The targets of aphids are the sieve elements, nutrient-rich cells in plants. As explained above, puncturing the sieve tubes implies that the sieve elements are damaged by sudden impalement of the stylet. Damage of sieve elements provokes a dual sieve-plate occlusion mechanism in dicotyledons that can extend over considerable distances from the site of wounding inside a plant (Furch et al., 2007, 2010; van Bel et al., 2014). An immediate sealing by protein plugs as a first response is followed by a slower deposition of callose along the sieve pores (Furch et al., 2007, 2008, 2009). Both reactions are Ca²⁺-dependent and respond to an increase of Ca²⁺ in the sieve-element lumen (Knoblauch et al., 2001, 2003, 2012; Furch et al., 2007, 2009; Hafke et al., 2009). Ca²⁺ increase may arise from influx of cell-wall Ca²⁺ via the imperfectly sealed wound inflicted by the stylet (Will and van Bel, 2006) or via Ca²⁺ channels in the sieve-element plasma membrane or in the sieve-element ER-membranes (Buchen et al., 1983; Sjolund and Shih, 1983; Furch et al., 2009; Hafke et al., 2009). Ca²⁺ influx further away from the site of wounding is triggered by electro-potential waves propagating along the sieve elements (Hafke et al., 2009; van Bel et al., 2014). Sieve-element depolarisations spread into adjacent cells (Rhodes et al., 1996; van Bel et al., 2014), which might explain the occlusion of neighboring sieve tubes during feeding of the generalist aphid species Macrosiphon euphorbiae and M. persicae (Medina-Ortega and Walker, 2015).

Sieve-tube occlusion by protein plugs was observed in legume sieve tubes that contain forisomes, giant spindle-like protein complexes (e.g., Lawton, 1978; van Bel and van Rijen, 1994; Knoblauch and van Bel, 1998). Forisomes, which seem to be composed of distinctly demarcated subunits (Schwan et al., 2009; Müller et al., 2014), expand in response to wounding and fully occlude the sieve tubes (Knoblauch et al., 2001, 2012). Their expansion is reversible (Knoblauch et al., 2001): forisomes re-condense after some time (30 min to a few hours) provided that the sieve elements are not severely damaged (Furch et al., 2007, 2008, 2009). Forisome expansion is induced by Ca²⁺ influx into the sieve elements (Knoblauch et al., 2001, 2003). It occurs, if the Ca²⁺ level in the sieve elements that is usually extremely low (50 nM; Furch et al., 2009), surpasses a certain threshold, most likely at membrane-located Ca²⁺ hotspots in sieve elements (Hafke et al., 2009). These hotspots arise, since Ca²⁺ ions do not quickly diffuse away from the Ca²⁺ channel apertures and tend to accumulate at sites where Ca²⁺ channels are aggregated. Ca²⁺ hotspots have strong local impact. Ca²⁺ hotspots visualized in transfer cells, for instance, co-localized with cell-wall apposition giving rise to cell wall protuberances (Zhang et al., 2015). The reversibility of the forisome dispersion may depend on the activity of Ca²⁺ pumps in sieve-element membranes as observed in other cell types (Kudla et al., 2010; Huda et al., 2013).

Forisomes are conglomerates of several types of SEO-(sieve-element occlusion) proteins (Noll et al., 2007; Pélissier et al., 2008; Tuteja et al., 2010; Rüping et al., 2010; Ernst et al., 2012; Zielonka et al., 2014; Müller et al., 2014). Remarkably, no known Ca²⁺-binding motifs were found so far in legume SEOs (Tuteja et al., 2010; Srivastava et al., 2016), but the pea forisome protein PsSEO-F1 showed a cleat-cut Ca²⁺-binding capability (Srivastava et al., 2016) so that identification of Ca²⁺ binding motifs in SEO protein structures may be a matter of time.

Other members of the SEO-protein family occur in a non-crystalline filamentous form in plant families other than legumes (Rüping et al., 2010; Anstead et al., 2012). Whether these SEO proteins present as heterodimers (Anstead et al., 2012; Jekat et al., 2013) bind Ca²⁺ ions and occlude the sieve plates in an aggregated state is a matter of debate. That mass flow is not blocked by dense aggregates of SEO-proteins near the sieve plates of intact A. thaliana plants was taken as evidence that filamentous SEO proteins lack occlusion properties (Froelich et al., 2011). However, this conclusion seems over-hasty, because the occluding capacities of SEO proteins only come to light, when the sieve tubes are damaged. Similar experiments with damaged A. thaliana (Jekat et al., 2013) and tobacco (Ernst et al., 2012) plants indicate SEO-mediated sieve-plate occlusion. Moreover, aphid feeding, recorded by using the electrical penetration graph technique, showed an instantaneous interruption in response to remote burning in a range of plant species. This reaction suggests that protein-mediated sieve-tube occlusion also occurs in plant species lacking forisomes (Will et al., 2009).

