Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Spodoptera littoralis oral secretions inhibit the activity of Phaseolus lunatus plasma membrane H+-ATPase

Abstract

Biotic stresses induced by herbivores result in diverse physiological changes in plants. In the interaction between the Lima bean (Phaseolus lunatus) and the herbivore Spodoptera littoralis, the earliest event induced by feeding on leaves is the depolarization of the plasma membrane potential (Vm), which is the results of both mechanical damage and insect oral secretions (OS). Although this herbivore-induced Vm depolarization depends on a calcium-dependent opening of potassium channels, the attacked leaf remains depolarized for an extended period, which cannot be explained by the sole action of potassium channels. Here we show that the plasma membrane H+-ATPase of P. lunatus leaves is strongly inhibited by S. littoralis OS. Inhibition of the H+-ATPase was also found in plasma membranes purified from leaf sections located distally from the application zone of OS, thus suggesting a long-distance transport of a signaling molecule(s). S. littoralis’ OS did not influence the amount of the plasma membrane H+-ATPase, whereas the levels of membrane-bound 14-3-3 proteins were significantly decreased in membranes purified from treated leaves. Furthermore, OS strongly reduced the in vitro interaction between P. lunatus H+-ATPase and 14-3-3 proteins. The results of this work demonstrate that inhibition of the plasma membrane H+-ATPase is a key component of the S. littoralis OS mechanism leading to an enduring Vm depolarization in P. lunatus wounded leaves.

Citation: Camoni L, Barbero F, Aducci P, Maffei ME (2018) Spodoptera littoralis oral secretions inhibit the activity of Phaseolus lunatus plasma membrane H+-ATPase. PLoS ONE 13(8): e0202142. https://doi.org/10.1371/journal.pone.0202142

Editor: Daniel Doucet, Natural Resources Canada, CANADA

Received: April 6, 2018; Accepted: July 27, 2018; Published: August 10, 2018

Copyright: © 2018 Camoni et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information file.

Funding: This research was sponsored by the University of Rome Tor Vergata and the University of Turin research funds.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Plants use a sophisticated sensory system to detect potential enemies and subsequently to translate and integrate such signals into appropriate biochemical and physiological responses. In plant-insect interactions, herbivory combines two different sites of the feeding process: the mechanical wounding of the infested tissue and the introduction of oral secretions (OS). OS from feeding insects contain herbivore-specific compounds with elicitor-like properties [1]. Following herbivory and the release of OS, there is an increased accumulation of cytosolic calcium [Ca2+]cyt and reactive oxygen species (ROS, e.g., H2O2) and NO release [2, 3]. Furthermore, changes of plasma membrane potentials (Vm), which are mainly caused by a calcium-dependent potassium channel activation [4] precede the cascade of events eventually leading to gene expression of defense responses [5–7].

Previous work has shown that the interaction between the Lima bean (Phaseolus lunatus) and the herbivore Spodoptera littoralis can be considered an ideal model to evaluate early and late responses of plants to herbivory [8–10]. The recent discovery of a putative β-galactofuranose polysaccharide in the OS of S. littoralis that is able to trigger early plant responses, and the finding that the responses to herbivory can be separated into a calcium-activated oxidative response and a K+-dependent Vm-activated jasmonate response associated with the release of volatile organic compounds (VOCs) [4], opens new questions on the role of the plasma membrane in early detection of biotic attacks. Moreover, upon herbivory, wounded leaves maintain Vm depolarized conditions that cannot be recovered by the activation of ion channels [2].

In plants, the plasma membrane H+-ATPase is responsible for the establishment of a proton electrochemical gradient across the plasma membrane that is utilized by channel and carrier proteins for the transport of ions and solutes [11]. Hence, variations in H+-ATPase activity can modulate membrane potential, thereby influencing the activities of voltage-gated channels and controlling ion flux at the plasma membrane [12].

In addition to its pivotal role in the regulation of basic aspects of plant cell function, H+-ATPase takes part in signaling events in response to diverse environmental stimuli, including pathogens [13]. The main regulatory mechanism of the plasma membrane’s H+-ATPase involves its interaction with 14-3-3 proteins, a family of eukaryotic regulatory proteins involved in the regulation of fundamental physiological processes in plants through phosphorylation-dependent interactions with client proteins [14, 15]. The association of 14-3-3 proteins with the C-terminal autoinhibitory domain of H+-ATPase brings about its displacement and consequent enzyme activation [16, 17]. The H+-ATPase binding site for 14-3-3s is generated upon phosphorylation of a conserved threonine residue within the sequence YTV, located at the very end of the C terminus [16]. To our knowledge, nothing is known on the effect of herbivores’ OS on the activity of plants’ H+-ATPase. Given the pivotal role of the plasma membrane H+-ATPase in the establishment and maintenance of the Vm, it is conceivable that this enzyme might be related to plant responses to insects’ OS.

