How phloem-feeding insects face the challenge of phloem-located defenses

1A key characteristic of higher-level plants (including monocots and dicots) is the existence of a vascular network that is composed of the phloem and xylem. The vascular system pervades the whole organism from root to shoot and distributes nutrients and water. The fact that sugars, amino acids, and other organic metabolites are available via phloem and xylem in significant amounts makes the vascular system a target for insect pests. Endogenous interference in the functioning of the vascular system may have disastrous consequences for a plant’s development. Using the interaction between plants and phloem-feeding insects as an example, offensive and defense strategies during the struggle for the valuable phloem content are explored.

2In higher-level plants, a long-distance transport system has evolved to translocate photoassimilates from a source (e.g., mature leaves) to a sink (e.g., roots) – the phloem. The angiosperm phloem is composed of sieve elements (SEs), companion cells (CCs), and phloem parenchyma cells (PPCs;; ). Mature SEs are elongate cells lacking certain cellular components (nucleus, vacuoles, ribosomes, and dictyosomes) but lined parietally with a thin mictoplasmic layer consisting of an endoplasmic reticulum (ER), plastids, a few inactive mitochondria and phloem-specific proteins (P-proteins; ). The CCs maintain SEs viability. A high density of pore-plasmodesma units (PPUs) and tight ER coupling between SE and CC underline an intimate symplastic connection across this boundary; the entire connection constitutes the SE-CC complex. The walls between the single SE-modules are transformed into sieve plates, perforated by plasmodesmata (PD) modified into sieve pores with a diameter up to 2.5 μm. These adaptations provide the basis of formation of long sieve tubes based upon single SE-modules, forming a tube-like symplastic continuum that serves translocation. The complex process of this translocation is regulated via highly active CCs.

3In general, the vascular system is a pressure system made up of two components (phloem and xylem) that effects the long-distance translocation of very heterogeneous constituents within higher-level plants. Xylem and phloem are parallel orientated vascular tissues in which pressure and tension gradients are built up in SEs and xylem vessels, respectively. In intact plants, the negative hydrostatic potential in xylem vessels is in balance with that inside SEs (e.g., ). The driving forces for translocation are, on the one hand, a longitudinal (axial) pressure gradient within phloem and xylem, and on the other, a lateral (radial) pressure gradient between the phloem and xylem. The balanced interaction between phloem and xylem is a basic requirement for long-distance transport. The longitudinal pressure gradient within the xylem is the result of water uptake in the root/rhizosphere and loss of water by transpiration. According to the classic Münch concept for phloem translocation, photoassimilates are amassed in the sieve tubes of source areas and escape from the sieve tubes in sink areas. The resulting turgor difference between source and sink drives the mass flow. Hence, in contrast to the xylem, the phloem exhibits a bidirectional translocation, as sink regions are found in the root and in the apex of the shoot.

4Phloem sap contains carbohydrates, proteins, and amino acids (Table 1) and makes SEs a favorite target for pathogens and pests (Figure 1A). An accession of SEs by pathogens/pests leads to varies impairments: (a) loss of nutrients, (b) disturbance of the translocation process, and (c) the infection by microbial pathogens (e.g., viruses, phytoplasmas, viroids;; ). Therefore, plants have evolved a range of defense mechanisms against pathogens and pests, which in turn possess mechanisms with which to counteract these defenses.

5Damage of sieve tubes disturbs the existing pressure conditions resulting in a turgor shift, which impact intracellular calcium levels and the redox state. Long-distance signaling by electropotential waves (EPWs) communicates local wounding to distant plant parts. This induces the occlusion of sieve tubes via the plugging of sieve pores and can be seen as primary defense response. Sieve-tube occlusion is assumed to prevent the loss of sieve-tube sap and to keep phytopathogens from invading through the injured site. Two groups of sieve-tube occlusion mechanisms can be found in plants: callose deposition and protein plugging (e.g.,; ).

