New phytoplasma effector: 50 shades of green.

Phytoplasmas are pathogenic bacteria that manipulate their plant hosts in dramatic ways. In issue 20 Vol. 184 of Cell, Huang and colleagues report on a phytoplasma effector protein that brings about manifold effects by a surprising mechanism. Are resistant plants on the horizon now?

Specialized 16SrX phytoplasmas induce diverse morphological and physiological changes in their respective fruit crops.

The host-pathogen combinations-Malus domestica (apple)/`Candidatus Phytoplasma mali´, Prunus persica (peach)/`Ca. P. prunorum´ and Pyrus communis (pear)/`Ca. P. pyri´ show different courses of diseases although the phytoplasma strains belong to the same 16SrX group. While infected apple trees can survive for decades, peach and pear trees die within weeks to few years. To this date, neither morphological nor physiological differences caused by phytoplasmas have been studied in these host plants. In this study, phytoplasma-induced morphological changes of the vascular system as well as physiological changes of the phloem sap and leaf phytohormones were analysed and compared with non-infected plants. Unlike peach and pear, infected apple trees showed substantial reductions in leaf and vascular area, affecting phloem mass flow. In contrast, in infected pear mass flow and physicochemical characteristics of phloem sap increased. Additionally, an increased callose deposition was detected in pear and peach leaves but not in apple trees in response to phytoplasma infection. The phytohormone levels in pear were not affected by an infection, while in apple and peach trees concentrations of defence- and stress-related phytohormones were increased. Compared with peach and pear trees, data from apple suggest that the long-lasting morphological adaptations in the vascular system, which likely cause reduced sap flow, triggers the ability of apple trees to survive phytoplasma infection. Some phytohormone-mediated defences might support the tolerance.

Species-Specific and Distance-Dependent Dispersive Behaviour of Forisomes in Different Legume Species.

Forisomes are giant fusiform protein complexes composed of sieve element occlusion (SEO) protein monomers, exclusively found in sieve elements (SEs) of legumes. Forisomes block the phloem mass flow by a Ca2+-induced conformational change (swelling and rounding). We studied the forisome reactivity in four different legume species-Medicago sativa, Pisum sativum, Trifolium pratense and Vicia faba. Depending on the species, we found direct relationships between SE diameter, forisome surface area and distance from the leaf tip, all indicative of a developmentally tuned regulation of SE diameter and forisome size. Heat-induced forisome dispersion occurred later with increasing distance from the stimulus site. T. pratense and V. faba dispersion occurred faster for forisomes with a smaller surface area. Near the stimulus site, electro potential waves (EPWs)-overlapping action (APs), and variation potentials (VPs)-were linked with high full-dispersion rates of forisomes. Distance-associated reduction of forisome reactivity was assigned to the disintegration of EPWs into APs, VPs and system potentials (SPs). Overall, APs and SPs alone were unable to induce forisome dispersion and only VPs above a critical threshold were capable of inducing forisome reactions.

Measurement of Electropotential Waves in Intact Phloem Sieve Elements Using Microelectrodes.

Electropotential waves (EPW) are involved in plant responses to both abiotic and biotic stresses. Three different types of EPWs have been identified: action potential, variation potential, and system potential, all of which have been indicated to participate in phloem-based communication between plant organs. In this chapter we describe in detail how to measure EPWs in plants, including how to access the phloem, and how to insert microelectrodes. Such experiments can be used, for example, to study the local and systemic signaling in response to diverse stimuli like microbial threat or herbivore attack.

Measurement of Electropotential Waves in Intact Sieve Elements Using Aphids as Bioelectrodes.