The natural toxins fusicoccin (FC) and okadaic acid (OKA) induce a Vm hyperpolarization by H+-ATPase activation and therefore are useful tools to investigate the regulatory mechanisms of the proton pump. FC activates the H+-ATPase by irreversibly stabilizing the H+-ATPase/14-3-3 complex [16–21], while the protein phosphatase inhibitor OKA [22] activates the proton pump by blocking the dephosphorylation of its C-terminal domain and consequently promoting 14-3-3 binding [18].

In this work, we performed in vivo and in vitro studies to investigate the ability of S. littoralis to affect the activity of the Lima bean proton pump. Here we show that S. littoralis’ OS inhibit interaction between the H+-ATPase and 14-3-3 proteins in Lima bean leaves.

Materials and methods

Chemicals

Fusicoccin (FC) was prepared according to Ballio et al. [23]. Okadaic acid was purchased from Calbiochem, (La Jolla, California). [γ-32P]ATP (specific activity 110 TBq/mmol) was from Perkin Elmer (Boston, MA). Protein kinase A, catalytic subunit, and thrombin were from Sigma-Aldrich (St. Louis, Missouri). Chemicals for gel electrophoresis were from Bio-Rad (Hercules, California). All other reagents were of analytical grade.

Plant and animal material

Lima bean (Phaseolus lunatus ‘Ferry Morse’ var. Jackson Wonder Bush) plants were grown in a growth chamber at 22 C, 80% humidity, under a 16 h light/8 h dark cycle for 2 weeks.

Eggs of Spodoptera littoralis Boisd. (Lepidoptera, Noctuidae) were kindly supplied by Syngenta (Switzerland) and larvae were fed on an artificial diet composed of 125 g bean flour, 2.25 g ascorbic acid, 2.25 g ethyl 4-hydroxybenzoate, 750 μl formaldehyde, 300 ml distilled water and 20 g agar previously dissolved in 300 ml distilled water. The ingredients (Sigma-Aldrich, St. Louis, MO, USA) were mixed with a blender and stored at 4°C for not more than 1 week. With the exception for VOC collection (see below), plants were fed for 2 h with third instar larvae reared from egg clutches in Petri dishes (6 cm diameter) in a growth chamber with 16 h photoperiod at 25°C and 60–70% humidity [24].

Collection of oral secretions

S. littoralis oral secretions (OS) were obtained from 5-day-old larvae which were allowed to feed on Lima bean leaves for 24 h. Regurgitation was caused by gently squeezing the larva with forceps behind the head. OS were collected into glass capillaries connected to an evacuated sterile vial (peristaltic pump). Secretions were stored at -20°C until analysis. Five microliters of OS in 5 mM Mes-NaOH (pH 6.0) were applied to the leaf with a microsyringe and the Vm of leaves was analyzed after 2 h. The OS quantity was assessed after several trials (from 0.5 to 10 μl) and was found the most appropriate to obtain reproducible experiments.

Plant treatments

Treatments were carried out by wounding the apex or primary leaves and then applying 10 μl OS or 10 μM FC to the apex or primary leaves. As a control, 10 μl deionized water was applied to wounded leaves without OS or FC application were used. For each treatment, at least three biological replicates were performed.

Membrane potential determination

Membrane potentials were determined in leaf segments. The transmembrane potential (Vm) was determined using glass micropipettes with a tip resistance of 4–10 MΩ and filled with 3 M KCl, as previously described [8, 25]. Based on topographical and temporal determination of Vm performed previously, the electrode was inserted between 0.5 and 1.5 mm from the wounded zone, where a significant Vm depolarization occurs after HW. The results of all Vm measurements are shown as the average number of at least 50 Vm measurements.