Not every protein clogging event in sieve tubes is Ca²⁺ dependent. In cucurbits, phloem protein 1 (PP1) and phloem protein 2 (PP2) aggregate to insoluble polymeric plugs in response to oxidation leading to sieve-pore occlusion in cut sieve tubes (Kleinig, 1975; Alosi et al., 1988; Golecki et al., 1998). Within a couple of minutes, interaction between PP1 and PP2 seals the cut surface of cucurbit plants by exudate gelling (Clark et al., 1997; Zimmermann et al., 2013). A similar coagulation of phloem exudate was observed shortly after excision of stylets from aphids feeding on V. faba (Fisher and Frame, 1984). It is hard to conceive that this gelling is due to dispersing forisomes, since their number is likely to be extremely low in the exudate (none to a few). Hence, the phenomenon infers oxygen-sensitive protein gelling in broadbean sieve-tube sap as well (Arsanto, 1986).

Oxygen-stimulated linkage of PP1 and PP2 may be corroborated by Ca²⁺-binding sites. Cucurbit sieve-tubes occlude several centimeters away from a site of burning (Furch et al., 2010). The distinct decrease in soluble PP1 and PP2-dimers in sieve elements there (Furch et al., 2010) is supportive of PP1–PP2 cross-linking. Latter may depend on a longitudinal electric potential wave evoked by burning (Furch et al., 2010). The electric potential wave induces a rise in Ca²⁺ concentrations along the sieve tubes (Furch et al., 2009). PP1 is a candidate for Ca²⁺-mediated sieve-element occlusion (Furch et al., 2010) in view of its potential Ca²⁺-binding sites (McEuen et al., 1981; Arsanto, 1986). PP2 is abundantly present in the sieve-tubes of A. thaliana (Batailler et al., 2012), but interaction with environmental factors remains unclear. Simultaneously, the Ca²⁺ wave may enhance the level of reactive oxygen species in sieve elements (Görlach et al., 2015; Evans et al., 2016). As yet it is difficult to discriminate between the potential cues of PP1–PP2 cross-linking there. Is either the rise in Ca²⁺ or ROS or is it a collaborative action?

Obviously, sieve-tube occlusion endangers food ingestion by aphids, because their nutrients are transported by mass flow through the sieve tubes. Sequestration of Ca²⁺ ions would prevent sieve-plate occlusion and preserve mass flow. Based upon this idea, it has been postulated that aphid saliva contains Ca²⁺-binding proteins (Will and van Bel, 2006). Ca²⁺-binding properties of aphid saliva were substantiated by experiments using the in vitro Ca²⁺ reactivity of forisomes from V. faba (Knoblauch et al., 2001, 2012; Will et al., 2007). Concentrated aphid saliva, as well as EDTA, showed similar condensation effects on isolated dispersed forisomes (Will et al., 2007). It has been argued that these in vitro experiments using salivary probes and forisomes (Will et al., 2007) lead to an enormous overestimation of the Ca²⁺-binding capacity of saliva proteins. It is difficult to rebut this critical point using quantitative arguments. The actual saliva concentration is probably much lower in sieve tubes, punctured by one or a few aphids. On the other hand, Ca²⁺ influx brought about by a single stylet puncture is much lower than the amount of Ca²⁺ ions administered in the in vitro forisome experiments (Will et al., 2007). Moreover, Ca²⁺ can be readily sequestered, since saliva is being secreted near the site of puncture.

Separation by SDS-PAGE and selective staining and radio-labeling disclosed the presence of several Ca²⁺-binding proteins in the saliva of M. viciae (Will et al., 2007). Later proteomic analysis of saliva from A. pisum showed the presence of regucalcin, a Ca²⁺-binding protein involved in Ca²⁺ homeostasis (Carolan et al., 2009). Regucalcin sequesters Ca²⁺ and activates Ca²⁺ pumps (Yamaguchi, 2005). This 43-kDa protein belongs to the SMP-30 superfamily and coincides in molecular size with a 43-kDa Ca²⁺-binding protein identified by one-dimensional SDS-PAGE in the saliva of M. viciae (Will et al., 2007). Regucalcin was not found in the saliva of cereal aphids (Rao et al., 2013), which once again illustrates the diversity of saliva between the aphid species.

