Doi:10.1016/j.plaphy.2005.03.003

Plant Physiology and Biochemistry 43 (2005) 567–572 A CFTR chloride channel activator prevents HrpN -induced cell death in Arabidopsis thaliana suspension cells David Reboutier a,b,*, Cécile Frankart a, Régine Vedel b, Mathias Brault a, Ronald G. Duggleby c, Jean Pierre Rona a, Marie Anne Barny b, François Bouteau a a LEM, EA 3514, Université Paris 7, Case 7069, 2, place Jussieu, 75251 Paris cedex 5, France b Laboratoire de Pathologie Végétale, UMR 217 Inra-INA-Paris VI, INA-PG, 16, rue Claude Bernard, 75231 Paris cedex 5, France c Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Qld. 4072, Australia Received 6 January 2005; accepted 17 March 2005 Abstract
Erwinia amylovora is a necrogenic bacterium that causes fire blight of the Maloideae subfamily of Roseacae, such as apple and pear. It provokes necrosis in aerial parts of susceptible host plants and the typical hypersensitive reaction in non-host plants. The secreted harpin,HrpN , is able by itself to induce an active cell death in non-host plants. Ion flux modulations were shown to be involved early in such processes but very few data are available on the plasma membrane ion channel activities responsible for the pathogen-induced ion fluxes. Weshow here that HrpN induces cell death in non-host Arabidopsis thaliana suspension cells. We further show that two cystic fibrosis trans- membrane conductance regulator modulators, glibenclamide and bromotetramisole, can regulate anion channel activities and HrpN -induced cell death.
2005 Elsevier SAS. All rights reserved.
Keywords: Anion channel; Arabidopsis thaliana; Cell death; CFTR; Harpin 1. Introduction
membrane potential and ion flux variations are among theearliest signaling events detectable in response to pathogens Erwinia amylovora is a bacterial pathogen that causes fire and elicitors However, the underlying ion chan- blight disease of apple, pear and other members of the nel activities have been recorded rarely. Indeed, most of the Rosaceae, its host plants. It secretes the HrpN electrophysiological studies on plant cells are performed with “hypersensitive response” (HR) elicitor HR cell death patch-clamp technique applied to protoplasts and it seems is a response of non-host plant to pathogen attack and con- that the cell wall removing (protoplasts preparation) alters sists of a rapid necrosis at the site of infection that cordons the cell response capacity to pathogen or elicitor off the pathogen and limits its spread There is a grow- Another approach to analysis of ion channels in intact cells ing consensus that HR is similar to animal programmed cell that retains their cell wall is the microelectrode voltage- death (PCD) and that ion channel regulation is a necessary clamp technique. This technique allows long-term recording of the free running membrane potential and whole cell ioncurrents, the internal medium remaining physiological (com-position non-controlled). We have shown previously, using single microelectrode voltage-clamp (DSEVC), that Arabi- AHAS, acetohydroxyacid synthase; AVD, apoptosis volume decrease; CFTR, cystic fibrosis transmembrane conductance regu- dopsis thaliana suspension cells respond to the fungal elici- lator; DMSO, dimethyl sulfoxide; DSEVC, discontinuous single electrode tor hypaphorine in the same way as root hairs, its natural tar- voltage-clamp; HR, hypersensitive response; KORC, K+ outward rectifying get Other studies demonstrated that suspension cultured current; MAPK, mitogen activated protein kinase; PCD, programmed cell cells are a powerful system of reduced complexity to analyze the signal transduction pathway induced by pathogens * Corresponding author. Tel.: +33 1 44 27 60 57; fax: +33 1 44 27 78 13.
E-mail address: [email protected] (D. Reboutier).
Thus, we used A. thaliana suspension cells to inves- 0981-9428/$ - see front matter 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2005.03.003 D. Reboutier et al. / Plant Physiology and Biochemistry 43 (2005) 567–572 tigate early signaling events induced by HrpN . We showed rents suggesting indirect effects on the channel proteins.
Regulation of anion fluxes was reported in pathogen-inducedplant cell death. For example, a large nitrate efflux is neces-sary to induce cell death in tobacco in response to cryptogeinMoreover, anion channel antagonists have beenshown to interfere with elicitor or pathogen-induced responsessuch as Ca2+ influx production of active oxygen speciesMAPK activation and phytoalexin synthesisIn animal cells, Maeno et al. showed that apop-tosis volume decrease (AVD: cell shrinkage) is a major hall-mark of PCD. This AVD is due to a strong activation of ioneffluxes. In plant cells, the increase of anion effluxes inresponse to pathogen elicitors is consistent with theAVD. However, in A. thaliana suspension cells, we observeda decrease in anion current (efflux decrease) in response toHrpN These data did not fit with the observations described above but are closely related to those reported forhepatoblastoma apoptosis In this last model, apoptosisis induced by the decrease in cystic fibrosis transmembraneconductance regulator (CFTR) anion currents (members ofABC transporter superfamily). In plant, CFTR modulators,were shown to be effective on slow anion channels of Viciafaba guard cells Moreover, Leonhardt et al. showed,using antibodies, that slow anion channels are, or are closely,controlled by a polypeptide exhibiting an epitope shared with Fig. 1. Effect of glibenclamide on anion currents.