6Callose is a linear β-1,3-glucan polymer with some 1,6 branches. β-1,3-glucans are produced as helical chains which, upon heating, form a gel. It is produced enzymatically in the presence of Ca2+ by callose synthases, located in the plasma membrane, and deposited extracellularly around sieve pores (Figure 1B) and PD in the form of collars as a reaction to chemical or mechanical stress. The pattern and timing of callose distribution and its physiological involvement suggest that callose plays an important part in plant cell growth and differentiation. In Arabidopsis, 12 callose synthase genes (CalS1–12) have been detected. CalS1 and CalS10 have been shown to be responsible for cell plate formation during cell division. CalS5, CalS9, CalS10, CalS11, and CalS12 play unique roles during microsporogenesis and pollination. CalS12 can be induced by pathogen infection and wounding, and CalS7 deposits callose in the phloem (Figure 1B; ). showed that callose deposition in the phloem, especially in the SEs, was greatly reduced in cs7 mutants. Callose accumulation was eliminated in the PDs of incipient sieve plates resulting in fewer sieve pores of cs7 mutants. They conclude that CalS7 is a phloem-specific callose synthase gene, and is responsible for callose deposition in developing SEs during phloem formation and in mature phloem induced by wounding. The physiological roles of the remaining CalS genes in Arabidopsis are still unknown.

7Callose deposition is a universally observed mode of sieve-plate occlusion. Reversible callose accumulation apparently plays a role in regulating cell-to-cell transport through sieve pores and PPUs. After a heat stimulus is applied to the leaf tip of Vicia faba plants, callose gradually builds up at sieve plates and PD. After reaching a maximum, callose is degraded at a lower rate than production. Callose appears to degrade more rapidly at PD (30–40 min) than at the sieve pores, where the level of callose deposition reaches its original state after 1–2 h. β-1,3-glucan endo-hydrolases is the enzyme that catalyses callose degradation (reviewed in ). It is present in large gene families in plants (e.g., 50 genes in Arabidopsis) and is located in the cell membrane and highly enriched at PD sites. The course of callose production/degradation is qualitatively similar between different plant species, but there are variations in the time scale.

8In addition to callose, sieve pores can be blocked rapidly by proteins. In electron microscopic images, SEs show protein networks that span the SE lumen and are attached to the cell periphery. Sieve tubes of grasses appear virtually empty but may have an occlusion mechanism based on precipitation of soluble proteins. A specific group of phloem proteins (P-proteins) enables rapid occlusion (within some seconds) to occur in sieve tubes of higher-level plants. Numerous aggregation forms (amorphous, granular, fibrillar, filamentous, tubular, or crystalline) of P-proteins that are thought to represent stages of P-protein differentiation denote an immense variation between plant species. The synthesis of P-proteins begins in immature, nucleate SEs, resulting in electron-dense proteinaceous structures. In young SEs, subunits accumulate within the cytoplasm, forming large P-protein bodies. As SEs mature the P-protein bodies disperse into smaller aggregates that move to the periphery of the cell.

9In cucurbits, phloem protein 1 (PP1) and phloem protein 2 (PP2) produce insoluble aggregates in response to oxidation by cross-linking, forming high-molecular-weight polymers that plug the sieve pores of injured sieve tubes. PP1 monomeric subunits have a predicted molecular mass of 95.4 kDa, but the apparent molecular size is dependent on the pH and oxidation state, as conformational isoforms exist that appear to be related to either the polymerized or unpolymerized, translocated forms of the protein. PP1 was immunolocalized in SE slime plugs and P-protein bodies, whereas the corresponding mRNA was shown to accumulate in CCs. Due to an interaction of PP1 and PP2 in presence of calcium and oxygen, sieve tubes and cut surfaces are rapidly occluded by gelling of the exudate. PP2-like proteins are lectins, sugar binding proteins, which have been identified in many angiosperms and are specifically expressed in SE/CC complexes, suggesting that PP2 may be a common component of P-proteins. It has been shown to interact with phloem sap proteins, potentially playing a role in the shuttling of glycoproteins between CC and SE. The phloem-specific PP2 homolog from Arabidopsis was shown to be anchored to P-proteins and other phloem organelles rather than being a structural component of P-proteins. These findings indicate that PP2 does not represent an essential part of the occlusion machinery. An insecticidal function for PP2 is described in vitro as well as in vivo. observed that snowdrop lectin from Galanthus nivalis, artificially applied or expressed in Nicotiana tabacum plants, reduced growth, decreased survival, and lowered reproduction in the aphid species Myzus persicae. Other lectins that showed insecticidal effects against aphids are a mannose-binding lectin, a garlic lectin, and concanavalin A. Similar effects were observed for protease inhibitors (PIs) applied to aphids via transgenic plants. observed that lectins negatively affect the activity of an aminopeptidase in rats. The identified aminopeptidase inside the aphid gut, which represents 15.6% of total gut proteins, is suggested to be a potential binding site for lectins.