The second Ca²⁺-binding protein, detected in A. pisum saliva, was an ARMET protein (Wang et al., 2015a) that was identified by proteomic analysis (Carolan et al., 2011). ARMET proteins are associated with the unfolded protein response (UPR) in ER stacks (Hampton, 2000; Apostolou et al., 2008). It was argued that salivary protein concentrations in sieve-tube sap are generally low, so that Ca²⁺ sequestration by salivary ARMET should be highly localized (Wang et al., 2015a). As sieve-element wounding is likely correlated with Ca²⁺ release from ER compartments via CICR-channels (Hafke et al., 2009; Evans et al., 2016), interference with Ca²⁺ trafficking at the sieve-element ER-membranes (Hafke et al., 2009) may be a feasible option for ARMET functioning.

Recently, a third potential mode of lowering the Ca²⁺ concentration in sieve elements by salivary proteins became apparent (Sinha et al., 2016). Ca²⁺ release from the sieve-element ER stacks (Buchen et al., 1983; Sjolund and Shih, 1983; Furch et al., 2009; Hafke et al., 2009) likely contributes to increased mictoplasmic Ca²⁺ levels in response to aphid attack. Mastoparan treatments of staminal hairs of Setcreasea purpurea demonstrated that IP₃-activated Ca²⁺ channels appear to be relevant for Ca²⁺ efflux from the ER compartments (Tucker and Boss, 1996), where the Ca²⁺ concentration is at least 10,000 times higher (Montero et al., 1995) than in the sieve-element lumen (Furch et al., 2009). As a speculation, an interference of salivary proteins with the phosphoinositide metabolism, would lead to a reduced IP₃ production, and thus would suppress the release of Ca²⁺ ions into the sieve-element lumen. D. noxia biotype-2 aphids that overexpress proteins inhibiting the key enzyme phospopholipase C, relevant for IP₃ formation, are virulent on aphid-resistant wheat plants (Sinha et al., 2016). However, it should be noted that, in contrast to overwhelming evidence for IP₃ involvement in Ca²⁺ release in animal cells, the presence of cytosolic IP₃ in plants has not been convincingly demonstrated as yet (Kudla et al., 2010).

The presence of Ca²⁺-binding proteins is not limited to aphids. An EF-hand motif – a helix-loop-helix structural domain in a large family of Ca²⁺-binding proteins – was found in a salivary protein of the green rice leafhopper Nephotettix cinticeps (Hattori et al., 2012), so that Ca²⁺-binding properties of saliva may be universal among plant sucking Hemiptera.

Ca²⁺ sequestration by salivary proteins with the objective to suppress sieve-tube occlusion may be wide-spread among aphid species. The sudden change in feeding behavior in response to remote heat shocks in diverse plant-aphid combinations (Will et al., 2009) hints at intensified saliva secretion to counteract imminent sieve-element occlusion. As a speculation, binding of Ca²⁺ by salivary proteins can be regarded as an adaptation of specialized feeders like A. pisum to those plants that highly rely on Ca²⁺-based occlusion. Penetration of A. pisum stylets did not trigger sieve-tube occlusion in V. faba (Walker and Medina-Ortega, 2012). This lack of reaction has been ascribed to the high specialization of pea aphids to legumes (Medina-Ortega and Walker, 2015). The saliva would sequester Ca²⁺ to such an extent that condensed forisomes are not sufficiently challenged (Will, 2016b). The latter conclusion was drawn, since feeding of generalists like M. persicae and M. euphorbiae indeed triggered forisome-mediated occlusion (Medina-Ortega and Walker, 2015). It is no surprise that Ca²⁺-channel blockers favored feeding of M. persicae by preventing sieve-element occlusion (Medina-Ortega and Walker, 2015), since the Ca²⁺-channel blocker gadolinium prevented forisome dispersion in sieve-element protoplasts (Hafke et al., 2007).

The second slower type of sieve-tube occlusion by callose partly overlaps in time with protein occlusion (Furch et al., 2007) and is executed by β-1,3 glucan depositions around the sieve plate pores, termed “callose plugs” (van Bel, 2005). These “plugs” reside unlike protein plugs in the extracellular space and are, in fact, callose collars apposed against the cell-wall areas bordering the plasma membrane corridor that crosses the sieve pores (Evert, 1990; Ehlers et al., 2000). As result of callose deposition, the mictoplasmic corridors between sieve elements become “strangled” and phloem transport stops. It should be noted that callose is not always deposited in the extracellular space. Intracellular callose plugs of plasmodesmata are delivered by multivesicular bodies as an early step in the hypersensitive response (An et al., 2006). Furthermore, callose plugs inside plasmodesmal corridors are involved in dormancy processes (Rinne et al., 2001).