Plasma membrane purification

Plasma membranes were purified from Lima bean leaves following the procedure described by Serrano [26], with minor modifications. Twenty-five g of treated or control leaves were cut and homogenized with 25 ml of a buffer containing 25 mM MOPS-BTP, 250 mM sucrose, 5 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.2% BSA, pH 7.8, filtered and centrifuged for 20 min at 8000 g at 4°C. The supernatant was then filtered and ultracentrifuged at 70000 g for 30 min. The pellet (microsomal fraction) was resuspended in 2 ml of 5 mM potassium phosphate buffer containing 0.2 mM PMSF, pH 7.8 and added to 14 ml of phase 7.2% Dextran T-500, 7.2% PEG-3350, 286 mM sucrose, 5.7 mM KCl, 5.7 K3PO4, pH 7.8. After mixing by repeated inversions, the samples were centrifuged at 2000 g for 15 min at 4°C. The upper phase, containing the plasma membrane fraction, was recovered, diluted twofold with the buffer 10 mM MOPS-BTP, 250 mM sucrose, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, pH 7.0 and ultracentrifuged for 45 min at 125000 g at 4°C. The pellet was resuspended in 2 ml of GTED 20 buffer (10 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 20% glycerol, pH 7.6) and stored at -80°C.

SDS-PAGE and Western blot

Immunodecoration of 14-3-3 and H+-ATPase was performed according to Muzi et al. [27]. In particular, plasma membrane proteins were separated by SDS-PAGE (Laemmli, 1970) using a mini-gel apparatus (Bio-Rad, Hercules, CA), then electroblotted onto a PVDF membrane with 39 mM glycine, 48 mM Tris, 0.1% SDS, 10% methanol. After blocking for 1 h in TTBS (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.05% Tween 20) with 5% no-fat dried milk at room temperature, the membrane was incubated with polyconal rabbit anti-14-3-3 antibodies recognizing all plant 14-3-3 isoforms or polyclonal anti-H+-ATPase antibodies directed against a conserved region in the C terminal domain. Following three washes with TTBS, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:10000; Bio-Rad) and decorated with Clarity ECL-Western Blotting Kit (Bio-Rad).

Overlay assay

The cDNA of the 14-3-3 isoform ω from Arabidopsis (14-3-3 PROTEIN G-BOX FACTOR14 OMEGA, AT1G78300), cloned into pGEX-2TK vector, was expressed in Escherichia coli, as previously described [28]. The expression system produces a GST-fused 14-3-3 containing a cAMP-dependent protein kinase phosphorylation site and a thrombin site between the two polypeptides. The 32P-labeled 14-3-3 ω was obtained as described by Pallucca et al. [28]. The specific activity of 32P-labeled 14-3-3 was 3.4 MBq/mg.

The overlay assay was performed according to Camoni et al. [29], with slight modifications. Two-phase partitioned plasma membranes (10 μg protein) were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane, using a semidry apparatus (2 h, 0.8 mA cm-2). The membrane was blocked with 5% milk (fatty-acid–free) in 25 mM Hepes-OH, 5 mM MgCl2, 75 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.04% Tween-20, pH 7.5 (buffer H) and then incubated overnight at 4°C in the same buffer containing 3% fatty-acid–free milk and 32P-labeled 14-3-3ω (8.0 kBq/ml). The membrane was then washed extensively with buffer H, dried and subjected to autoradiography. The densitometric analysis was performed using ImageJ image-processing software [30]. The densitometric values are expressed as a percentage of the maximum integrated densitometric value (the product of the area and mean grey value).

Protein content and H+-ATPase activity

Protein concentration was determined by the method set out by Bradford [31], using bovine serum albumin as a standard. H+-ATPase activity was assayed by measuring the release of inorganic phosphate, after Serrano [26]. In particular, 15 μg of plasma membrane vesicles from Lima bean leaves were incubated for 30 min in 500 μl of incubation buffer (50 mM Tris-MES, 5 mM MgSO4, 5 mM KNO3, 0.2 mM (NH4)6Mo7O24, pH 6.5) in the presence of 2 mM ATP. The reaction was stopped by the addition of 1 ml of phosphate reagent (0.5% SDS, 0.5% (NH4)6Mo7O24, 2% (v/v) H2SO4) and 0.01% ascorbate. The phosphate released during the reaction was determined by measuring the absorbance at 740 nm. The phosphate concentration was calculated by interpolation with a calibration curve obtained with different concentrations of potassium phosphate. For each sample, the residual activity in the presence of 0.2 mM of the H+-ATPase inhibitor ortovanadate was determined and subtracted from the obtained values for the calculation of H+-ATPase specific activity.

Statistical analysis

The data given represent the mean of four independent experiments. Statistical significance was assessed by unpaired Student’s t-tests. All values are expressed as means ± s.e.m.

The minimal data set necessary to replicate the study findings is reported in S1 Dataset.

