Journal of Experimental Botany, Vol. 66, No. 2 pp. 533–539, 2015
doi:10.1093/jxb/eru487 Advance Access publication 24 December, 2014
Review Paper
Aphid salivary proteases are capable of degrading sieve-tube
proteins
Alexandra C. U. Furch1, Aart J. E. van Bel2 and Torsten Will3,*
1 Institute of General Botany and Plant Physiology, Friedrich-Schiller-University, Dornburger Str. 159, D-07743 Jena, Germany
Department of General Botany, Justus-Liebig-University, Heinrich-Buff-Ring 17, D-35392 Giessen, Germany
3 Institute of Phytopathology and Applied Zoology, Centre for BioSystems, Land Use and Nutrition, Justus-Liebig-University, HeinrichBuff-Ring 26–32, D-35392 Giessen, Germany
2 * To whom correspondence should be addressed. E-mail: [email protected]
Received 31 October 2014; Revised 12 November 2014; Accepted 13 November 2014
Abstract
Sieve tubes serve as transport conduits for photo-assimilates and other resources in angiosperms and are profitable targets for piercing–sucking insects such as aphids. Sieve-tube sap also contains significant amounts of proteins
with diverse functions, for example in signalling, metabolism, and defence. The identification of salivary proteases in
Acyrthosiphon pisum led to the hypothesis that aphids might be able to digest these proteins and by doing so suppress
plant defence and access additional nitrogen sources. Here, the scarce knowledge of proteases in aphid saliva is briefly
reviewed. In order to provide a better platform for discussion, we conducted a few tests on in vitro protease activity
and degradation of sieve-tube sap proteins of Cucurbita maxima by watery saliva. Inhibition of protein degradation by
EDTA indicates the presence of different types of proteases (e.g. metalloproteses) in saliva of A. pisum. Proteases in
the watery saliva from Macrosiphum euphorbiae and A. pisum were able to degrade the most abundant phloem protein,
which is phloem protein 1. Our results provide support for the breakdown of sieve-element proteins by aphid saliva in
order to suppress/neutralize the defence responses of the plant and to make proteins of sieve-tube sap accessible as
a nitrogen source, as is discussed in detail. Finally, we discuss whether glycosylation of sieve-element proteins and the
presence of protease inhibitors may confer partial protection against the proteolytic activity of aphid saliva.
Key words: Aphid, aphid–plant interaction, aphid saliva, protease, protease inhibitor, sieve-tube occlusion, sieve-tube protein.
General functions of saliva in aphid–plant
interaction
Aphids are serious agricultural pest insects causing direct
damage to crops as phloem feeders and indirect yield losses
by virus transmission. Viruses are secreted together with
aphid saliva during cell penetration by the stylet (Powell et al.,
1995; Martin et al., 1997). Along the stylet track, plant cells
are regularly probed (Tjallingii and Hogen Esch, 1993) by
ingesting small amounts of cell sap after secretion of watery
saliva into penetrated cells, (Martin et al., 1997), which possibly serves for orientation (Hewer et al., 2010, 2011). Stylet
movement is accompanied by pulsed secretion of gel saliva
(Miles et al., 1964). After secretion, gel saliva hardens and
forms a tubular structure, through which the stylet is pushed
forward (Kimmins, 1986; Will et al., 2012). After the stylet
has pierced the sieve-element plasma membrane, continuous
secretion of watery saliva precedes regular intervals of saliva
secretion during sap ingestion (Prado and Tjallingii, 1994).
During ingestion, saliva may no longer reach the sieve-element lumen, but rather mixes with ingested phloem sap in
the tip region of the stylet, the so-called common duct (Uzest
et al., 2010).
Abbreviations: EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyanate; O-GlcNAc, O-linked β-N-acetylglucosamine; PP1, phloem protein 1; PP2,
phloem protein 2; RFU, relative fluorescence unit; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; SEO, sieve-element occlusion
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
534 | Furch et al.