Since sieve pores are evolutionary and ontogenic “descendants” of plasmodesmata (Evert, 1977, 1990), one may expect that fundamental traits such as callose homeostasis have strong similarities between both structures and that plasmodesmal physiology might tell us much about sieve-pore physiology. Unlike the continuous callose collars in the sieve pores, plasmodesmata have a ring of callose around both neck regions (sphincters) of the cytoplasmic corridors that connect neighboring cells (Radford et al., 1998). The permanence of callose rings around the sphincters of standard plasmodesmata is a matter of debate (Levy and Epel, 2009). In sieve elements, callose depositions – necessary for the formation of sieve pores and PPUs (Evert, 1977, 1990; Barratt et al., 2011) – permanently surround the mictoplasmic corridors (through the sieve pores) between sieve elements after maturation (Ehlers et al., 2000). Plasmodesmal fluxes are inversely correlated with the degree of callose deposition (e.g., Zavaliev et al., 2011; Tilsner et al., 2016), which suggests a strong resemblance with sieve-pore functionality.

According to recent reviews (Zavaliev et al., 2011; De Storme and Geelen, 2014; Kumar et al., 2015; Tilsner et al., 2016), callose deposition results from an equilibirum imposed by the activities of plasma transmembrane β-1,3 glucan synthases or callose synthases (GSLs) and plasma-membrane anchored extracellular β-1,3 glucanases or glucan endo-1,3-hydrolases (BGs).

Callose synthases present a multigene family of large (200–220 kDa) plasma-membrane spanning proteins with both the N- and C-terminus residing in the cytoplasm (Farrokhi et al., 2006; Brownfield et al., 2007). They are typically clustered in two transmembrane regions connected by an extensive cytoplasmic loop, including an UDP-glucose catalytic site and a glysosyltransferase domain (see for reviews: De Storme and Geelen, 2014; Tilsner et al., 2016). Together with the N-terminal region, the hydrophilic loop acts as a site for interaction with regulatory molecules due to several glycosylation and phosphorylation domains (Verma and Hong, 2001). Callose synthases are probably incorporated into complex protein structures (CalS holoenzyme complexes; Amor et al., 1995; Hong et al., 2001), that include several proteins participating in the polymerization of the substrate UDP-glucose and the delivery of glucan chains into the apoplasmic space. Three genes were found to be directly associated with plasmodesmal callose deposition. Most likely, GSL 8 is responsible for callose deposition in plasmodesmata in a wide variety of tissues (Guseman et al., 2010), GSL7 is involved in sieve-pore shaping during sieve-element development (Barratt et al., 2011; Xie et al., 2011), and GSL12 has a major role in adjusting the functional diameter of plasmodesmata (Vaten et al., 2011). Their distribution over the cell types seems to indicate that all three genes are engaged in callose homeostasis in sieve elements.

Glucanases are the functional counterpart of callose synthases. In Arabidopsis, the glucanase family comprises 50 representatives of β-1,3-glucan-degrading enzymes (Doxey et al., 2007). Thus far, three of them were found to be associated with plasmodesmal regions and were characterized: one in leaves (Zavaliev et al., 2013) and two in roots (Benitez-Alfonso et al., 2013). They are likely anchored by a C-terminal glycophosphatidylinositol (Gaudioso-Pedraza and Benitez-Alfonso, 2014) to the outer leaflet of the plasma membrane and thus face the inner side of the cell wall. β-1,3-glucanases are produced in the Golgi system and secreted into the apoplasmic space via exocytosis (De Storme and Geelen, 2014). In conclusion, it is of paramount importance to note that the regulation of callose deposition is located in entirely different cell compartments. Callose synthases operate in the cytosol, glucanases in the extracellular space.

Pioneer experiments of Erwee and Goodwin (1983) demonstrated that the plasmodesmal diameter is under the control of Ca²⁺ ions. A rise in Ca²⁺ concentration, probably from the ER stores (Tucker, 1988; Tucker and Boss, 1996), coincides with enhanced callose deposition (Tucker and Boss, 1996; Holdaway-Clarke et al., 2000) and, hence, increased obstruction of symplasmic transports (Sager and Lee, 2014). As a result, abrupt changes in intercellular Ca²⁺ concentration have an immediate effect on intercellular communication. The molecular mechanisms that control this crucial Ca²⁺ effect on callose synthesis has yet to be thoroughly investigated (Zavaliev et al., 2011; De Storme and Geelen, 2014; Kumar et al., 2015; Tilsner et al., 2016).