Results

The H+-ATPase activators fusicoccin and okadaic acid counteract the Spodoptera littoralis OS-induced plasma membrane depolarization in Lima bean leaves

The OS of S. littoralis induce a fast and persistent Vm depolarization of Lima bean leaves [8]. In order to investigate the possible role of the plasma membrane H+-ATPase on the Vm depolarization induced by S. littoralis OS, we evaluated the effect of OS perfusion on the Lima bean leaf Vm in the presence of either fusicoccin (FC) or okadaic acid (OKA). These natural toxins are known to induce a Vm hyperpolarization by H+-ATPase activation. The proton pump activation by FC is the consequence of the irreversible stabilization of the H+-ATPase/14-3-3 complex [16–19, 21], whereas the protein phosphatase 1-2A inhibitor OKA [22] activates the H+-ATPase by blocking the dephosphorylation of its C-terminal domain, consequently promoting 14-3-3 binding [18].

As expected, perfusion of S. littoralis OS induced a rapid and marked significant (P < 0.05) Vm depolarization in the Lima bean leaf, with respect to the starting conditions (Fig 1A). The addition of either FC or OKA to OS-depolarized leaves induced a significant (P < 0.05) Vm hyperpolarization, with respect to the OS-depolarized Vm value, by partially (FC) or fully (OKA) restoring the initial Vm potential (Fig 1A). When FC and OKA were perfused through Lima bean leaves, both drugs induced, to a different extent, a significant (P< 0.05) Vm hyperpolarization (Fig 1B), with respect to the starting condition. Intriguingly, the simultaneous administration of both of the inhibitors and S. littoralis OS partly reduced the OKA effect (Fig 1C), with respect to the sole administration of OKA (Fig 1B), whereas the combined effect of OS + FC (Fig 1C) was almost identical to the effect of the sole FC administration (Fig 1B). These data suggest that the proton pump could be a possible target of OS action.

thumbnail
Download:
Fig 1. Effect of Spodoptera littoralis oral secretions (OS), okadaic acid (OKA) and fusicoccin (FC) on Phaseolus lunatus plasma membrane potential (Vm).

A, Perfusion of OS causes a strong and significant (P < 0.05) Vm depolarization of P. lunatus mesophyll cell Vm, which is partly recovered by the addition of FC and totally restored to the initial Vm values by the addition of OKA. B, The perfusion of P. lunatus mesophyll cells with either FC or OKA causes a strong and significant (P < 0.05) Vm hyperpolarization, with a significantly higher OKA-dependent Vm hyperpolarization. C, The simultaneous perfusion of P. lunatus mesophyll cells with either OS + FC or OS + OKA shows inhibitory effects of the two toxins on the Vm depolarizing action of OS. Metric bars indicate standard deviation.

https://doi.org/10.1371/journal.pone.0202142.g001

Spodoptera littoralis OS inhibit the activity of the Lima bean plasma membrane H+-ATPase

In order to investigate whether the OS-induced Vm depolarization is also caused by the direct inhibition of the proton pump, we studied the in vivo effect of OS treatment on the phosphohydrolitic activity of the plasma membrane H+-ATPase. Lima bean leaves were treated with either OS or FC. Then plasma membranes were isolated by two-phase partitioning and the H+-ATPase activity was assayed in vitro. Treatment with OS consistently inhibited (-51%) the phosphohydrolitic activity of the H+-ATPase (Fig 2), with respect to the enzyme isolated from mock-treated leaves. When Lima bean leaves were simultaneously treated with OS and FC, the OS inhibitory effect was still present, albeit to a lower extent (-28%) (Fig 2).

thumbnail
Download:
Fig 2. In vivo OS treatment inhibition of the of P. lunatus plasma membrane H+-ATPase activity.

Apices of P. lunatus leaves were treated with either OS or FC and the vanadate-sensitive ATPase phosphohydrolitic activity was assayed as described in Materials and Methods. A. ATPase activity of whole leaf plasma membranes purified from controls and OS-treated plants without (-FC) and with the use of fusicoccin (+FC). B, ATPase activity of proximal half leaf plasma membranes purified from controls and OS-treated plants without (-FC) and with the use of fusicoccin (+FC). C, ATPase activity of plasma membranes purified from the distal half of the leaf plasma membranes purified from controls and OS-treated plants without (-FC) and with the use of fusicoccin (+FC). Data given are the mean of four independent experiments. Metric bars indicate standard deviation. Asterisk indicates significant (P< 0.05) differences.

https://doi.org/10.1371/journal.pone.0202142.g002

Previous studies have demonstrated the existence of an OS-induced signal that travels through the phloem and induces the Vm depolarization of plant tissues in a systemic way [4]; therefore, we tested whether OS could induce H+-ATPase inhibition distantly from the site of OS application. To this purpose, Lima bean leaf apices were treated with OS or FC. After 5 h incubation, the leaves were cut in two parts, a proximal (towards the apex) and a distal (towards the petiole) portion, and the plasma membranes were extracted and purified. The plasma membrane H+-ATPase activity was similarly inhibited by OS both in the proximal and the distal half (Fig 2) of the Lima bean leaves.

