestation was also
evaluated in TGR-1551 and ‘Bola de Oro’. Cell collapse was
only observed in TGR-1551 12 h after aphid infestation in
cells punctured by A. gossypii and around the stylet
penetration point (Fig. 1G). No sign of this event was
observed in ‘Bola de Oro’.
All these results led to the consideration that A. gossypii
clearly induced a fast programmed cell death restricted to
the aphid-infested area in the resistant genotypes (‘AR 5’,
PI 161375, PI 414723, and TGR-1551). None of these
events was observed in the susceptible genotypes (‘Bola de
Oro’, ‘PMR 5’, and ‘PMR 6’), even after extended infestation periods (72 h).
Specificity of the plant hypersensitive response
To evaluate the specificity of such a response, melon plants
of TGR-1551 and ‘Bola de Oro’ genotypes and the nearisogenic melon lines ‘AR 5’ and ‘PMR 5’ were infested with
B. tabaci and M. persicae, two additional hemipteran
species that also feed from the phloem.
Stylet tracks were observed through the intercellular
spaces of the epidermal and mesophyll tissues of leaves
infested with B. tabaci in all genotypes (‘AR 5’, ‘Bola de
Oro’, ‘PMR 5’, and TGR-1551). However, no active cell
response was observed, with the exception of slight callose
deposits located in vascular tissues 72 h after whitefly infestation and this was observed in all the genotypes tested.
When leaves were infested with M. persicae, stylet tracks,
callose and lignin depositions, and autofluorescence were
observed 24 h after infestation in epidermal and mesophyll
cells in all the melon genotypes tested (‘AR 5’, ‘Bola de
Oro’, ‘PMR 5’, and TGR-1551). To compare this response
to the one observed after A. gossypii infestation, the
dynamics of callose and lignin depositions and the apparition of autofluorescence were evaluated following the same
methodology described above. All these events were observed in both the resistant and the susceptible genotypes
Hypersensitive response to Aphis gossypii in melon | 3273
and an important delay in their apparition was also
observed. Thus, callose deposition was observed in resistant
and susceptible genotypes 12 h after infestation (Fig. 2A),
and autofluorescence in cells (Fig. 2B) and lignin deposition
(Fig. 2C) were not observed until 24 h after aphid
infestation. The number of affected cells was lower after
infestation with M. persicae than with A. gossypii in the
resistant accessions (Fig. 2A).
The induction of plasmalemma damage and the increase
of peroxidase activity was evaluated 24 h and 72 h after
B. tabaci and M. persicae infestations in the same genotypes
and no sign of these events were observed.
Probing activity of A. gossypii that triggers the
hypersensitive response in TGR-1551
Electrical penetration graph techniques were used to determine the probing activities of A. gossypii that triggers the
hypersensitive response in the resistant melon TGR-1551.
‘Bola de Oro’ was used as the susceptible control. The
probing activities evaluated were related to the number of
intracellular punctures made by A. gossypii before reaching
the phloem. Callose deposition 1 h after aphid removal was
used as a response indicator to A. gossypii infestation because of its early and easy observation. M. persicae probing
activities were also tested using this technique.
A clear response to intracellular punctures made by A.
gossypii was observed in the aphid-resistant genotype. This
response differed in relation to the aphid probing activities
registered by means of the EPG device. No callose deposition in cell walls was observed when aphids did not
make intracellular punctures. When a single intracellular
puncture was recorded, callose deposition in the cell walls
was observed in 34 out of the 53 leaf samples tested. The
number of leaf samples showing callose deposition after five
or more intracellular punctures increased to 34 out of the 38
leaf samples tested. In many samples, callose deposits were
observed not only in cells directly punctured by the aphid
stylet but also in surrounding cells. None of the stylet
activities of this aphid on ‘Bola de Oro’ leaves induced
callose deposition. Furthermore, none of the stylet activities
of M. persicae induced any response to cell puncture in any
of the tested genotypes.
Discussion
Wright et al. (2000) and Garcia-Brugger et al. (2006),
described the loss of plasmalemma integrity, cell collapse,
and the increase in peroxidase activity as being tightly
related to the resistance mechanisms of the hypersensitive
response. These events are clearly observed in leaf cells of
aphid-resistant melon genotypes soon after A. gossypii
infestation. Therefore, this aphid species induces a hypersensitive response in melon genotypes carrying the Vat gene.