Watery saliva contains a variety of proteins (Madhusudhan
and Miles, 1998; Carolan et al., 2009; Will et al., 2009; Cooper
et al., 2011; Rao et al., 2013), some of which are species specific, while others appear to be universally present, such as
the putative sheath protein and glucose–methanol–choline
(GMC) oxidoreductase (Rao et al., 2013). In particular,
detoxifying enzymes such as peroxidase and phenoloxidase
occur in the saliva of diverse aphid species (Madhusudhan
and Miles, 1998; Harmel et al., 2008; Cooper et al., 2011).
In addition, saliva of various aphid species contains proteinaceous effectors, which appear species specific and adapted to
specific host plants (summarized in Will et al., 2013). RNA interference techniques and in planta expression of effectors indicate
a relevance for aphid–plant interaction by facilitating ingestion,
but functions on the molecular level have not been identified to
date (Pitino et al., 2011; Pitino and Hogenhout, 2013).
Calcium-binding proteins, detected in saliva of the aphid
species Megoura viciae (Will et al., 2007) and in the saliva
and salivary glands of Acyrthosiphon pisum (Carolan et al.,
2009, 2011), may belong to the group of effectors, as they
presumably interfere with calcium-induced sieve-element
occlusion (SEO; see Will et al., 2013). Apart from saliva proteins involved in preventing, suppressing, or manipulating
host plant defence, two potential proteases of the M1 and
M2 family, respectively, have been detected in A. pisum saliva
and salivary glands by a proteomic approach (Carolan et al.,
2009, 2011). In contrast, no structural homologues have been
detected in the saliva of other aphid species (Sitobion avenae
and Metopolophium dirhodum; Rao et al., 2013).
This review aims to provide some (experimental) information on the protein degradation capacity of the saliva of
A. pisum and M. euphorbiae. Moreover, potential targets of
salivary proteases, their role in aphid–plant interaction, and
the plant defence against protein breakdown are discussed.
Potential target proteins for salivary
proteases from aphids
Proteomes of sieve-tube sap have been analysed, for example
for Brassica napus, Ricinus communis, and Cucurbita maxima (Barnes et al., 2004; Giavalisco et al., 2006; Lin et al.,
2009). In the exudate of C. maxima, ~1100 different proteins
have been identified (Lin et al., 2009). Of those, two structural proteins—phloem protein 1 (PP1) and phloem protein 2
(PP2; Read and Northcote, 1983a, b; Thompson and Schulz,
1999; Walz et al., 2002; Atkins et al., 2011)—were the most
abundant. PP1 is a 96 kDa filamentous protein with diameters of 7–13 nm. PP2 is a monomeric (25 kDa) or dimeric
(46 kDa) lectin that covalently interacts with PP1 (Read and
Northcote, 1983a; Alosi et al., 1988). PP1 and PP2 are linked
by oxidation in the presence of free oxygen (Alosi et al., 1988)
or reactive oxygen species (Gaupels et al., 2008a). These conglomerates form either protein depositions on the sieve plates
after severe metabolic disturbance (Gaupels et al., 2008a) or
gelled exudate plugs on the cutting surface of cucurbit vascular bundles (Alosi et al., 1988; Clark et al., 1997; Furch et al.,
2010; Zimmermann et al., 2013).
Other abundant proteins in the protein plugs of C. maxima
are allocated to the SEO protein family (Ernst et al., 2012).
Members of the SEO gene family (Rüping et al., 2010; Zielonka
et al., 2014) were first described as a group of occlusion proteins arranged as highly organized spindle-like forisome bodies in Medicago truncatula (Pélissier et al., 2008). SEO genes
encode forisome subunits in legumes (e.g. Müller et al., 2010;
Rüping et al., 2010). In the plant species Arabidopsis thaliana, Nicotiana tabacum, and C. maxima (Anstead et al., 2012;
Ernst et al., 2012; Jekat et al., 2012; Zielonka et al., 2014) that
do not belong to the Fabaceae, SEO genes encode non-forisome proteins, occurring as loosely organized protein depositions or filaments (Müller et al., 2010; Anstead et al., 2012).