Spodoptera littoralis OS inhibits the association of 14-3-3 proteins with the Lima bean plasma membrane

The inhibitory effect of OS on H+-ATPase activity can be potentially ascribed either to the inhibition of gene expression or to a post-translational mechanism involving the inhibition of 14-3-3 binding to the enzyme. We therefore analyzed H+-ATPase and 14-3-3 levels in the plasma membrane by western blot analysis in both control and OS-treated Lima bean leaves. The amount of H+-ATPase was not affected by the OS treatment either in the presence or absence of FC (Fig 3). On the other hand, the levels of membrane-bound 14-3-3 proteins were significantly decreased by the treatment with OS, also without the presence of FC (-50% by densitometric analysis, Fig 3). However, FC-treated leaves showed a significant (P < 0.05) increase of 14-3-3 levels when compared to leaves that were not treated with FC. Therefore, OS treatment, while ineffective on H+-ATPase content, was found to inhibit (-34%) association between 14-3-3 and the plasma membrane.

thumbnail
Download:
Fig 3. In vivo OS treatment inhibits the association of P. lunatus 14-3-3 proteins with the plasma membrane.

A, SDS-PAGE analysis of plasma membranes of controls and OS-treated plants without (-FC) and with the use of fusicoccin (+FC) blotted onto nitrocellulose and probed with anti-H+-ATPase. B, SDS-PAGE analysis of plasma membranes of controls and OS-treated plants without (-FC) and with the use of fusicoccin (+FC) blotted onto nitrocellulose and probed with anti-14-3-3 antibodies. C, Densitometric analysis of H+-ATPase in controls (white bars) and OS-treated (black bars) plants without (-FC) and with the use of fusicoccin (+FC). D, Densitometric analysis of 14-3-3 proteins in controls and OS-treated plants without (-FC) and with the use of fusicoccin (+FC). Data are the mean of four independent experiments. Metric bars indicate standard deviation. C and D, expressed as percentage of maximum densitometric value. Asterisk indicates significant (P< 0.05) differences.

https://doi.org/10.1371/journal.pone.0202142.g003

Spodoptera littoralis OS inhibit the interaction between the H+-ATPase and 14-3-3 proteins in Lima bean

In order to verify whether OS were able to inhibit the association between H+-ATPase and 14-3-3 proteins, an in vitro overlay assay was performed. Plasma membranes from OS-treated Lima bean leaves were subjected to SDS–PAGE, blotted onto a nitrocellulose membrane and probed with 32P-labeled 14-3-3. OS treatment significantly (P < 0.05) reduced the association between the H+-ATPase and 14-3-3 proteins, both in the absence (-43%) and in the presence (-35%) of FC (Fig 4). These results suggest that the H+-ATPase might be a possible target of OS action.

thumbnail
Download:
Fig 4. In vivo OS inhibition of the interaction between H+-ATPase and 14-3-3 proteins.

A, Autoradiography and overlay assay performed using plasma membranes purified from control (C) and OS-treated P. lunatus leaves without (-FC) and after the addition (+FC) of 10 μM FC. B, Densitometric analysis of H+-ATPase controls (white bars) and OS-treated leaves (black bars) without (-FC) and with the use of fusicoccin (+FC). Data given are the mean of four independent experiments. Metric bars indicate standard deviation.

https://doi.org/10.1371/journal.pone.0202142.g004

Discussion

The plasma membrane is the first line of defense against pathogens and herbivores. In plant-herbivore and plant-microorganism interactions, the plasma membrane responds with a Vm depolarization, depending on the nature of the biotic attack [32]. This Vm depolarization is associated with a Ca2+-dependent opening of K+ channels [4]; however, Vm depolarization has been found to persist for several hours after the application of OS following mechanical damage or after an herbivore attack [8, 9, 32]. Evidently, other factors besides K+ channel activity must be involved in order to sustain such a persistent Vm depolarization upon herbivory. For the first time, we provide evidence that the sustained Vm depolarization occurring after the administration of herbivore’s OS is associated with the inhibition of the plant plasma membrane H+-ATPase.