Similar Ca²⁺-controlled mechanisms are supposed to regulate mass flow through sieve tubes (Kauss, 1987). The uneven deployment of Ca²⁺ channels along sieve elements renders credence for a strong relationship between Ca²⁺ level and callose deposition (Furch et al., 2009). The modified plasmodesmata of sieve elements (sieve pores and pore-plasmodesm units or PPUs, the symplasmic connections with the companion cells) possess constitutive callose linings (Evert, 1990; Ehlers et al., 2000). Sieve pores and PPUs perfectly co-localize with Ca²⁺ hotspots (Furch et al., 2009; Hafke et al., 2009; see section Ca²⁺-binding proteins for a hotspot definition). Aggregates of Ca²⁺ channels along the sieve-element plasma membrane allow changes in Ca²⁺ level focused at sites, where they are physiologically relevant for immediate action such as callose deposition and protein dispersion (Hafke et al., 2009). Such a phenomenon is by no means unique. In developing transfer cells, cytosolic Ca²⁺ plumes co-localize with the deposition of cell-wall protrusions, which may consist in part of callose to provide a plastic matrix for embedment of stiffer cell-wall components (Zhang et al., 2015).

In disturbed, yet undamaged sieve elements, supplementary callose depositions at sieve pores and PPUs disappear within a couple of hours after disturbance (Furch et al., 2007, 2010). The quick build-up and slower breakdown of callose in sieve elements raises questions regarding the control mechanisms. The basic level of callose deposition may be balanced by the counteraction of callose synthases and glucanases (Zavaliev et al., 2011; De Storme and Geelen, 2014; Kumar et al., 2015).

The rapidity of callose build-up in response to wounding (seconds to minutes) suggests a regulation on the protein level. Ca²⁺ released into the sieve element mictoplasm may readily bind to the CalS complex (Hong et al., 2001) with the putative participation of ATP and calmodulin (Levy and Epel, 2009) under favorable redox conditions (see Zavaliev et al., 2011). As a speculation, a rapid rise in intracellular Ca²⁺ will boost the activity of the callose synthases to an extent that cannot be counteracted by glucanase activity and additional callose will be deposited. If however, Ca²⁺ is removed from the local cytosol to the ground level as will happen in undamaged sieve elements (Furch et al., 2007), glucanases have the chance to reduce the callose layer to the original thickness in sieve elements.

It is possible that callose synthases and glucanases are upregulated at the transcript level during peaks of callose turnover. It should be realized, however that the situation in enucleate sieve elements differs radically from that in nucleate cells. Gene expression of callose synthases and glucanases must take place in cells adjacent to the sieve elements, most probably the companion cells, from which the glucanases are released into the apoplasmic space by exocytosis (De Storme and Geelen, 2014).

In conclusion, the previous considerations infer altogether at least two potential modes of callose suppression by aphid saliva. As callose sealing is considered to be a general, but quantitatively species-specific (Saheed et al., 2009) defense mechanism against aphids and other sucking insects (Hao et al., 2008), Ca²⁺ sequestration by salivary proteins may enable the aphids to prevent callose deposition by suppression of Ca²⁺-stimulated callose-synthase activity. When thick callose depositions are found in sieve tubes as a response to aphid attack, Ca²⁺ sequestration has likely been insufficient to prevent callose synthesis (Saheed et al., 2009). Ca²⁺-binding proteins may also disturb the equilibrium between callose build-up and degradation so that stimulation of glucanase action is conceivable. Breakdown of callose depositions by salivary glucanases is unlikey, since they would be introduced into the luminal compartment of sieve elements, out of reach of the callose located in the cell-walls. It is much more logical; therefore, that callose breakdown is facilitated by upregulation of plant glucanases (Van der Westhuizen et al., 1998) probably making use of salivary effectors. In keeping with this idea, infestation with R. padi induced a transcript abundance of three β-1,3-glucanases in 15 barley breeding lines (Mehrabi et al., 2016). In Mp55-expressing Arabidopsis plants, callose deposition in response to aphid infestation is reduced (Elzinga et al., 2014), but the molecular action of Mp55 is yet to be identified.