In addition to the above-mentioned proteins, numerous antioxidant defence-related proteins were found in cucurbit exudates (Alosi et al., 1988; Walz et al., 2002, 2004). We tackled
the question of whether cucurbit sieve-element proteins can
be degraded by aphid saliva components by conducting some
experiments, as reported in the following section.
Saliva of A. pisum contains different
proteases
Whereas Cherqui and Tjallingii (2000) were unable to identify
proteases in aphid saliva by functional approaches, later proteomic studies provided evidence for two different proteases, an
M1 zinc metalloprotease and an angiotensin-converting enzyme
(M2 metalloprotease), in the saliva of A. pisum (Carolan et al.,
2009). Metalloproteases have been found in the saliva of other
insects such as the phytophagous thrips Frankliniella occidentalis (Stafford-Banks et al., 2014) and the blood-feeding tick
Ixodes scapularis (Decrem et al., 2008). They are believed to
counteract protein-mediated defence responses of their hosts
(Francischetti et al., 2003; Carolan et al., 2009; Stafford-Banks
et al., 2014). No homologies to metalloproteases from A. pisum
were detected in the saliva of the two cereal aphid species
S. avenae and M. dirhodum (Rao et al., 2013).
To verify the presence of proteases in saliva of A. pisum, we
used a fluorescence-based functional assay (Voss et al., 1996).
Over a period of 2000 s, degradation of fluorescein isothiocyanate (FITC)-labelled albumin led to an increase in fluorescence that increased rapidly to a maximum intensity of ~2300
relative fluorescence units (RFUs) in ~500 s (Fig. 1). In the
presence of 5 mM EDTA, fluorescence reached a maximum of
~1000 RFUs after 600 s (Fig. 1). Thus, the increase of fluorescence is strongly reduced in the presence of EDTA, a chelator
of divalent cations. The breakdown time profiles demonstrate
the presence of proteases in aphid saliva; the EDTA-reduced
breakdown of albumin is indicative for involvement of metalloproteases (Parvathy et al., 1997; Windle and Kelleher, 1997) and
non-metalloproteases in protein degradation. A similar reduction of protease activity by EDTA, observed for the tick I. scapularis, was also attributed to the presence of metalloproteases
in its saliva (Francischetti et al., 2003). Non-metalloproteases
were not identified in previous studies, but likely candidates are
among the high numbers of unidentified proteins in the saliva/
salivary glands of A. pisum (Carolan et al., 2009, 2011).
Aphid salivary proteases degrade sieve-tube proteins | 535
As for their function, angiotensin-converting enzymes and
proteases of the M1 type bind zinc at their active catalytic
domain (Lausten et al., 2001; Kim et al., 2003), while calcium regulates their enzymatic activity (Goto et al., 2007).
Although both protease types are putatively exopeptidases
(Iturrioz et al., 2001; Naqvi et al., 2005), solely cleaving the
terminal amino acids from the albumin body, some M1 metalloproteases in Escherichia coli and angiotensin-converting
enzymes in Musca domestica may possess endopeptidase
activity as well (Lamango et al., 1996). One may argue
that exopeptidases would hardly degrade albumin due to
the globular structure of albumin (Voss et al., 1996) which
would limit the increase in fluorescence as a consequence of
fluorescence quenching. In contrast, albumin degradation
by endopeptidases may produce more and larger fragments,
leading to lower quenching and, hence, a stronger increase
in detected fluorescence. Based on our results, M1 metalloproteases and angiotensin-converting enzyme of A. pisum
could both be engaged in the degradation of FITC-labelled
albumin. Finally, activities of metalloproteases from other
classes must be taken into consideration in future research,
since EDTA is not a specific inhibitor of M1 and M2
metalloproteases.