The first evidence of the direct involvement of H+-ATPase in response to the insect’s OS was the ability of FC or OKA to recover the membrane Vm depolarization, demonstrating that activation of the plasma membrane H+-ATPase can counteract the Vm depolarizing effects of OS. Interestingly, some differences between the OKA and FC effect could be observed. In fact, the protein phosphatase inhibitor OKA [18], which activates the H+-ATPase by blocking dephosphorylation of its C-terminal domain and consequently promoting 14-3-3 binding [19], completely restored the hyperpolarized Vm in OS-depolarized leaves, whereas FC was only able to partially suppress the OS effect. However, when OKA and OS were administrated simultaneously, the protein phosphatase inhibitor was not able to completely hamper the OS’ depolarizing effect. On the contrary, FC fully blocked the effects of the OS when administrated simultaneously. This effect can be ascribed to the ability of FC to irreversibly stabilize the H+-ATPase/14-3-3 complex [16–19] and suggests that the proton pump is involved in the OS-mediated Vm depolarization.

Upon herbivory, a wave of Vm depolarization spreads from the wounded zone to the rest of the leaf [8]. This event is associated with a hypothetical phloem mobile signal [2] that travels through active plasmodesmata [4]. The finding that the H+-ATPase activity of the plasma membrane was similarly inhibited by OS both in the proximal and the distal half of Lima bean leaves, indicates that OS can trigger the inhibition of the proton pump in intact (unwounded) tissues as well, as a result of a long-distance transport, in agreement with the hypothesis of a moving signaling molecule [2 and references cited therein].

It is well known that H+-ATPase is the main target of 14-3-3 proteins at the plasma membrane and its activation status is strictly correlated to membrane-bound 14-3-3 levels [18, 33]. As expected, 14-3-3 levels increased in plasma membranes purified from FC-treated leaves, since the toxin stabilizes 14-3-3 binding to the H+-ATPase [19]. It is well established that the H+-ATPase/14-3-3 interaction depends on the phosphorylation status of a Thr residue within a binding sequence located at the extreme end of the H+-ATPase C-terminus [16, 34]. Hence, the reduction in 14-3-3 proteins binding to H+-ATPase upon OS treatment could be ascribed to a Thr dephosphorylation. However, other mechanisms could be involved in the dissociation of 14-3-3 proteins induced by OS. In fact, it has been recently shown that the peptide hormone RALF and the flagellin fragment flg22 induce phosphorylation of Ser-899 in the H+-ATPase C terminus, which can inhibit the association of 14-3-3 proteins with the phosphorylated binding site Tyr-pThr-Val [11, 35, 36].

To conclude, the present results, along with previous data, make it possible to better depict the early events taking place in a plant upon herbivory. Chewing insects such as S. littoralis release OS into wounded plant tissues and induce a calcium-dependent opening of K+ channels that causes a strong and sudden Vm depolarization. OS also inhibit the H+-ATPase activity by inhibiting the phosphorohydrolitic activity of the proton pump. Moreover, OS inhibit the association between 14-3-3 proteins and H+-ATPase, resulting in a reduced activity of the proton pump (with a reduced extrusion of H+ from the cytosol). It is likely that upon recognition of elicitors present in the OS the plant activates a signaling cascade that potentially de-phosphorylates the ATPase leading to its dissociation with the 14-3-3 proteins and reduced activity and subsequent effect on Vm. Overall, these events cause a Vm depolarization (Fig 5) that triggers late events that induce the typical plant response to herbivory [2].

thumbnail
Download:
Fig 5. Early events upon S. littoralis herbivory on Lima bean leaves.

Herbivory delivers oral secretions (OS) in wounded plant tissues that induce a calcium-dependent opening of K+ channels. The increased cytosolic K+ concentration causes a strong and sudden Vm depolarization. OS also inhibits the H+-ATPase activity by hampering the association between 14-3-3 and H+-ATPase proteins. The inhibitory action of OS causes a reduced phosphohydrolitic activity of the proton pump with a reduced extrusion of H+ from the cytosol. Overall, these events concur to the K+-dependent Vm depolarization.

https://doi.org/10.1371/journal.pone.0202142.g005

Further studies will assess the immuno-purification of the ATPase from the plasma membranes and then the interaction assays on the purified protein. However, the overlay assay used in this study is recognized as an appropriate assay to study the H+-ATPase/14-3-3 interaction, by allowing to finely measure the affinity of the enzyme, which is strictly dependent to its phosphorylation status, towards a recombinant14-3-3 protein, as previously reported [16, 17, 19, 21].