the mammalian CFTR. Lastly, AtMRP5, a protein of the ABC (A) Anion currents measured under control conditions and after adding 10 µMglibenclamide in the culture medium. Voltage pulses were –200 mV. Hol- transporter superfamily, which has a high similarity to CFTR ding potential was V . (B) Dose-dependent decrease of anion currents as a and which is sensitive to glibenclamide, was suggested to con- function of glibenclamide concentrations. Data represent the maximal effect.
trol ion channels In this study, our aim was to investi- They were obtained from at least three independent experiments and were gate the putative involvement of the anion current decrease in fitted by a 5-parameter double exponential. The error bar corresponds to onestandard error.
HrpN -induced cell death on A. thaliana suspension cells (non-host plant) by using CFTR modulators, glibenclamide, cell CFTR-Cl– currents are inhibited at a half-maximal a sulfonylurea (SU) molecule, as an inhibitor and bromotet- concentration of 20 µM apoptosis is induced at 1 mM ramisole as an activator, according to the hypothesis devel- Thus, we tested the effect of different concentrations of glibenclamide on anion currents. The deactivating currentspreviously characterized as anion currentsare sensitive to glibenclamide in a dose-dependent 2. Results
manner The glibenclamide concentration for half-maximum inhibition of anion current observed in our model HrpN at 5 µg ml–1 (0.13 µM), a classically used HrpN is about 7 µM of the same order of magnitude as concentration increases cell death in comparison with those observed for slow-type anion currents on plant cells the cells treated with negative control According to Thus, we tested the effect of 10 µM glibenclamide on our hypothesis we thus tested in our model the effect of glib- cell death. After 24 h, glibenclamide increased cell death, enclamide and bromotetramisole. In animal systems, the con- mimicking the HrpN -induced cell death Although centrations of glibenclamide tested are highly variable: whole- glibenclamide is effective on anion currents in our modeland in V. faba guard cells we checked if the glibenclamide-induced cell death might be due to other known Modulation of HrpN -induced cell death by anion channel modulators glibenclamide or SU effects, i.e. K+ channel inhibition Increase in cell death after a 24 h treatment with glibenclamide, HrpN alone or herbicidal activity Glibenclamide (10 µM) does not or mixed with bromotetramisole. Variations are given as a percentage withrespect to the control level. Data correspond to mean values ± S.D. and n is induce a change in K+ outward rectifying current (KORC) activity of A. thaliana suspension cells (D I state for a + 80 mV voltage step = 1.3 ± 9%, n = 6, data not shown). Yet in plants, the herbicidal activity of SU family Cell death (%) 21 ± 3 (n = 6) 26 ± 4 (n = 3) acts through AHAS inhibition AHAS catalyses the for- D. Reboutier et al. / Plant Physiology and Biochemistry 43 (2005) 567–572 Fig. 2. Effect of glibenclamide on AHAS activity.
Inhibition of A. thaliana AHAS activity. Duplicate measurements were madeat each inhibitor concentration and the graph shows all values as separatepoints. From the data shown, the inhibition constant (Ki) was determined.
The arrow indicates the AHAS inhibition at 10 µM.
mation of 2-acetolactate and 2-aceto-2-hydroxybutyrate asthe first step in the biosynthesis of the branched-chain aminoacids glibenclamide-induced cell death involves this type of herbi-cidal effect or acts through ion channel regulation, we havetested whether glibenclamide could inhibit AHAS from A.
thaliana. The residual AHAS activity at 10 µM glibencla-mide, compared to the corresponding control, was greater than95% for A. thaliana AHAS. The inhibition constant (Ki) cal-culated for AHAS, 146 ± 7 µM is much higher thanthose of known herbicidal sulfonylureas which have inhibi- Fig. 3. Effect of bromotetramisole on anion currents.
tion constants in the 10–100 nM range for A. thaliana AHAS (A) Anion currents measured under control conditions and after adding 5 µM bromotetramisole in the culture medium. Voltage pulses were –200 mV. Hol-ding potential was V . (B) Dose-dependent increase of anion currents as a In contrast to glibenclamide, the anion currents down regu- function of bromotetramisole concentrations. Data represent the maximal lated by HrpN are stimulated by bromotetramisole in a effect. They were obtained from at least three experiments and were fitted by dose-dependent manner The bromotetramisole half- a 5-parameter double exponential. The error bar corresponds to one standard maximum activation concentration we observed for anion cur- rent in our model is about 20 µM Bromotetrami- sole appeared thus as a useful tool to check if the increase of ever, this decrease fits well with the PCD observed on hepa- anion efflux could interfere with HrpN -induced cell death.