Electropotential waves (EPWs) are thought to transmit sudden and profound physiological changes between plant organs. The recording of EPWs can be performed via extracellular or intracellular probes. Both approaches have advantages and disadvantages. Since the phloem is responsible for long distance transport of the most forms of EPWs, the direct measurement in sieve elements is preferable. The conductance using glass microelectrodes inserted into free lying sieve elements is described in Chapter 34 . In this chapter the measurement of EPWs by using aphids as bioelectrodes is described in detail.The electrical penetration graph technique (EPG) takes advantage of the flexible mouthparts (stylet) of aphids, which specifically penetrate into sieve elements. The use of aphids as bioelectrodes enables multiple electrode recordings and long-distance observations of EPWs. Importantly, this method allows for noninvasive, intracellular measurements.

Collection of Phloem Sap in Phytoplasma-Infected Plants.

Phytoplasmas colonize specifically the phloem sieve elements (SEs) of plants and influence effectively the plant physiology. To study and understand the interaction of phytoplasmas and host plants an access to the cellular, microscale volume of SEs is demanded. Different methods are suitable to collect phloem sap of phytoplasma-infected plants. The two most common methods are the EDTA-facilitated exudation and the stylectomy. For the EDTA-facilitated method, the cut end of a leaf is placed into an EDTA solution. The EDTA prevents and avoids the Ca2+ dependent (re-) occlusion of SEs by binding Ca2+ ions and the mass flow of SEs is restarted which results in an outflow of the SE content into the EDTA bathing solution. The advantage is on the one hand a simple application and secondly, feasible for all plant species.The stylectomy method requires piercing-sucking insects like any aphids. During phloem-sap ingestion, the stylet is severed by a microcautery device or a laser from the insect body. Due to the high turgor pressure of the SEs the phloem sap is forced out through the remaining stylet and can be collected with a glass capillary, for example. The stylectomy delivers pure phloem sap, however, the collected volumes are in the range of nano liters and the temporal and staff costs are tremendous. A third method is the spontaneous exudation in phytoplasma-infected apple trees providing only in springtime large volumes of vascular sap after cutting along the bark. For the spontaneous exudation the proportion of phloem sap is unclear. Thus, this third method still needs a closer examination in prospective surveys.

Barley yellow dwarf virus Infection Leads to Higher Chemical Defense Signals and Lower Electrophysiological Reactions in Susceptible Compared to Tolerant Barley Genotypes.

Barley yellow dwarf virus (BYDV) is a phloem limited virus that is persistently transmitted by aphids. Due to huge yield losses in agriculture, the virus is of high economic relevance. Since the control of the virus itself is not possible, tolerant barley genotypes are considered as the most effective approach to avoid yield losses. Although several genes and quantitative trait loci are known and used in barley breeding for virus tolerance, little is known about molecular and physiological backgrounds of this trait. Therefore, we compared the anatomy and early defense responses of a virus susceptible to those of a virus-tolerant cultivar. One of the very early defense responses is the transmission of electrophysiological reactions. Electrophysiological reactions to BYDV infection might differ between susceptible and tolerant cultivars, since BYDV causes disintegration of sieve elements in susceptible cultivars. The structure of vascular bundles, xylem vessels and sieve elements was examined using microscopy. All three were significantly decreased in size in infected susceptible plants where the virus causes disintegration of sieve elements. This could be associated with an uncontrolled ion exchange between the sieve-element lumen and apoplast. Further, a reduced electrophysiological isolation would negatively affect the propagation of electrophysiological reactions. To test the influence of BYDV infection on electrophysiological reactions, electropotential waves (EPWs) induced by leaf-tip burning were recorded using aphids as bioelectrodes. EPWs in infected susceptible plants disappeared already after 10 cm in contrast to those in healthy susceptible or infected tolerant or healthy tolerant plants. Another early plant defense reaction is an increase in reactive oxygen species (ROS). Using a fluorescent dye, we found a significant increase in ROS content in infected susceptible plants but not in infected tolerant plants. Similar results were found for the phytohormones abscisic acid and three jasmonates. Salicylic acid levels were generally higher after BYDV infection compared to uninfected plants. Heat stimulation caused an increase in jasmonates. By shedding light on the plant defense mechanisms against BYDV, this study, provides further knowledge for breeding virus tolerant plants.