10Sieve elements of Fabaceae contain elongate protein bodies called forisomes. Forisomes consist of fibrils and were previously classified as “non-dispersive P-protein bodies”. They were suspected to undergo structural transformations, from a crystalloid state with co-aligned fibrils to a “slime-body” with dispersed fibrils. The transition is a rapid and reversible conformational change in which forisomes shorten longitudinally while expanding radially with a several-fold volume increase. Forisomes disperse upon wounding and occlude sieve tubes, leading to a stop of mass flow observed in artificial sieve tubes. Furthermore, showed a cooling rate dependent transport interruption and parallel forisome dispersion in intact V. faba plants. Dispersion is triggered by an increase of free calcium (Ca2+) in sieve tubes, although as of yet, no Ca2+-binding sites have been detected in forisomes. As observed in vitro, high Ca2+ concentration (>50 μM) is also needed to disperse forisome in vivo. After burning the tip of a V. faba leaf, the elevation of Ca2+ concentration in most regions of sieve tubes inside the respective leaf was demonstrated to be below the threshold that is necessary for forisome dispersion. Only in the close vicinity of the Ca2+-channel pore Ca2+-level goes beyond the threshold and increases up to 100 μM (Figure 1B; ). Therefore, it was concluded that forisomes are directly associated with Ca2+ release sites. An association between forisomes and the ER (where the highest frequencies of Ca2+ channels were observed) was found. It was observed that the more intimately forisomes were associated with the ER or the plasma membrane of SEs, the greater was the probability of dispersion.

11Scanning electron microscopic images show that forisomes are composed of largely identical subunits named forisomettes. Indicated by transmission electron microscopy studies, forisomettes consist of strictly ordered arrays of a number of forisome proteins. At least three proteins are involved in formation of forisomettes, called sieve element occlusion 1 (SEO1), SEO2, and SEO3, and were identified in Medicago truncatula. SEOs are also present in plant families that do not possess forisomes, e.g., Rosaceae, Solanaceae, and Brassicaceae. Two Arabidopsis thaliana genes (At3g01670 and At3g01680) encode SEO proteins assigned AtSEOR1 and AtSEOR2. Both phloem filament proteins are required for formation of filaments that are arranged as complex network inside SEs. Whether the formation of dense SEO filament deposits at sieve plates stops phloem mass flow (Figure 1B; ) or mass flow remains intact is a matter of debate. Aphids of the species Myzus persicae that feed on AtSEOR1 and AtSEOR2 mutants without SEO filament formation show no benefit from the absence of filaments. Thus, conclude that SEOs are not involved in plant defense against phloem-feeding insects. In fact, aphids perform worse when compared to aphids on control plants, indicated by reduced reproduction and shortened reproduction period. The authors suggest that reduced fitness could be associated with lower nitrogen supply due to reduced protein content, but no data about amino acid concentration or protein content in the phloem sap of these plants are available. A further explanation could be that the absence of SEO filaments influences parameters in sieve tubes relevant for aphid feeding, e.g., turgor pressure, reducing nutrition supply and leading to the observed reduction of aphid reproduction. Although, describe that the phenotype SEO mutants does not differ to the wildtype this allows no conclusion about the state of sieve tubes.

12Callose deposition and protein plugging operate in parallel. A burning stimulus elicits distant occlusion in V. faba with rapid forisome dispersion and a slower subsequent callose deposition. While forisomes reconstitute into the condensed state, constriction of sieve pores by callose deposition reaches its maximum level. It is suggested, therefore, that plants possess a universal safety design for sieve-tube occlusion, one that proceeds rapidly and involves P-protein and a slower and more long-lasting one based on callose.

13The distant-induced occlusion was associated with the passage of a damage-induced EPW. EPWs communicate sudden and profound physiological changes over long-distances. EPWs trigger a release of Ca2+ that results in callose deposition and protein plugging. Ca2+ originates from the apoplast via opened plasma membrane channels or from the ER acting as an intracellular Ca2+ storage.