toblastoma cells by Kim et al. These authors showed We tested bromotetramisole at 5 µM a low concentration that that a decrease of CFTR anion current is a necessary step of is able to increase anion currents. At this concentration, bro- PCD development. To test the hypothesis that a decrease in motetramisole counteracted the HrpN -induced cell death anion current is involved in cell death, we used CFTR modu- lators. Glibenclamide is believed to bind the CFTR channelallowing its block Glibenclamide is also a well-knowninhibitor of ATP-dependent K+ channels used as a therapeu- 3. Discussion
tic agent to treat type 2 diabetes If the increase in KORCinduced by HrpN was involved in cell death, the puta- tive inhibition of KORC by glibenclamide should favor sur- death symptom of aggression by an avirulent pathogen.
vival of cells. However, in our model, glibenclamide is effec- We showed that HrpN could induce cell death in A. thaliana tive on anion currents but failed to block the KORC.
suspension cells, a model non-host plant. The extent of Glibenclamide also did not inhibit significantly AHAS HrpN -induced cell death was in the same range as observed and thus failed to induce any herbicidal effect as reported for with another harpin, HrpZ, on A. thaliana suspension cells other SU. Thus, the glibenclamide-induced cell death of A. thaliana suspension cells, mimicking the HrpN -induced cell anion currents (anion effluxes) This result is not in accor- death, most likely involves the inhibition of anion channels.
dance with previous studies suggesting that anion effluxes The protecting effect of bromotetramisole (5 µM) against the are a part of the pathogen or elicitor induced responses lethal effect of HrpN reinforces this hypothesis. Bromotet- D. Reboutier et al. / Plant Physiology and Biochemistry 43 (2005) 567–572 ramisole is an uncompetitive inhibitor specific for alkaline both preparations were compared on a 12% SDS- phosphatase and tyrosine phosphatase. It inhibits a constitu- polyacrylamide gel and quantified using the method of Brad- tive, membrane associated, phosphatase activity and stabi- represent 75% of the total protein prepara- lizes the phosphorylated form of CFTR, thus increasing the activity of CFTR chloride channel In our model, thestimulation of anion current by bromotetramisole is compat- ible with such effects since phosphorylation is required forcomplete activation of slow anion channel A putative Cell cultures (25 ml) were incubated for 15 min with 0.05% phosphorylated form of anion channels could avoid the Evans blue after 24 h of treatment with an effector then HrpN -induced decrease in anion current, thus counteract- washed with 200 ml of deionized water to remove the excess ing cell death. In conclusion, our data suggest that the anion and unbound dye. Dye bound to dead cells was solubilized in channel involved in HrpN -induced cell death is, or is con- 50% methanol with 1% SDS for 60 min at 50 °C and quan- trolled by, a protein exhibiting analogy with the CFTR and tified at 595 nm The data represent an increase in cell highlight the importance of ion channel regulation during the death determined with regard to basal cell death control level.
The control levels correspond to treatment with negative con-trol for HrpN, and/or the solvent used with other effectors,water for bromotetramisole and methanol for glibenclamide.
4. Methods
The final concentration of methanol in all assays was 0.1%(v/v).
4.1. Cell culture and voltage-clamp experiments 4.4. Acetohydroxyacid synthase (AHAS) assays A. thaliana L. (ecotype Columbia) suspension cells were cultured at 24 ± 2 °C, under continuous white light (40 µE m– A. thaliana AHAS was purified as described previously Assays were performed at 30 °C in a mixture containing 2 s–1) with rotation shaking, in a 1-l round bottom flask con-taining 350 ml Gamborg culture medium (main ions after 50 mM pyruvate, 1 mM thiamine diphosphate, 10 mM MgCl2 4 days of culture: 9 mM K+, 11 mM NO – The pH of and 10 µM flavin adenine dinucleotide in 100 mM potassium the culture medium was maintained at 5.8. Cells were sub- phosphate buffer (pH 7.8). A colorimetric assay was cultured weekly by a 10-fold dilution. The experiments were employed. Glibenclamide was dissolved in the organic sol- vent DMSO and added to the assay to a maximum concen- For electrophysiological measurements, the cells were tration of 400 µM. The final concentration of DMSO in all impaled in the culture medium as previously described The microelectrode resistance was 40–50 MX whenfilled with 600 mM KCl. Individual cells were voltage-clamped using an Axoclamp 2B amplifier (Axon Instru- Acknowledgements
ments, Foster City, CA, USA) for discontinuous single elec-trode voltage-clamp experiments Voltage and current We would like to thank the referees for helpful remarks, were digitized with a personal computer fitted with a Digi- D. Expert and H. El-Maarouf for critical reading of the manu- data 1320A acquisition board (Axon Instruments). The elec- script. Y.T. Lee supplied the sample of A. thaliana AHAS.
trometer was driven by pClamp software (pCLAMP8, Axon This work was supported by funds from Inra and from the Instruments). Experiments were performed at 22 ± 2 °C.
MENRT to EA 3514 and ACI 5078. D. Reboutier was sup-ported by a grant from MENRT.
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