Fig. 1. Detection of protease activity in watery saliva from Acyrthosiphon
pisum. Protease activity assays were conducted using fluorescein
isothiocyanate (FITC)-labelled albumin (Sigma-Aldrich, St Louis, MO, USA)
that is suitable as a substrate for a wide range of proteases. Its globular
structure results in low fluorescence of undigested FITC-labelled albumin
due to fluorescence quenching (Voss et al., 1996). Saliva samples were
collected and processed as described earlier (Will et al., 2007). To identify
the presence of metalloproteases (Carolan et al., 2009, 2011), protease
activity of aphid saliva was measured in Tris-buffered saline (TBS), pH 7.2
with/without 5 mM EDTA (Francischetti et al., 2003) as a chelating agent
for binding divalent cations (e.g. Ca2+, Mg2+, and Zn2+). Each sample
contained 20 μl of aphid saliva from ~4000 aphids, 180 μl of buffer, and
5 μg ml–1 FITC-labelled albumin. The blank sample contained aphid diet
(Will et al., 2007) instead of aphid saliva. A Infinite® F200 microplate reader
(Tecan, Männedorf, Switzerland) was applied for fluorescence detection
with an excitation wavelength of 485 nm and an emission wavelength of
530 nm. Samples were incubated in black 96-well plates at 25 °C and
were shaken for 1 s prior to each measurement. The relative fluorescence
intensity was calculated from 20 measurements per well and was amplified
×50. The entire measuring time encompassed 2000 s with intervals of 25
s. Measurements of relative fluorescence units (RFUs) of the blank were
subtracted from RFUs of saliva samples.
Impact of salivary (metallo)proteases on
plant defence and aphid feeding
Breakdown of sieve-tube sap proteins involved in defence,
signalling, and/or occlusion may lead to suppression of plant
defence (Carolan et al., 2009). A similar role has been attributed to metalloproteases in ticks and thrips, where salivary
proteases were believed to degrade defence-related proteins
in their respective hosts (Francischetti et al., 2003; StaffordBanks et al., 2014).
SEO proteins in Fabaceae (Pélissier et al., 2008) and in
A. thaliana (Anstead et al., 2012), as well as PP1 and PP2
in cucurbits (Walz et al., 2004; Ernst et al., 2012) are potential targets for salivary proteases to prevent food deprivation
(Will et al., 2008). The underlying molecular mechanisms of
occlusion by SEO proteins as well as PP1 and PP2 seem to be
diverse (Read and Northcote, 1983a; Knoblauch et al., 2001;
Furch et al., 2009; Jekat et al., 2013). Degradation of occlusion proteins may therefore be an option for generalist feeders to enable ingestion (e.g. Myzus persicae and Macrosiphum
euphorbiae) rather than for specialists such as A. pisum. The
latter aphid species does not trigger the dispersion of SEO
proteins in Vicia faba (Walker and Medina Ortega, 2012),
presumably by preventing an increase of calcium in the sievetube lumen during stylet penetration (Will et al., 2013). In
contrast, M. euphorbiae induces sieve-tube occlusion in
V. faba (Medina-Ortega and Walker, 2014).
The question was raised of whether degradation of sievetube proteins by salivary proteases enables aphids to access
an amino acid source (Carolan et al., 2009) to supplement
the limited amount of essential amino acids in sieve-tube sap
(Gündüz and Douglas, 2009). During ingestion, saliva mixes
with sieve-tube sap inside the aphid’s stylet tip (Tjallingii,
2006). This could speed up degradation of ingested peptides
and proteins, and thus facilitate the digestion in the aphid’s
stomach as in A. pisum (Rahbé et al., 1995).
The breakdown strategy might work best for dicotyledons
because high protein levels were reported for the sieve-tube
sap of dicotyledonous plants [e.g. R. communis 2–5 mg ml–1
(Schobert et al., 1998), C. maxima 19–100 mg ml–1 (Richardson
et al., 1982; Schobert et al., 1998; Zimmermann et al., 2013)].