14Phloem-feeding insects belong to the order Hemiptera. Of these, important pests are planthoppers (suborder Auchenorrhyncha) and leafhoppers (suborder Clypeorrhyncha) as well as aphids and whiteflies (suborder Sternorrhyncha). The most currently available information about interaction with plants is on aphids, which make them a model organism for phloem feeders. Phloem-feeding insects possess specialized mouthparts, so-called stylets, with which they are able to obtain nutrition from plant tissues that are located deep inside the plant (Figure 1A). To access their food source, phloem-feeding insects secrete saliva that potentially interacts with defense mechanisms located in the sieve tube.

15The thin stylets of phloem-feeding insects are formed of four subunits, and their diameter and length are species dependent. The two outer mandibular parts contain nerve canals; the inner maxillary parts form the nutrition channel and a saliva channel that merge to a common duct at the tip of the stylet. An insect penetrates the plant with its stylet and moves the stylet toward the sieve tubes (Figure 1A). The stylet moves through the apoplast without causing significant damage to plant cells. Plant cells of different cell types are regularly penetrated along the stylet track. Aphids take up small cell sap samples when penetrating; this sample is most likely analyzed by chemosensilla in the precibarium as observed for leafhoppers. Beside acceptance of host plants this behavior could allow aphids to orient themselves inside the plant and helps them to detect sieve tubes, whereat parameters like sucrose and pH (Table 1) are suggested to be indicators for sieve tube penetration. After a sieve tube is penetrated, ingestion starts.

16Before plant penetration with their stylets, during stylet movement through the apoplast, penetration of cells, and ingestion, saliva is secreted by planthoppers, leafhoppers, aphids as well as whiteflies. Saliva has been suggested to play a key role in the interaction of insect pests and their respective host plants (reviewed in ). Aphids in particular function as model organisms for studying both phloem-feeding insects and the role and functions of saliva inside the plant. Like other hemiptera, aphids possess two types of saliva, one gel-like and one watery, although recently the protein composition of both types was shown to overlap partly.

17Gel saliva forms a salivary flange on the plant surface prior to plant penetration (Figure 1A; ), which is suggested to stabilize the stylet before initiating stylet penetration of the plant. When the stylet moves, small amounts of gel saliva are secreted; these harden and are then penetrated by the stylet. This continuous secretion of gel saliva leads to the formation of a solid salivary sheath that envelops the stylet and is left in situ after it is withdrawn from plant tissues. Sheath formation was assumed to be associated with the oxidation of protein sulphydryl groups, e.g., present in the amino acid cysteine. observed in this context that salivary sheath formation is disturbed under anoxic conditions. A protein that possesses a high content of cysteine was identified in saliva of the pea aphid Acyrthosiphon pisum by and termed “sheath protein” (SHP). It is assumed that formation of disulfide bonds leads to SHP aggregation and formation of the solid sheath. The fact that most phytophagous hemiptera were observed to form a salivary sheath during the feeding process, implies biological relevance but specific functions are unknown.

18It is suggested that gel saliva functions as a lubricant to facilitate stylet movement and that the sheath protects the stylet against mechanical forces and chemicals. Furthermore, postulated that the salivary sheath prevents the induction of defense responses in these conduits. In contrast, some enzyme components of the gel saliva are assumed to trigger plant defense responses by forming so-called pathogen-induced molecular patterns (PIMPs; Figure 1C). Potential candidates for PIMP production are cell-wall-degrading enzymes, such as cellulase and pectinase, which were detected in aphid saliva. Whether the protein or peptide components of gel saliva act in a similar manner to, e.g., flg22 from bacteria as pathogen-associated molecular patterns (PAMPs; Figure 1C) that trigger plant defense responses in a gene-to-gene interaction can be speculated. It may be that aphid gel saliva on one hand induces plant defense in cortex cells along the stylet track and on the other hand suppresses defense inside penetrated sieve tubes (Figure 1C;; ).