Callose and lignin depositions in cell walls and the appearance of autofluorescence, widely related to the synthesis of
phenol compounds (Nicholson and Hammerschmidt, 1992),
are commonly observed in general responses to cell damage
in many plants (Russo and Bushnell, 1989). Actually,
callose deposition has also been reported in compatible
interactions between plants and phloem-feeding insects
(Walling, 2008). Like Shinoda (1993), callose depositions
have also been observed in epidermal and mesophyll cell
walls of susceptible genotypes, but they were only evident
72 h after aphid infestation. By contrast, in melon genotypes carrying the Vat gene, callose deposits in cell walls
were observed just 20 min after A. gossypii infestation,
affecting not only whole cells close to the stylet tracks but
also the surrounding cells. In addition to the rapid
accumulation of callose deposits, phenol and lignin synthesis were also observed very soon after aphid infestation, 40
min and 4 h 30 min, respectively. Moreover, an increase in
peroxidase activity and plasmalemma damage were observed just 10 min after aphid infestation around the
probing site. Therefore, all these events observed at
a microscopic level and tightly restricted to the infested area
should be associated with a hypersensitive response (Walters, 2003) which seems to be associated with the Vat gene.
A. gossypii resistance in the melon genotypes tested is
controlled by the Vat gene. These genotypes have very
different genetic backgrounds, but their response to A.
Fig. 2. Microphotographs of melon leaves of an aphid resistant melon, TGR-1551, infested with M. persicae. Callose deposition in
parenchyma cells was observed 12 h after infestation (A, black arrows). The appearance of autofluorescence (B, black arrow) and lignin
deposition (C, black arrow) were observed 24 h after infestation. Legend, e, epidermis; m, mesophyll; t, trichome; v, vascular tissues;
bar¼50 lm.
3274 | Sarria et al.
gossypii infestation was identical in all of them. In addition,
this response was also observed in ‘AR 5’ and was absent in
‘PMR 5’, its near-isogenic line, which reinforces the idea
that the Vat gene is associated with this type of hypersensitive response. Since the Vat gene putatively belongs to the
R gene family (Brotman et al., 2002), this gene should play
an important role in the hypersensitive response observed.
Several authors have reported the increase of ROS, the
induction of the activity of enzymes such as phenylalanine
amonia-lyase and the activation of different signalling pathways associated with plant defence after aphid infestation in
other species different from melon (Argandoña et al., 2001;
Moran and Thompson, 2001; Chaman et al., 2003; Martı́nez
de Ilarduya et al., 2003; De Vos et al., 2005; Moloi and van
der Westhuizen, 2006; Gao et al., 2007). However, only two
authors have reported the occurrence of a hypersensitive
response against aphids. Lyth (1985) observed macroscopic
necrotic spots related to a hypersensitive response around the
aphid feeding site in apple cultivars resistant to Dysaphis
plantaginea Passerini. Belefant-Miller et al. (1994) described
a microscopic hypersensitive response in resistant barley to
Diuraphis noxia Mordvilko through the evaluation of cell collapse and the induction of autofluorescence related to the
appearance of phenols.
Aphids regularly puncture cells found along the stylet
pathway and ingest cytosolic samples, thus detecting cues
present in peripheral (non-vascular) plant cells that are
probably important in stimulating settling and parturition
(Powell et al., 2006). The evaluation of the probing and
feeding behaviour of A. gossypii exposed to resistant melon
genotypes using the EPG techniques revealed that aphid
behaviour is affected only during the first probes 30 min
after the beginning of EPG recordings (Garzo et al., 2002).
The nature and the rapid speed of events related to the
microscopic hypersensitive response could explain the
difficulty that aphids encounter in trying to reach the
phloem to feed on plants carrying the Vat gene. For
instance, callose deposition, which rapidly enlarges and
reinforces the cell walls (Cohen et al., 1990; Shinoda, 1993;
Kuzuya et al., 2006), and phenol compounds, related to the
deterrence of aphids (Dreyer and Jones, 1981; Cichocka
et al., 2000), could impede the normal aphid stylet
penetration activities in the leaf tissues. In addition, passive
ingestion of reactive oxygen species during aphid probing
could also affect the insect midgut tissues, as suggested by
Smith and Boyko (2007). In conclusion, physical and chemical changes induced by aphids in epidermal and mesophyll
cells could be associated with the problems that aphids have
in reaching the vascular tissues in melon genotypes carrying
the Vat gene (Garzo et al., 2002). Thus, these early events
could partially explain the A. gossypii rejection of Vat
melon genotypes after the first probes.