References

  1. 1. Maffei ME, Arimura GI, Mithoefer A. Natural elicitors, effectors and modulators of plant responses. Natural Product Reports. 2012;29(11):1288–303. pmid:22918379
  2. 2. Zebelo AS, Maffei ME. Role of early signalling events in plant–insect interactions. J Exp Bot. 2015;66:435–48. pmid:25429000
  3. 3. Maffei ME, Mithöfer A, Boland W. Before gene expression: early events in plant-insect interaction. Trends in Plant Science. 2007;12(7):310–6. pmid:17596996
  4. 4. Bricchi I, Occhipinti A, Bertea CM, Zebelo SA, Brillada C, Verrillo F, et al. Separation of early and late responses to herbivory in Arabidopsis by changing plasmodesmal function. Plant Journal. 2013;73(1):14–25. pmid:22775399
  5. 5. Arimura G, Ozawa R, Maffei ME. Recent Advances in Plant Early Signaling in Response to Herbivory. Int J Mol Sci. 2011;12(6):3723–39. pmid:21747702
  6. 6. Arimura GI, Maffei ME. Calcium and secondary CPK signaling in plants in response to herbivore attack. Biochem Biophys Res Comm. 2010;400(4):455–60. pmid:20816754
  7. 7. Maffei ME, Mithöfer A, Boland W. Insects feeding on plants: Rapid signals and responses preceding the induction of phytochemical release. Phytochemistry. 2007;68(22–24):2946–59. pmid:17825328
  8. 8. Maffei M, Bossi S, Spiteller D, Mithöfer A, Boland W. Effects of feeding Spodoptera littoralis on lima bean leaves. I. Membrane potentials, intracellular calcium variations, oral secretions, and regurgitate components. Plant Physiol. 2004;134(4):1752–62. pmid:15051862
  9. 9. Maffei ME, Mithöfer A, Arimura GI, Uchtenhagen H, Bossi S, Bertea CM, et al. Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide. Plant Physiology. 2006;140(3):1022–35. pmid:16443697
  10. 10. Arimura GI, Kopke S, Kunert M, Volpe V, David A, Brand P, et al. Effects of feeding Spodoptera littoralis on lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission. Plant Physiology. 2008;146(3):965–73. pmid:18165324
  11. 11. Falhof J, Pedersen JT, Fuglsang AT, Palmgren M. Plasma Membrane H+-ATPase Regulation in the Center of Plant Physiology. Molecular Plant. 2016;9(3):323–37. PubMed PMID: WOS:000371975400003. pmid:26584714
  12. 12. Hedrich R, Schroeder JI. THE PHYSIOLOGY OF ION CHANNELS AND ELECTROGENIC PUMPS IN HIGHER-PLANTS. Annual Review of Plant Physiology and Plant Molecular Biology. 1989;40:539–69. PubMed PMID: WOS:A1989U857900020.
  13. 13. Elmore JM, Coaker G. The Role of the Plasma Membrane H(+)-ATPase in Plant-Microbe Interactions. Molecular Plant. 2011;4(3):416–27. pmid:21300757
  14. 14. Aducci P, Camoni L, Marra M, Visconti S. From cytosol to organelles: 14-3-3 proteins as multifunctional regulators of plant cell. Iubmb Life. 2002;53(1):49–55. PubMed PMID: WOS:000175201700007. pmid:12018408
  15. 15. Denison FC, Paul AL, Zupanska AK, Ferl RJ. 14-3-3 proteins in plant physiology. Seminars in Cell & Developmental Biology. 2011;22(7):720–7. PubMed PMID: WOS:000296578000009. pmid:21907297
  16. 16. Fuglsang AT, Visconti S, Drumm K, Jahn T, Stensballe A, Mattei B, et al. Binding of 14-3-3 protein to the plasma membrane H+-ATPase AHA2 involves the three C-terminal residues Tyr(946)-Thr-Val and requires phosphorylation of Thr(947). Journal of Biological Chemistry. 1999;274(51):36774–80. PubMed PMID: WOS:000084279200105. pmid:10593986
  17. 17. Visconti S, Camoni L, Fullone MR, Lalle M, Marra M, Aducci P. Mutational analysis of the interaction between 14-3-3 proteins and plant plasma membrane H+-ATPase. Journal of Biological Chemistry. 2003;278(10):8172–8. PubMed PMID: WOS:000181466800055. pmid:12509351
  18. 18. Camoni L, Iori V, Marra M, Aducci P. Phosphorylation-dependent interaction between plant plasma membrane H+-ATPase and 14-3-3 proteins. Journal of Biological Chemistry. 2000;275(14):9919–23. PubMed PMID: WOS:000086345600006. pmid:10744665