19Aphid watery saliva is secreted intracellularly, either when the stylets briefly puncture cells during probing or immediately before and during sap ingestion (Figure 1C; ), and recent studies on Aphis gossypii indicate that watery saliva is secreted into the apoplast as well. Gel saliva and watery saliva contain many different proteins of a broad molecular weight range (e.g.,;,;;;; ). The main classes of proteins that were identified with a proteomic approach in the species Acyrthosiphon pisum are proteases, detoxifying enzymes and proteins that potentially interact with plant signaling cascades, so-called effectors. Salivary proteins appear to move from the SE where they were secreted into adjacent SEs, which suggests that saliva functions are not restricted to an aphid-penetrated SE. Thus, the activity of aphids in a population puncturing SEs downstream from an SE already punctured by another aphid may be facilitated by suppressed defense responses. The aphids may therefore benefit from the saliva secretions of other individuals. The observation that feeding is locally stimulated on potato for the aphid species Myzus persicae and Macrosiphon euphorbiae, respectively, 96 h after first infestation supports this hypothesis.

20Sieve tubes lack most organelles and gene expression machinery but possess a variety of defense components, both physical and chemical mechanisms. Ca2+ represents a core of both groups (Figure 1B). The high concentration gradient of Ca2+ between apoplast and SE lumen leads to an influx of Ca2+ into the SE lumen during penetration of the SE membrane by a thin glass capillary, which induces occlusion (Figure 1B; ). During SE penetration by an aphid stylet this transient event is assumed to be suppressed initially by gel saliva that is secreted prior to penetration and seals the penetration site (Figure 1C; ). did not observe forisome dispersion in penetrated SEs prior to secretion of watery saliva and concluded that SE occlusion does not represent a defense mechanism against aphids, which is also suggested by. Nevertheless, findings of support the hypothesis that sealing of the stylet penetration site by gel saliva already mediates suppression of occlusion mechanisms (Figure 1C). The risk of triggering SE occlusion also occurs when aphids start to remove solute from the SE lumen, as this too potentially decreases turgor in SEs (Table 1) and thus activates potential mechano-sensitive Ca2+-channels that results, e.g., in forisome dispersion. For this reason, in a second step prior to ingestion, aphids secrete watery saliva (Figure 1C) that contains proteins that bind Ca2+; these proteins were detected by functional analysis for the aphid species Megoura viciae and were shown to counteract SE occlusion. Because aphids of different species change to watery saliva secretion if an occlusion event is induced during ingestion, the phenomenon of counteracting SE occlusion by secreting watery saliva is likely widespread. In vitro experiments have demonstrated that this change of behavior is triggered by a decrease of turgor pressure inside the sieve tubes that is the consequence of SE occlusion.

21In the saliva of Acyrthosiphon pisum, a Ca2+-binding protein was detected by mass spectrometry and was identified as regucalcin. The molecular mass of this protein, 43 kDa, is comparable to that of a previously detected Ca2+-binding protein. Regucalcin is a member of the senescence marker protein-30 (SMP-30) family that helps sequester signaling molecules such as Ca2+. In addition, regucalcin maintains intracellular Ca2+ homeostasis by activating Ca2+ pumps in the plasma membrane, ER, and mitochondria of many animal cell types. Moreover, in animals regucalcin has an inhibitory effect on the activation of Ca2+/calmodulin-dependent enzymes and protein kinase C. Thus, an inhibition of signaling cascades due to Ca2+-binding by saliva proteins appears likely, although little information about the molecular level of defense signaling inside sieve tubes is available. A recent study by did not detect the presence of regucalcin in the saliva of cereal aphids and the authors suggest that different protein compositions of watery saliva of various aphid species may illustrate the insects’ adaptation to various host plants.

22Other aphid species than Megoura viciae and Acyrthosiphon pisum, were not screened for Ca2+-binding proteins in their watery saliva. A Ca2+-binding protein was also identified in saliva of the green rice leafhopper; that this insect’s saliva was secreted into sieve tubes may indicate the presence of comparable mechanisms in different groups of phloem-feeding insects. Previously, demonstrated that the brown planthopper Nilaparvata lugens activates callose synthases during plant infestation but is able to unplug sieve pores by activating β-1,3-glucanases. Whether aphids also influence filament formation of SEO proteins or influence callose degradation is currently unknown.

23Several detoxifying proteins in the saliva of aphids were identified by enzymatic essays and novel approaches, including mass spectrometry. The detoxification of phenols by the secretion of polyphenoloxidase and peroxidase was reported for Sitobion avenae. The degradation of hydrogen peroxide could most likely interfere with defense signaling because hydrogen peroxide represents an activator of Ca2+ channels in the plasma membrane.