The presence of proteases in the alimentary tract of aphids is an established fact (Rahbé et al., 1995; Cristofoletti et al., 2003, 2006; Pyati et al., 2011). It was suspected for a long time that aphid saliva as well contains several proteases, although the first functional assays were not successful (Cherqui and Tjallingii, 2000). Some proteases were recently identified using proteomics on aphid saliva and salivary gland extracts of A. pisum (Carolan et al., 2009, 2011). In mammalian systems, the M1 zinc metalloprotease and M2 metalloproteases (angiotensin-converting enzymes, ACEs; Wang et al., 2015b) bind zinc at their catalytic domain (Lausten et al., 2001; Kim et al., 2003), while Ca²⁺ regulates their catalytic activity (Goto et al., 2007). ACEs remove dipeptides from short oligopeptides (Ehlers and Riordan, 1989) which contrasts the action of M1 metalloproteases cleaving the terminal amino acids from proteins (Itturioz et al., 2001; Naqvi et al., 2005). It is not excluded that salivary proteases are species-specific in aphids: the metalloproteases detected in A. pisum (Carolan et al., 2009, 2011) were not found in the saliva of S. avenae and M. dirhodum (Rao et al., 2013). Like Ca²⁺-binding proteins, metalloproteases were also found in the saliva of other insect groups such as the phytophagous thrips Frankliniella occidentalis (Stafford-Banks et al., 2014) and the blood-feeding tick Ixodes scapularis (Francischetti et al., 2003; Decrem et al., 2008).

The putative proteolytic activity of saliva was verified by two functional assays (Furch et al., 2015). Albumin was degraded by salivary probes of A. pisum in the presence or absence of EDTA. Rapid albumin breakdown, in particular in the absence of EDTA, indicated the involvement of metalloproteases in protein degradation (Furch et al., 2015). Mixing salivary probes of A. pisum and M. euphorbiae, respectively, with sieve-tube exudate of Cucurbita maxima demonstrated that PP1 and PP2 in the protein-rich exudate were degraded with time (Furch et al., 2015). This is supportive of protease-mediated breakdown of sieve-element proteins by salivary proteases. PP1, the protein specialized in occlusion, was more rapidly broken down than PP2, which appeared to be recalcitrant to breakdown by proteases in the alimentary tract (Rahbé et al., 1995). In conclusion, proteases may act as an auxiliary tool to remove proteinaceous occlusions.

A third functional aspect of protease activity pertains to aphid nutrition (Carolan et al., 2009; Furch et al., 2015). As sieve-tube sap is poor in several essential amino acids (Gündüz and Douglas, 2009), aphids are mostly short of these indispensable compounds. The deficiency is compensated by endosymbiotic bacteria that transform non-essential into essential amino acids (Baumann et al., 1995). Amino acids from degraded proteins in sieve tubes could enhance the aphid’s diet quality. The breakdown strategy may be more successful in dicots than in monocots given the higher protein contents in sieve-tube sap of dicots (2–100 mg ml^-1: Richardson et al., 1982; Schobert et al., 1998; Zimmermann et al., 2013) as compared to monocots (0.1–1.0 mg ml^-1: Fisher et al., 1992; Schobert et al., 1998; Gaupels et al., 2008).

It is unclear, if protein breakdown by salivary proteases merely has a non-selective character as indicated by in vitro breakdown of proteins by salivary proteases (Furch et al., 2015). In view of the presence of so many other vital proteins in the saliva, however, additional selective protein-breakdown machinery seems logical. Selective protease activity is usually associated with ubiquitin tagging accompanied by several accessory enzymes (van der Hoorn, 2008) and the final transfer of the ubiquitin-protein complex to the proteasome. The question arises, whether ubiquitin as a first indicator of selective protein degradation occurs in sieve elements or aphid saliva. After the first detection of ubiquitin in Ricinus communis sieve-tube exudate (Schobert et al., 1995), proteomics disclosed the presence of at least 116 components involved in proteasome-associated protein degradation in the sieve-tube sap of cucurbits (Lin et al., 2009) This indication for a complete proteolytic system in sieve elements was consistent with the presence of ubiquination compounds in the sieve-tube sap of rice (Aki et al., 2008) and rapeseed (Giavalisco et al., 2006) and the likeliness of proteasomes in sieve elements (Ingvardsen and Veierskov, 2001). In contrast, native ubiquitin nor proteasomal components have not been found in aphid saliva thus far (Chaudhary et al., 2015). Should selective protein degradation be executed by salivary proteases, they likely make use of the sieve-element breakdown machinery.