  19. 19. Würtele M, Jelich-Ottmann C, Wittinghofer A, Oecking C. Structural view of a fungal toxin acting on a 14-3-3 regulatory complex. Embo Journal. 2003;22(5):987–94. PubMed PMID: WOS:000181406300001. pmid:12606564
  20. 20. Hager A. Role of the plasma membrane H+-ATPase in auxin-induced elongation growth: historical and new aspects. J Plant Res. 2003;116(6):483–505. pmid:12937999
  21. 21. Camoni L, Visconti S, Aducci P. The Phytotoxin Fusicoccin, a Selective Stabilizer of 14-3-3 Interactions? Iubmb Life. 2013;65(6):513–7. PubMed PMID: WOS:000320777300004. pmid:23568668
  22. 22. Bialojan C, Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases—Specificity and kinetics. Biochemical Journal. 1988;256(1):283–90. PubMed PMID: WOS:A1988R045200041. pmid:2851982
  23. 23. Ballio A, Chain EB, De Leo P, Erlanger BF, Mauri M, Rouola A. Fusicoccin: a new wilting toxin produced by Fusicoccum amygdali Del. Nature. 1964;203:297.
  24. 24. Zebelo SA, Maffei ME. The Ventral Eversible Gland (VEG) of Spodoptera littoralis triggers early responses to herbivory in Arabidopsis thaliana. Arthr Plant Interact. 2012;6:543–51.
  25. 25. Zebelo SA, Maffei ME. Signal transduction in plant–insect interactions: From membrane potential variations to metabolomics. In: Volkov A, editor. Plant Electrophysiology. Berlin Heidelberg: Springer-Verlag; 2012. p. 1–30.
    • 26. Serrano R. H+-ATPASE FROM PLASMA-MEMBRANES OF SACCHAROMYCES-CEREVISIAE AND AVENA-SATIVA ROOTS—PURIFICATION AND RECONSTITUTION. Methods in Enzymology. 1988;157:533–44. PubMed PMID: WOS:A1988R318900040. pmid:2906717
    • 27. Muzi C, Camoni L, Visconti S, Aducci P. Cold stress affects H+-ATPase and phospholipase D activity in Arabidopsis. Plant Physiology and Biochemistry. 2016;108:328–36. PubMed PMID: WOS:000386989700035. pmid:27497302
    • 28. Pallucca R, Visconti S, Camoni L, Cesareni G, Melino S, Panni S, et al. Specificity of epsilon and Non-epsilon Isoforms of Arabidopsis 14-3-3 Proteins Towards the H+-ATPase and Other Targets. Plos One. 2014;9(3). PubMed PMID: WOS:000332483600083. pmid:24603559
    • 29. Camoni L, Visconti S, Marra M, Aducci P. Adenosine 5 '-monophosphate inhibits the association of 14-3-3 proteins with the plant plasma membrane H+-ATPase. Journal of Biological Chemistry. 2001;276(34):31709–12. PubMed PMID: WOS:000170613500033. pmid:11423544
    • 30. Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with ImageJ. Biophot Int. 2004;11:36e42.
    • 31. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. pmid:942051
    • 32. Bricchi I, Bertea CM, Occhipinti A, Paponov IA, Maffei ME. Dynamics of Membrane Potential Variation and Gene Expression Induced by Spodoptera littoralis, Myzus persicae, and Pseudomonas syringae in Arabidopsis. PLoS One. 2012;7(10).
    • 33. Olivari C, Meanti C, De Michelis MI, Rasi-Caldogno F. Fusicoccin binding to its plasma membrane receptor and the activation of the plasma membrane H+-ATPase—IV. Fusicoccin induces the association between the plasma membrane H+-ATPase and the fusicoccin receptor. Plant Physiology. 1998;116(2):529–37. PubMed PMID: WOS:000072021600011. pmid:9489010
    • 34. Svennelid F, Olsson A, Piotrowski M, Rosenquist M, Ottman C, Larsson C, et al. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell. 1999;11(12):2379–91. PubMed PMID: WOS:000084529600011. pmid:10590165
    • 35. Nühse TS, Bottrill AR, Jones AME, Peck SC. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant Journal. 2007;51(5):931–40. PubMed PMID: WOS:000249424100016. pmid:17651370
    • 36. Haruta M, Sabat G, Stecker K, Minkoff BB, Sussman MR. A Peptide Hormone and Its Receptor Protein Kinase Regulate Plant Cell Expansion. Science. 2014;343(6169):408–11. PubMed PMID: WOS:000330039300045. pmid:24458638