When the cell response was evaluated after different stylet
activities of A. gossypii in the resistant genotype TGR-1551,
callose deposition in cell walls was not induced in samples
where A. gossypii did not make intracellular punctures, but
was observed in most of the samples where this aphid
species made at least one intracellular puncture. Thus,
recognition of A. gossypii infestation seems to occur in the
cytoplasm and should be related to a component (elicitor)
of the aqueous saliva that this aphid injects during the brief
punctures (Martin et al., 1997). These results agree with the
idea that the Vat gene encodes a cytosolic protein (Brotman
et al., 2002).
Localized callose depositions were observed next to
vascular bundles 72 h after infestation with B. tabaci in
aphid resistant (‘AR 5’ and TGR-1551) and susceptible
(‘Bola de Oro’ and ‘PMR 5’) melon genotypes. Whiteflies
rarely puncture cells other than those in phloem tissues
(Johnson and Walker, 1999), and actually, callose deposition was not observed in epidermal and mesophyll tissues.
The deposition of callose in cells after B. tabaci infestation
could be explained as a result of the general plant response
to compatible phloem-feeding insects (Walling, 2000). When
‘AR 5’, ‘Bola de Oro’, ‘PMR 5’, and TGR-1551 were
infested with M. persicae, the aphid induced deposition of
callose in the cell walls of epidermal and mesophyll tissues
12 h after infestation in all the genotypes as well as the
appearance of autofluorescence and the deposition of lignin
24 h after infestation. In contrast to whitefly, M. persicae
induced this response in cells of any leaf tissue, possibly due
to cell probes carried out by aphids before reaching the
phloem. Since C. melo is not a suitable host for this aphid
species, and these events were observed in resistant and
susceptible genotypes they should be triggered in response
to cell damage and should be associated with general plant
defence. According to these results, the fast programmed
cell death observed in aphid-resistant genotypes should be
specific to the probing and feeding activities of A. gossypii.
The specificity of the plant response to A. gossypii
infestation could help to explain the observation that the
Vat gene also controls resistance to virus transmission
(Lecoq et al., 1980; Soria et al., 2003). The rapid hypersensitive response at the epidermal and mesophyll levels,
together with the drastic changes induced in cell metabolism, could affect the cellular redox status, limiting the virus
particles released from the aphid stylet as suggested by
Martı́n et al. (2003). However, in case some virus particles
were inoculated through intracellular salivation, virus
replication and cell-to-cell movement of the viral particles
could be affected by some of the events related to the
hypersensitive response. The role of callose in limiting viral
infection is well-known; the extracellular callose deposition
around plasmodesmata (callose collar) could constrain the
cell-to-cell transport of viral particles (Tu and Hiruki, 1971;
Allison and Shalla, 1974; Pennazio et al., 1981; Carrington
et al., 1996; Iglesias and Meins, 2000; Iriti et al., 2006). On
the other hand, and as observed through EPGs techniques,
the hypersensitive response was not observed in all the
samples where A. gossypii made intracellular punctures,
which could indicate that the cell response could fail or be
delayed on a few occasions. This could explain the overcoming of virus transmission resistance described in melon
genotypes with the Vat gene in experiments carried out
under laboratory conditions (Pitrat and Lecoq, 1980; Soria
et al., 2003; Sarria et al., 2008).
Hypersensitive response to Aphis gossypii in melon | 3275
Klingler et al. (2001) reported that resistance to
A. gossypii and to virus transmission seems to be controlled
by a single gene or two tightly-linked genes. Our results
support the hypothesis that the very fast micro-hypersensitive response possibly triggered by the Vat gene after the
plant cell had been punctured by the A. gossypii stylet in
aphid-resistant genotypes could impede the release, replication, and/or spread of the virus transmitted by the aphid in
a non-persistent manner. However, further studies should
be encouraged to investigate the nature of the aphid
infestation and the role of this response in virus transmission resistance.
Chen JQ, Rahbé Y, Delobel B, Sauvion N, Guillaud J, Febvay G.
1997. Melon resistance to the aphid Aphis gossypii: behavioural
analysis and chemical correlations with nitrogenous compounds.
Entomologia Experimentalis et Applicata 85, 33–44.
Chen JQ, Rahbé Y, Delobel B. 1999. Effects of pyrazole compounds from melon on the melon aphid Aphis gossypii. Phytochemistry 50, 1117–1122.
Cichocka E, Leszczyński B, Goszczyński W. 2000. Effect of
phenolic compounds on acceptance of broad bean cultivars by black
bean aphid Aphis fabae Scop. In: Aphids and other homopterous
insects, Vol. 7. Olsztyn: Polish Academy of Sciences, 169–176.
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collaboration in all the experiments. This work was financed
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