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First published online 28 August 2007
doi: 10.1242/jcs.011098

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Antagonistic action of harpin proteins: HrpW_ea from Erwinia amylovora suppresses HrpN_ea-induced cell death in Arabidopsis thaliana

¹ LEM, EA 3514, Université Paris Diderot, Case 7069, 2 place Jussieu, 75251 Paris cedex 5, France
² Laboratoire de Pathologie Végétale, UMR 217 INRA-INA-Paris VI, INA-PG, 16 rue Claude Bernard, 75231 Paris cedex 5, France
³ General Microbiology, Department of Biological and Environmental Sciences, P.O.B. 56, 00014 University of Helsinki, Finland

View larger version (9K): [in this window] [in a new window]   Fig. 1. Effects of Erwinia amylovora wild-type (wt), hrpWea, hrpNea and hrpNea-hrpWea mutant strains on electrolyte leakages from A. thaliana foliar disks. Disks were infiltrated with bacterial suspensions of 3x108 cells/ml. Values are means of three replicates and error bars represent s.e.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effect of increasing concentrations of HrpWea (A), or HrpNea (B), on cell death of A. thaliana suspension cells. Desalting buffer did not modify cell viability. Values are means of four replicates and error bars represent s.e.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Effect of 200 nM HrpWea or HrpNea on H2O2 production and on cytosolic Ca2+ level ([Ca2+]cyt) of A. thaliana suspension cells. (A) Time course of H2O2 accumulation in the medium of cells treated with 200 nM HrpWea, 200 nM HrpNea or the corresponding volume of desalting buffer. Half dilution of cell suspension with distilled water triggered hypo-osmotic stress used as positive control. (B) Percentage of increase of H2O2 accumulation, at 1 hour, in the medium of cells treated with 200 nM HrpWea or HrpNea, alone or in combination with 10 µM DPI or by the corresponding volume of desalting buffer. Values are given as a percentage with respect to untreated cells (100%). Values are means of eleven replicates. Error bars represent s.e. (C) Changes in [Ca2+]cyt were measured by using transgenic A. thaliana cells expressing aequorin protein. Cells suspended in culture medium containing 1 mM Ca2+, were treated with 200 nM HrpWea or HrpNea or the corresponding volume of desalting buffer. Hypo-osmotic stress induced by diluting the cell suspension 1:1 with distilled water was used as a positive control. Five independent experiments were carried out, with similar results.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Modulations of membrane potential (Vm), K+ outward rectifying currents (KORCs) and anion currents of A. thaliana cells in response to 200 nM HrpWea or HrpNea. (A) Free-running Vm observed in response to HrpWea, HrpNea or the desalting buffer. (B) Mean values of the Vm variations. (C) Effect of 200 nM HrpWea on KORCs recorded at +80 mV (leak substracted). The activated currents were measured when the maximum effect of HrpWea was reached. Vh, holding potential; Vm, membrane potential. (D) Current-voltage relationships. The steady-state current amplitudes were measured at membrane potentials ranging from –200 to +80 mV. (E) Mean amplitudes of steady state KORCs (at 80 mV) in response to 200 nM HrpWea, HrpNea alone, HrpNea and 10 mM TEA or the corresponding desalting buffer volume. Values are given as a percentage with respect to current value before treatment. (F) Effect of 200 nM HrpWea on anion current recorded at –200 mV. Currents were measured when the maximum effect of HrpWea was reached. (G) Corresponding current-voltage curves. (H) Mean amplitudes of anion currents in response to 200 nM HrpWea alone, HrpWea and 10 µM Glibenclamide or 40 µM 9AC, 200 nM HrpNea or the corresponding desalting buffer volume. Values are given as a percentage with respect to current value before treatment. All values are mean of at least five independent experiments. Error bars represent s.e.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Inhibitory effect of HrpWea on cell death triggered by HrpNea. (A) Inhibition of HrpNea-induced cell death by increasing concentration of HrpWea (from 0.002 nM to 200 nM) in A. thaliana suspension cells. (B) Increasing concentrations of HrpNea (from 0.002 nM to 200 nM) do not modify HrpWea-induced cell death in A. thaliana suspension cells. Values shown in both A and B are means of four replicates and error bars represent s.e.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Inhibitory effect of HrpWea on H2O2 production and ion channel modulations triggered by HrpNea. (A) Inhibition of 200 nM HrpNea-induced H2O2 production by 0.2 nM HrpWea. Values are given as a percentage with respect to untreated cells (100%). Values are means of four replicates. (B) Inhibition of 200 nM HrpNea-induced KORC activation by 0.2 nM HrpWea. (C) Inhibition of 200 nM HrpNea-induced anion current decrease by 0.2 nM HrpWea. Values are given as a percentage with respect to currents recorded before treatment. Values are means of at least four replicates. Error bars represent s.e.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Increasing concentrations of HrpWea (from 0.002 nM to 200 nM) do not modify 200 nM HrpZpph-induced cell death in A. thaliana suspension cells. Values are means of three replicates and error bars represent s.e.

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