The presence of polyphenoloxidases (Peng and Miles, 1988; Miles and Oertli, 1993; Madhusudhan and Miles, 1998; Urbanska et al., 1998; Cherqui and Tjallingii, 2000; Harmel et al., 2008; Cooper et al., 2011; Chaudhary et al., 2015) and peroxidases in saliva (Miles and Peng, 1989; Cherqui and Tjallingii, 2000; Chaudhary et al., 2015) is interpreted as a reductive weapon against plant phenols and reactive oxygen species (Leszczynski et al., 1985; Moloi and van der Westhuizen, 2006). A similar function has been attributed to oxidoreductases (Miles and Oertli, 1993) and GMC-oxidoreductases (Carolan et al., 2009; Nicholson et al., 2012; Rao et al., 2013). Given the differences in phenol localization (epidermis, cortex, mesophyll, sieve tubes) and phenol quantities between plant species, some of the oxidases may be most effective against toxic substances along the stylet pathway and others against phenols or other toxic substances residing inside the sieve tubes. Peroxidases may also play a role in the breakdown of hydrogen peroxide, a booster of Ca²⁺-channel gating (Lecourieux et al., 2006), and hence may reduce the occlusion of sieve tubes. The same may be true for GMC-oxidoreductases, which possess ROS (reactive oxygen species)-scavenging properties. However, the low pH optima of these enzymes raise some reserve as for their efficiency in view of the alkaline sieve-tube milieu (Will, 2016b). The ROS-scavengers possibly interact with the native ROS-scavenging system in sieve tubes (Walz et al., 2002).

Evidence has emerged that the redox status impacts on callose synthesis. In Arabidopsis mutants defective in the thioredoxin-m3 gene (TRX-m3), GFP diffusion out of the sieve elements was reduced in comparison with wild-type plants. These mutants accumulated ROS and contained higher levels of callose in root tips (Benitez-Alfonso et al., 2009). Overexpression of TRX-m3, by contrast, resulted in enhanced intercellular transport compared to wild-type plants. Moreover, reduced macromolecular trafficking in wild-type plants treated with oxidants (Benitez-Alfonso et al., 2009) and in Arabidopsis mutants defective in the production of glutathione (Cairns et al., 2006) inferred that oxidants stimulate callose production. Consequently, oxidoreductases and other ROS scavengers in aphid saliva may limit the cellular damage caused by ROS (e.g., lipids and proteins), but also impair callose production by withdrawal of oxidants. This conclusion may be premature as in Arabidopsis ise1 mutants in which the ROS level exceeds that in wild-type plants, intercellular transport is stimulated (Stonebloom et al., 2009). It seems beyond doubt that redox homeostasis regulates intercellular trafficking probably by interaction with callose metabolism. It is unclear, whether ROS directly act upon callose deposition or if Ca²⁺ channels are gated by ROS as demonstrated for hydrogen peroxide (Lecourieux et al., 2006).

Salivary dehydrogenases and glucose oxidases possibly interfere with plant defense systems regulated by jasmonic acid (JA) and salicylic acid (SA) during aphid infestation (Louis and Shah, 2013). The suppressed JA-controlled defense responses of A. thaliana to Brevicoryne brassicae infestation (Kusnierczyk et al., 2011) could be due to a diminished production of JA (Takemoto et al., 2013) conferred by the above-mentioned enzymes. It is unclear, if the aphid-imposed modulation of genes engaged in SA-synthesis (Zhu-Salzman et al., 2004) depends on the cross-talk with the JA pathway (Pieterse et al., 2012) or on so-called salivary effectors.

Chaudhary et al. (2015) detected several other defense modulators in the saliva of M. euphorbia, mainly interfering with regulatory enzymes of the oxidative burst, a hallmark in plant immunity. In addition, lipase-like proteins in the same secretome may function as virulence factors to promote aphid colonization (Chaudhary et al., 2015).

In contrast to the protein classes discussed above, the majority of salivary proteins may not interfere in a direct disruptive manner with events in cell walls or inside sieve elements. They probably interact in an unknown fashion with the protein network involved in plant defense. Effectors act in a broad spectrum of pathogens and are generally defined as molecules that alter function and/or structure of host cells (Hogenhout et al., 2009). This definition includes a group of proteins that effect on aphid fecundity and behavior. Their effect on suppression or elicitation of plant defense, respectively, are inferred from the rates of aphid colonization or fecundity (Bos et al., 2010; Pitino et al., 2011; Atamian et al., 2013; Elzinga and Jander, 2013; Pitino and Hogenhout, 2013; Rodriguez and Bos, 2013; Elzinga et al., 2014; Rodriguez et al., 2014; Chaudhary et al., 2015).