Monocotyledonous plants possess lower amounts of protein
[e.g. barley 0.1–0.4 mg ml–1 (Schobert et al., 1998; Gaupels
et al., 2008b) and wheat 1 mg ml–1 (Fisher et al., 1992).
The functionality of salivary proteases is speculative,
because direct interaction between salivary proteins and
sieve-tube proteins was only tested once for the aphid species
M. viciae in combination with forisomes from V. faba without
a focus on protease activity (Will et al., 2007). To test proteolysis of sieve-tube proteins by salivary proteases, we collected sieve-tube exudate from C. maxima (the exudate is easy
to collect) and mixed it with concentrated aphid saliva from
A. pisum (having a known salivary proteome) or M. euphorbiae (a natural pest on cucurbits). Sieve-tube protein degradation was followed by polyacrylamide gel electrophoresis.
Analysis of SDS–PAGE by calculation of trace quantity
shows that the protein band intensity of the PP2 monomer
(PP2mm) did not decrease with time when phloem exudate
536 | Furch et al.
was mixed with saliva from M. euphorbiae and A. pisum,
respectively (Fig. 2A, B), indicating that lectins are difficult to degrade. This interpretation finds support in the fact
that artificially fed lectin was found intact in honeydew of
A. pisum (Rahbé et al., 1995), implying that gut proteases are
unable to digest lectins (Rahbé et al., 1995). In contrast to
controls with aphid diet (Fig. 2C), the intensity of PP1 bands
declined with time in the presence of aphid saliva.
High degradation rates of PP1 occurred with saliva from
M. euphorbiae (Fig. 2A) as a possible adaptation to its natural
host. Less degradation was observed with saliva from A. pisum
(Fig. 2A), that does not feed on Cucurbitaceae. Degradation
of PP1 may prevent formation of protein plugs (Furch et al.,
2009) because PP1 and PP2 need to be cross-linked for plug
formation (Read and Northcote, 1983a), whereas PP1 is the
primary structural protein (Walker and Thaine, 1971; Walker,
1972). Another option is that M. euphorbiae degrades the
plugs after their formation, which would require a high proteolytic activity. This mode of action concurs with the observation that M. euphorbiae induces sieve-tube occlusion in
V. faba, but resumes ingestion some time after sieve-element
penetration (Medina-Ortega and Walker, 2014).
Are proteins in sieve-tube sap protected
from degradation by salivary proteases?
Given the protease activity in saliva of A. pisum (Figs 1,
2B) and M. euphorbiae (Fig. 2A), sieve-tube proteins seem
Fig. 2. Proteolytic degradation of sieve-tube proteins from Cucurbita maxima by aphid saliva. To test the proteolytic activity of aphid watery saliva from
M. euphorbiae (A) and A. pisum (B) on sieve-tube proteins of C. maxima (21–28 d after germination), 5 μl of saliva from ~4000 aphids per species was
mixed with 0.5 μl of sieve-tube sap. Total protein amount per sample was ~4.2 μg for M. euphorbiae and 8.5 μg for A. pisum (FluoroProfile Protein
Quantification Kit, Sigma-Aldrich). Sieve-tube sap was sampled according to a protocol described by Zimmermann et al. (2013). The samples were
incubated for 0, 60, and 120 min, respectively, in a Thermomixer Comfort (Eppendorf, Hamburg, Germany) at 37 °C and 1000 rpm (Williams et al., 1994),
and were directly frozen in liquid nitrogen and stored subsequently at –80 °C. As a negative control (C), phloem sap was mixed with artificial diet, used for
saliva collection (Will et al., 2007). Samples of pure sieve-tube sap (PS) were taken as the reference in all three treatments. One-dimensional SDS–PAGE
of the samples was carried out according to Laemmli (1970) by using a 4% stacking gel and a 12% separation gel in a MiniProtean 3 Electrophoresis
System (Bio-Rad Laboratories, Hercules, CA, USA) with Precision Plus Protein Standard-All Blue (Bio-Rad) as a protein size marker. SDS–polyacrylamide
gels were stained with colloidal Coomassie Blue (Roti-Blue; Carl-Roth, Karlsruhe, Germany), documented with the Gel Doc XR system (Bio-Rad), and
analysed by means of Quantity One 1-D analysis software (Bio-Rad). Three replicates were made for each aphid species (A. pisum and M. euphorbiae)
and the negative control. The trace quantity, the quantity of a band as measured by the area under its intensity profile curve, of PP1 (*), PP2 dimer
(PP2dm; **), and PP2 monomer (PP2mm; ***), was calculated. Trace quantity of pure sieve-tube sap samples was defined as 100% and the percentage
of trace quantity of further samples was calculated. Units are intensity/mm.
