Aphids, which constitute one of the most important groups of agricultural pests, ingest nutrients from sieve tubes, the photoassimilate transport conduits in plants. Aphids are able to successfully puncture sieve tubes with their piercing mouthparts (stylets) and ingest phloem sap without eliciting the sieve tubes' normal occlusion response to injury. Occlusion mechanisms are calciumtriggered and may be prevented by chemical constituents in aphid saliva injected into sieve tubes before and during feeding. We recorded aphid feeding behavior with the electrical penetration graph (EPG) technique and then experimentally induced sieve tube plugging. Initiation of sieve tube occlusion caused a change in aphid behavior from phloem sap ingestion to secretion of watery saliva. Direct proof of ''unplugging'' properties of aphid saliva was provided by the effect of aphid saliva on forisomes. Forisomes are proteinaceous inclusions in sieve tubes of legumes that show calcium-regulated changes in conformation between a contracted state (below calcium threshold) that does not occlude the sieve tubes and a dispersed state (above calcium threshold) that occludes the sieve tubes. We demonstrated in vitro that aphid saliva induces dispersed forisomes to revert back to the nonplugging contracted state. Labeling Western-blotted saliva proteins with 45 Ca 2؉ or ruthenium red inferred the presence of calcium-binding domains. These results demonstrate that aphid saliva has the ability to prevent sieve tube plugging by molecular interactions between salivary proteins and calcium. This provides aphids with access to a continuous flow of phloem sap and is a critical adaptation instrumental in the evolutionary success of aphids.calcium-binding ͉ plant-aphid interaction ͉ plugging ͉ saliva proteins ͉ sieve element

Aphids feed from sieve tubes deep inside the host plant. Therefore, aphids must be able to recognize their host plant(s) and to direct their stylets which must be long and thin enough to reach and puncture the sieve tubes at a particular site. Sieve tubes in angiosperms are longitudinal arrays of sieve element/companion cell modules which are highly sensitive to disturbance of any kind. The sieve tubes dispose of elaborate sealing mechanisms such as protein plugging and callose sealing which are triggered by a rise in calcium in the sieve tubes. Aphids seem to have developed a range of physical and chemical measures to limit the amount of calcium influx in response to stylet puncturing. Loss of sieve-element turgor pressure induced by stylet insertion is minimized by the minute stylet volume. Turgor-dependent Ca(2+) influx, possibly mediated by mechano sensitive Ca(2+) channels, must therefore be limited. The components of the sheath and watery saliva play a pivotal role in establishing the physical and chemical constraints on the rise of calcium. Most likely, sheath saliva prevents the influx of calcium from the apoplast by sealing the stylet puncture site while watery saliva may prevent plugging and sealing of sieve plates by potential interaction with SE sap ingredients.

Due to the high content of nutrient, sieve tubes are a primary target for pests, e.g., most phytophagous hemipteran. To protect the integrity of the sieve tubes as well as their content, plants possess diverse chemical and physical defense mechanisms. The latter mechanisms are important because they can potentially interfere with the food source accession of phloem-feeding insects. Physical defense mechanisms are based on callose as well as on proteins and often plug the sieve tube. Insects that feed from sieve tubes are potentially able to overwhelm these defense mechanisms using their saliva. Gel saliva forms a sheath in the apoplast around the stylet and is suggested to seal the stylet penetration site in the cell plasma membrane. In addition, watery saliva is secreted into penetrated cells including sieve elements; the presence of specific enzymes/effectors in this saliva is thought to interfere with plant defense responses. Here we detail several aspects of plant defense and discuss the interaction of plants and phloem-feeding insects. Recent agro-biotechnological phloem-located aphid control strategies are presented.

Aphids produce gel saliva during feeding which forms a sheath around the stylet as it penetrates through the apoplast. The sheath is required for the sustained ingestion of phloem sap from sieve elements and is thought to form when the structural sheath protein (SHP) is cross-linked by intermolecular disulphide bridges. We investigated the possibility of controlling aphid infestation by host-induced gene silencing (HIGS) targeting shp expression in the grain aphid Sitobion avenae. When aphids were fed on transgenic barley expressing shp double-stranded RNA (shp-dsRNA), they produced significantly lower levels of shp mRNA compared to aphids feeding on wild-type plants, suggesting that the transfer of inhibitory RNA from the plant to the insect was successful. shp expression remained low when aphids were transferred from transgenic plants and fed for 1 or 2 weeks, respectively, on wild-type plants, confirming that silencing had a prolonged impact. Reduced shp expression correlated with a decline in growth, reproduction and survival rates. Remarkably, morphological and physiological aberrations such as winged adults and delayed maturation were maintained over seven aphid generations feeding on wild-type plants. Targeting shp expression therefore appears to cause strong transgenerational effects on feeding, development and survival in S. avenae, suggesting that the HIGS technology has a realistic potential for the control of aphid pests in agriculture.