Corrigendum: Systemic Induction of NO-, Redox-, and cGMP Signaling in the Pumpkin Extrafascicular Phloem upon Local Leaf Wounding.

[This corrects the article on p. 154 in vol. 7, PMID: 26904092.].

Systemic Induction of NO-, Redox-, and cGMP Signaling in the Pumpkin Extrafascicular Phloem upon Local Leaf Wounding.

Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima). Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint toward a wound-induced shift in the redox status of the EFP. Nitric oxide (NO) is another important player in stress-induced redox signaling in plants. Therefore, we analyzed NO-dependent protein modifications in the EFP. Six to forty eight hours after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1) and Cyclophilin 18 (CYP18) as well as the 26.5 kD isoform of Phloem Protein 2 (PP2) were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP) in the EFP and discuss the possible function of this second messenger in systemic NO and redox signaling within the EFP.

Herbivore-Triggered Electrophysiological Reactions: Candidates for Systemic Signals in Higher Plants and the Challenge of Their Identification.

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 long-distance communication in higher plants.

Direct and individual analysis of stress-related phytohormone dispersion in the vascular system of Cucurbita maxima after flagellin 22 treatment.

• The stress-related phytohormones, salicylic acid (SA) and abscisic acid (ABA), and the three jasmonates, jasmonic acid (JA), cis-12-oxo-phytodienoic acid (cis-OPDA), and (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), were investigated in phloem and xylem exudates of Cucurbita maxima. • Phloem and xylem exudates were separately collected and analysed via liquid chromatography-mass spectrometry. • We show direct evidence for all three jasmonates, ABA, and SA in both phloem and xylem exudates of C. maxima. JA and JA-Ile concentrations are higher in xylem (JA: c(xylem) ≈ 199.5 nM, c(phloem) ≈ 43.9 nM; JA-Ile: c(xylem) ≈ 7.9 nM, c(phloem) ≈ 1.6 nM), whereas ABA and SA concentrations are higher in phloem exudates (ABA: c(xylem) ≈ 37.1 nM, c(phloem) ≈ 142.6 nM; SA: c(xylem) ≈ 61.6 nM, c(phloem) ≈ 1319 nM). During bacteria-derived flagellin 22 (flg22)-triggered remote root-to-shoot signalling, phytohormone concentration changed rapidly both in phloem and xylem. • The unequal distribution of phytohormones suggests that phloem and xylem have distinct roles in defence responses. Our data shed light on systemic phytohormone signalling and help explain how plants cope with environmental challenges by lateral exchange between phloem and xylem. Our analysis is a starting point for further investigations of how phytohormones contribute to phloem- and xylem-based defence signalling.

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

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.

Interaction of xylem and phloem during exudation and wound occlusion in Cucurbita maxima.

Collection of cucurbit exudates from cut petioles has been a powerful tool for gaining knowledge on phloem sap composition without full notion of the complex exudation mechanism. Only few publications explicitly mentioned that exudates were collected from the basal side of the cut, which exudes more copiously than the apical side. This is surprising since only exudation from the apical side is supposedly driven by phloem pressure gradients. Composition of carbohydrates and pH values at both wounding sides are equal, whereas protein concentration is higher at the basal side. Apparently, exudation is far more complex than just the delivery of phloem sap. Xylem involvement is indicated by lower protein concentrations after elimination of root pressure. Moreover, dye was sucked into xylem vessels owing to relaxation of negative pressure after cutting. The lateral water efflux from the vessels increases turgor of surrounding cells including sieve elements. Simultaneously, detached parietal proteins (PP1/PP2) induce occlusion of sieve plates and cover wound surface. If root pressure is strong enough, pure xylem sap can be collected after removal of the occlusion plug at the wound surface. The present findings provide a mechanism of sap exudation in Cucurbita maxima, in which the contribution of xylem water is integrated.

Alamethicin-induced electrical long distance signaling in plants.