24Proteins that appear to interact directly with plant defense signaling are glucose dehydrogenase and glucose oxidase that were detected in the aphid species Myzus persicae and Acyrthosiphon pisum. Both potentially interfere with jasmonic acid (JA)-regulated defense responses that were shown to be induced during infestation of Arabidopsis by Brevicoryne brassicae. noticed that endogenous JA production was less for Acyrthosiphon pisum infested broad bean plants. Furthermore, aphids appear to be able to modulate genes in the salicylic acid (SA) pathway. Cross-talk between JA and SA defense pathways may allow aphids to suppress specific plant defense responses as has been previously described for whiteflies by. The role of SA and JA in plant-aphid interaction is reviewed by.

25Effectors are defined as proteins and/or small molecules that modify cell structure and function inside the host of a pathogen. Aphid species were shown to pursue similar strategies and secrete effectors as components of their saliva (Figure 1C). Effectors in saliva were first shown for Megoura viciae by identifying proteins that bind Ca2+. Later it was shown that C002, a salivary protein that is secreted into the sieve tubes by Acyrthosiphon pisum, plays an important role in aphid feeding. If C002 is silenced by RNA interference (RNAi) in Acyrthosiphon pisum, aphids’ life spans are reduced because they have problems reaching the sieve tubes and are thus unable to sustain ingestion. Silencing of C002 homolog in Myzus persicae by feeding on transgenic plants showed lower aphid reproduction rates than usual but no overall change in survival rates. When Myzus persicae fed on MpC002-expressing plants, an enhanced fecundity was observed, while the reproduction rates of Myzus persicae feeding on plants that express C002 from Acyrthosiphon pisum are not influenced. Further effectors with beneficial effect on aphid reproduction and thus on colonization are PIntO1 and PIntO2. Orthologs of C002, PIntO1and PIntO2 were detected in salivary gland transcriptome of multiple aphid species and appear to be specific for the respective aphid species. A recent study of the effector Me23 shows that effectors are specific not only to aphid but also to plant by demonstrating that the fecundity of Macrosiphon euphorbiae was enhanced when aphids fed on Me23 expressing Nicotiana benthamiana and not on Me23 expressing tomato. Above described effectors may be able to facilitate ingestion by suppressing plant defense responses, perhaps by interfering with signal cascades as described for different fungi (reviewed in ) and appear to contribute to aphid-plant compatibility. In addition to effectors that promote aphid colonization, some effectors induce plant defense responses. MP10 and MP42 were shown to reduce fecundity when expressed in plants, possibly interacting with plant receptors of the NBS-LRR superfamily and thus triggering plant defense responses. The identified aphid resistance genes Mi-1.2 in tomato and Vat in melon belong to the NBS-LRR receptor family (reviewed in ).

26As described, plant defense can be overwhelmed by aphids. For this reason, e.g., agro-biotechnological control strategies support plant defense by inserting additional insecticidal compounds into the sieve tubes. Approaches are the expression of PIs and antimicrobial peptides (AMPs) that naturally do not belong to the target plants defense system (reviewed in ). PIs can be used to target proteases detected in the watery saliva and alimentary tract of aphids; there the PIs may prevent the digestion of proteins within the sieve-tube sap (Table 1). Although for a long time researchers did not believe that aphids were able to use proteins as a source of nutrition, new findings show that aphids can digest proteins in sieve-tube sap. The use of AMPs represents an approach that targets the endosymbiotic bacteria of aphids, assuming that disrupting these bacteria would negatively affect aphid fitness. The primary (obligate) endosymbiotic bacteria Buchnera aphidicola improves the quality of aphid diet by supplying it with essential amino acids that are absent in sieve-tube sap. Other endosymbiotic bacteria improve aphid fitness by giving resistance to pathogenic fungi (e.g., ) or increasing thermo tolerance (e.g., ). Although described methods of controlling aphids address different levels of interaction of pests and their respective host plants, a common goal is disrupting plant accession and nutrition uptake. Expressing defense agents in the sieve tubes is an effective way of accomplishing this disruption. Defense agents negatively affect fitness parameters (e.g.,;; ), such as growth, reproduction, and survival, which may reduce infestations among plants.

27The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.