Aphid salivary proteases degrade sieve-tube proteins | 537
to be prone to degradation as a result of aphid infestation.
An intact set of proteins is mandatory for the fulfilment of
the entire spectrum of tasks of sieve tubes (e.g. van Bel et al.,
2011, 2014), which calls for protection from proteolysis.
Glycosylation could be one of the tools used for protection.
Glycosylation is a post-translational modification, responsible for correct folding (Pfeil, 2002) and stabilization of the
tertiary structure of many proteins (Wyss et al., 1995). There
is evidence in favour of preventive measures against proteolysis (Russel et al., 2009). O-linked β-N-acetylglucosamine
(O-GlcNAc)-glycosylated proteins were detected in the sievetube sap of C. maxima by labelling with an anti-O-GlcNAc
monoclonal antibody (Taoka et al., 2007). In-gel detection
revealed several proteins with low glycosylation labelling and
an intensively labelled protein with a molecular weight of
~28 kDa, most probably PP2.
Protease inhibitors may present an alternative or parallel
mode of protecting sieve-tube proteins. Protease inhibitors of
sieve-tube sap were reported to protect plants against phytophagous insects (Christeller et al., 1998; Ryan, 2000; Walz
et al., 2004). An appreciable set of different protease inhibitors, such as phloem-serpin-1, aspartic, tryptic, and chymotryptic protease inhibitors, and cystatin, which all act against
different types of proteases, were found in the sieve-tube sap
of Cucurbitaceae (Walz et al., 2004). Infestation by the aphid
species Schizaphis graminum increased transcripts of several
protease inhibitor genes in Sorghum (Zhu-Salzman et al.,
2004). Moreover, infestation with M. euphorbiae or M. persicae increased protease inhibitor-I and -II transcripts in
tomato (De Ilarduya et al., 2003). In poplars, Kunitz trypsin
inhibitor 3 expression is increased as a response to aphid colonization (Voelckel et al., 2004). This on-demand expression
of protease inhibitors potentially enables a flexible reallocation of resources inside the plant.
In conclusion, plants appear to possess mechanisms to
protect sieve-tube proteins from degradation. At the same
time, degradation experiments (Fig. 2) show that plant
defence mechanisms such as protein glycosylation and protease inhibitor production may fall short for full protection
from proteolysis by saliva proteases at an initial state of aphid
infestation. Degradation of sieve-element proteins may therefore be a dynamic balancing act between proteolytic attack
and defence.
Acknowledgements
TW was funded by the Justus-Liebig-University via the programme
‘Forschungsförderung
für
Nachwuchswissenschaftlerinnen
und
Nachwuchswissenschaftler’. We thank Dr Tom Wilkinson (School of
Biology, University College Dublin, Ireland) for the aphid clone A. pisum
LL01. Furthermore, we thank Christina Holschuh for excellent technical
assistance with the experiments conducted, and Matthias R. Zimmermann
for support in literature research.
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