As a first salivary protein, C002 was termed “effector”. C002 discovered in the saliva of A. pisum and is required for continuous aphid feeding (Mutti et al., 2008). When C002 is transiently overexpressed in plants that were subsequently infested by M. persicae, aphid fecundity is increased (Bos et al., 2010). In reverse, suppression of C002 expression by RNA interference is lethal in A. pisum (Mutti et al., 2006) and causes reduced fecundity in M. persicae (Pitino et al., 2011). Interestingly, C002 is only required for feeding on plants, not on diets (Mutti et al., 2008), which implies a role in compatibilty. C002 may have significance for the host range of an aphid species. The ability of M. persicae to feed on a broad range of host plants may be due to the presence of a repeated 7-amino acid motif in C002 of M. persicae that is absent in C002 of A. pisum (Pitino and Hogenhout, 2013).

Aphid colonization is also promoted by two further salivary proteins detected in M. persicae (Pitino and Hogenhout, 2013). These effector proteins [PIntO1, (Mp1) and PintO2 (Mp2)] are acting in a plant species-specific manner (Pitino and Hogenhout, 2013). The effector Mp55 also suppresses plant defense. Mp55-expressing A. thaliana plants were more attractive to aphids in choice assays, whereas Mp55 silencing in aphids led to reduced reproduction rates (Elzinga et al., 2014).

Likewise, saliva of M. euphorbiae contains two proteins, Me10 and Me23 that promote aphid colonization, as they seem to suppress plant defense when expressed in Nicotiana benthamiana (Atamian et al., 2013). Me23 possesses a conserved glutathione peroxidase domain and might interfere with plant defense responses, while the function of Me10 is unknown (Chaudhary et al., 2015). They are both phosphorylated, but the molecular significance of these phosphorylation sites is elusive (Chaudhary et al., 2015). When expressed in tomato, only Me10 increased aphid fecundity underlining once again the species-specificity of effectors (Atamian et al., 2013), the more as expression of the Me10-homologue Mp58 resulted in a decreased fecundity in Nicotiana tabacum and Arabidopsis (Elzinga et al., 2014). As for the function of these proteins, the fundamental question remains as whether the increased fecundity of aphids is due to the suppression of plant defense or to a promoted efficiency of aphid feeding.

By contrast, some salivary proteins such as Mp10 and Mp42 from M. persicae suppress aphid reproductive performance and appear to elicit plant defense reactions (Bos et al., 2010). In planta overexpression of Mp10 and Mp42 in N. benthamiana reduced aphid feeding (Bos et al., 2010). Further work revealed that Mp10 and Mp42 are engaged in elicitation of plant defense in distinct ways at different subcellular locations. Transient overexpression activated JA and SA signaling pathways, while Mp42 did not (Rodriguez and Bos, 2013; Rodriguez et al., 2014).

Recently, a macrophage migration inhibitory factor (MIF) was identified in the saliva of A. pisum using 2D-DIGE-MALDI-TOF/MS (Vandermoten et al., 2014; Naessens et al., 2015) that sheds some light on effector action. In mammals MIFs are pro-inflammatory cytokins that regulate immune responses (Calandra and Roger, 2003). Of five members of a MIF multigene family in A. pisum (Dubreuil et al., 2014), just one (ApMIF1) is present in the saliva (Naessens et al., 2015; Reymond and Calandra, 2015). This protein was postulated to suppress the immune response of plants by inhibiting the expression of defense-related genes (Naessens et al., 2015).

Beside proteins that possess a secretory signal sequence and were classified as salivary components, a number of proteins that have been identified in salivary glands are lacking such a sequence (Carolan et al., 2011). It has been a matter of dispute, if such proteins belong to the salivary proteome. Recently, eleven proteins from Buchnera aphidicola proteins were detected in the saliva of M. euphorbia, four of which were attributed to gel saliva only (Chaudhary et al., 2014). One of the identified proteins was the chaperone GroEL, a heat shock protein engaged in protein folding, which is abundant in B. aphidicola (Baumann et al., 1996). Several GroEL expression studies in plants demonstrated that GroEL is recognized intra- and extracellularly and functions as a microbe associated molecular pattern (MAMP) triggering an oxidative burst and the expression of a number of immunity marker genes (Chaudhary et al., 2014). This suggests that GroEL has to be regarded as a biologically relevant contaminant that elicits plant defense. Aphids reacted to increased GroEL expression by diminished fecundity (Chaudhary et al., 2014; Elzinga et al., 2014). The investigators interpreted the plant defense response as being targeted to B. aphidicola, which impacts negatively on the reproduction of the aphids (Chaudhary et al., 2014). Removal of B. aphidicola activity indeed led to a delayed aphid development and a considerable decrease of reproduction rates (Sasaki et al., 1991).

It is of major importance to investigate the route followed by GroEL into the salivary glands. Such studies may give a clue, as if proteins without a secretory sequence are produced by the gland cells or are imported into the glands after being produced by other organs.