Systemic signals induced by wounding and/or pathogen or herbivore attack may be realized by either chemical or mechanical signals. In plants a variety of electrical phenomena have been described and may be considered as signal-transducing events; such as variation potentials (VPs) and action potentials (APs) which propagate over long distances and hence are able to carry information from organ to organ. In addition, we recently described a new type of electrical long-distance signal that propagates systemically, i.e. from leaf to leaf, the 'system potential' (SP). This was possible only by establishing a non-invasive method with micro-electrodes positioned in sub-stomatal cavities of open stomata and recording apoplastic responses. Using this technical approach, we investigated the function of the peptaibole alamethicin (ALA), a channel-forming peptide from Trichoderma viride, which is widely used as agent to induce various physiological and defence responses in eukaryotic cells including plants. Although the ability of ALA to initiate changes in membrane potentials in plants has always been postulated it has never been demonstrated. Here we show that both local and long-distance electrical signals, namely depolarization, can be induced by ALA treatment.

Remote-controlled stop of phloem mass flow by biphasic occlusion in Cucurbita maxima.

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 approximately 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 Ca(2+)-dependent occlusion mechanisms is attributed to Ca(2+) influx during EPW passage; the reversibility of the occlusion is explained by removal of Ca(2+) ions.

System potentials, a novel electrical long-distance apoplastic signal in plants, induced by wounding.

Systemic signaling was investigated in both a dicot (Vicia faba) and a monocot (Hordeum vulgare) plant. Stimuli were applied to one leaf (S-leaf), and apoplastic responses were monitored on a distant leaf (target; T-leaf) with microelectrodes positioned in substomatal cavities of open stomata. Leaves that had been injured by cutting and to which a variety of cations were subsequently added caused voltage transients at the T-leaf, which are neither action potentials nor variation potentials: with respect to the cell interior, the initial polarity of these voltage transients is hyperpolarizing; they do not obey the all-or-none rule but depend on both the concentration and the type of substance added and propagate at 5 to 10 cm min(-1). This response is thought to be due to the stimulation of the plasma membrane H(+)-ATPase, a notion supported by the action of fusicoccin, which also causes such voltage transients to appear on the T-leaf, whereas orthovanadate prevents their propagation. Moreover, apoplastic ion flux analysis reveals that, in contrast to action or variation potentials, all of the investigated ion movements (Ca(2+), K(+), H(+), and Cl(-)) occur after the voltage change begins. We suggest that these wound-induced "system potentials" represent a new type of electrical long-distance signaling in higher plants.

Systemic signalling in barley through action potentials.

Using apoplastic voltage- and ion selective microprobes, in barley leaves action potentials (APs) have been measured, which propagate acropetally as well as basipetally from leaf to leaf or from root to leaf following the application of mild salt stress (e.g. 30-50 mM KCl or NH(4)Cl) or amino acids (e.g. 1 mM glutamic acid or 5 mM GABA). Voltage changes were biphasic, followed an 'all-or-none' characteristic, and propagated at 20-30 cm min(-1) irrespective of the direction. With the salt-induced APs, a strong initial depolarization is the main AP-releasing factor that first causes Ca(2+) influx and then anion efflux. Ca(2+) influx coincides with an initial slower depolarization, the rapid anion efflux causes the typical voltage 'break-through'. Subsequently, K(+)-efflux starts after the depolarizing voltage has passed the K(+) equilibrium potential (inversion of the K(+) driving force). Glutamic acid and GABA induce APs not through membrane depolarization, but presumably by binding to a putative receptor or to ligand-gated Ca(2+)-conducting channels, respectively, followed by Ca(2+) induced activation of anion efflux. APs are accompanied by transient apoplastic pH increase (about 1 unit), and by cytoplasmic pH decrease (about 0.5 units). The apoplastic pH change is interpreted as an indicator of stress, the cytoplasmic pH change as a prerequisite for defence related gene activation. Since APs are released by agents added in a moderate concentration range, it is suggested that they may serve as first and fast systemic signals following attack from pathogens.