We explored the idea of whether electropotential waves (EPWs) primarily act as vehicles for systemic spread of Ca(2+) signals. EPW-associated Ca(2+) influx may trigger generation and amplification of countless long-distance signals along the phloem pathway given the fact that gating of Ca(2+)-permeable channels is a universal response to biotic and abiotic challenges. Despite fundamental differences, both action and variation potentials are associated with a sudden Ca(2+) influx. Both EPWs probably disperse in the lateral direction, which could be of essential functional significance. A vast set of Ca(2+)-permeable channels, some of which have been localized, is required for Ca(2+)-modulated events in sieve elements. There, Ca(2+)-permeable channels are clustered and create so-called Ca(2+) hotspots, which play a pivotal role in sieve element occlusion. Occlusion mechanisms play a central part in the interaction between plants and phytopathogens (e.g. aphids or phytoplasmas) and in transient re-organization of the vascular symplasm. It is argued that Ca(2+)-triggered systemic signalling occurs in partly overlapping waves. The forefront of EPWs may be accompanied by a burst of free Ca(2+) ions and Ca(2+)-binding proteins in the sieve tube sap, with a far-reaching impact on target cells. Lateral dispersion of EPWs may induce diverse Ca(2+) influx and handling patterns (Ca(2+) signatures) in various cell types lining the sieve tubes. As a result, a variety of cascades may trigger the fabrication of signals such as phytohormones, proteins, or RNA species released into the sap stream after product-related lag times. Moreover, transient reorganization of the vascular symplasm could modify cascades in disjunct vascular cells.

SUMMARY Ca 2+-binding proteins in the watery saliva of Megoura viciae counteract Ca 2+ -dependent occlusion of sieve plates in Vicia faba and so prevent the shut-down of food supply in response to stylet penetration. The question arises whether this interaction between aphid saliva and sieve-element proteins is a universal phenomenon as inferred by the coincidence between sieve-tube occlusion and salivation. For this purpose, leaf tips were burnt in a number of plant species from four different families to induce remote sieve-plate occlusion. Resultant sieve-plate occlusion in these plant species was counteracted by an abrupt switch of aphid behaviour. Each of the seven aphid species tested interrupted its feeding behaviour and started secreting watery saliva. The protein composition of watery saliva appeared strikingly different between aphid species with less than 50% overlap. Secretion of watery saliva seems to be a universal means to suppress sieve-plate occlusion, although the protein composition of watery saliva seems to diverge between species.

In stressed plants, electrophysiological reactions (elRs) are presumed to contribute to long-distance intercellular communication between distant plant parts. Because of the focus on abiotic stress-induced elRs in recent decades, biotic stress-triggered elRs have been widely ignored. It is likely that the challenge to identify the particular elR types (action potential [AP], variation potential, and system potential [SP]) was responsible for this course of action. Thus, this survey focused on insect larva feeding (Spodoptera littoralis and Manduca sexta) that triggers distant APs, variation potentials, and SPs in monocotyledonous and dicotyledonous plant species (Hordeum vulgare, Vicia faba, and Nicotiana tabacum). APs were detected only after feeding on the stem/culm, whereas SPs were observed systemically following damage to both stem/culm and leaves. This was attributed to the unequal vascular innervation of the plant and a selective electrophysiological connectivity of the plant tissue. However, striking variations in voltage patterns were detected for each elR type. Further analyses (also in Brassica napus and Cucurbita maxima) employing complementary electrophysiological approaches in response to different stimuli revealed various reasons for these voltage pattern variations: an intrinsic plasticity of elRs, a plant-specific signature of elRs, a specific influence of the applied (a)biotic trigger, the impact of the technical approach, and/or the experimental setup. As a consequence, voltage pattern variations, which are not irregular but rather common, need to be included in electrophysiological signaling analysis. Due to their widespread occurrence, systemic propagation, and respective triggers, elRs should be considered as candidates for longdistance communication in higher plants.

The relationships between damage-induced electropotential waves (EPWs), sieve tube occlusion, and stop of mass flow were investigated in intact Cucurbita maxima plants. After burning leaf tips, EPWs propagating along the phloem of the main vein were recorded by extra- and intracellular microelectrodes. The respective EPW profiles (a steep hyperpolarization/depolarization peak followed by a prolonged hyperpolarization/depolarization) probably reflect merged action and variation potentials. A few minutes after passage of the first EPW peak, sieve tubes gradually became occluded by callose, with maximum synthesis occurring ∼10 min after burning. Early stop of mass flow, well before completion of callose deposition, pointed to an occlusion mechanism preceding callose deposition. This obstruction of mass flow was inferred from the halt of carboxyfluorescein movement in sieve tubes and intensified secretion of aqueous saliva by feeding aphids. The early occlusion is probably due to proteins, as indicated by a dramatic drop in soluble sieve element proteins and a simultaneous coagulation of sieve element proteins shortly after the burning stimulus. Mass flow resumed 30–40 min after burning, as demonstrated by carboxyfluorescein movement and aphid activities. Stop of mass flow by Ca2+-dependent occlusion mechanisms is attributed to Ca2+ influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca2+ ions.