Harpins and ion channels modulations

Abstract

Harpins are type three secretion system (TTSS) effectors. While few harpins are thought to be translocators of TTSS effectors through the host plasma membrane during plant/bacteria interactions, functions of many harpins remain for the moment mysterious. We recently showed that the HrpW_ea harpin from Erwinia amylovora, at subnamolar concentration, was able to decrease defense responses triggered by another harpin from this bacteria, HrpN_ea. This antagonism could be the result of opposed anion channels modulations triggered by HrpW_ea and HrpN_ea. At upper concentrations HrpW_ea alone, or in combination with HrpN_ea, was able to induce cell death. This form of cell death involves strong ion channel activation and shares similarity with apoptosis volume decrease (AVD), a form of programmed cell death well described in animal cells. All these results suggest different ways for harpins to trigger cell death and highlight the role of ion channels during cell death processes.

The type III secretion system (TTSS), which is present in most plant and animal pathogenic Gram-negative bacteria, is determinant for many interactions since it allows delivery of specific bacterial proteins to host cell.1 Bacterial TTSS effectors are highly diverse and collectively manipulate host cell metabolism to improve bacterial nutrition or to encounter cell defense mechanisms. Among TTSS effectors, harpins constitute a unique family of TTSS effectors that are not found in mammalian pathogens. While their function still remains unclear during bacteria / host plants interactions, harpins are supposed to be able to trigger HR cell death and associated defense responses when infiltrated in nonhost plants.1

We recently showed by using Erwinia amylovora mutant strains and purified proteins that the HrpW_ea harpin, at subnanomolar concentrations could antagonize cell death and AOS production induced by HrpN_ea, another harpin, secreted by Erwinia amylovora.2 Our results showed that this antagonism is probably the result of opposed ion channels modulations triggered by HrpW_ea and HrpN_ea (Fig. 1A and 1B) and allowed us to hypothesize that HrpW_ea could be important for E. amylovora initial survival in nonhost plants by decreasing plant cell defenses.2 In animal system, YopB, a TTSS translocator of Yersinia, is important for pro-inflammatory signaling response in epithelial cells infected with Y. pseudotuberculosis.3 YopB could stimulate signaling through a cytoplasmic domain following insertion into the membrane or through an interaction with a receptor in the membrane. However, three Yop effectors, YopE, YopH, and YopJ, counteracted this response in infected cells. It is speculated that YopB signaling induces changes in the cell that are required for an efficient translocation process and that once proper amounts of Yops are translocated, the process would be shut down to avoid further cell damage caused by excessive signaling. In the same way, we could speculate that signaling induced by HrpN_ea, a putative translocator of E. amylovora TTSS effectors through the host plasma membrane,4,5 which posses a cytoplasmic domain which harbors a protein binding site,6 is shut down by subnanomolar concentrations of HrpW_ea, allowing a decrease of basal immunity to prolong colonization. Although phenotype of a hrpW_ea mutant strain indicates that HrpW_ea likely acts as a cell death inhibitor when secreted by E. amylovora,2 HrpW_ea, at higher concentrations, was also reported to induce hypersensitive response (HR) on tobacco7,8 and a cell death concomitant with strong anion channels activation on A. thaliana cells.2 This strong activation of anion channels could be responsible for an anion efflux and the cell death triggered by HrpW_ea could be similar to cell death activated in tobacco by the fungal elicitor cryptogein. In this last model, cryptogein induced cell death via anion channel mediated nitrate efflux.9 In addition to nitrate efflux, cryptogein induces K⁺ efflux10 via activation of K⁺ outward rectifying channels (KORC, unpublished data). The cell death triggered by cryptogein could thus be provoked by a mechanism similar to apoptosis volume decrease (AVD) described in animal cells.11 According to this model, PCD could be induced by simultaneous activation of KORC and anion channels in response to an apoptosis inducer. This increase in ion conductance leads to a strong cell volume decrease and activation of a caspase,11 which could be reduced by anion or K⁺ channel inhibitors. Pharmacological blocking of anion efflux prevents the cryptogein-induced cell death and the accumulation of transcripts encoding vacuolar-processing enzymes, a family of proteases showing caspase-1 activity and previously reported to contribute to the disruption of vacuole integrity observed during the HR.9 Such experiments were unsuccessful in our model due to lethal effect of anion channel blockers12 (unpublished data), in contrary of what was observed for tobacco cells.13 However, simultaneous treatment of A. thaliana suspension cells with HrpN_ea and HrpW_ea at 200 nM induced a strong KORC and anion channel activation (Fig. 2) that could be responsible for large electrolyte leakage, similarly to what was observed during cryptogein treatment in plant cells or during AVD in animal cells. This treatment also induces a stronger cell death than cell death observed in response to 200 nM HrpN_ea or 200 nM HrpW_ea alone.2 We thus tested whether the ion channel activations observed could be involved in cell death triggered by simultaneous addition of HrpN_ea and HrpW_ea. Yet, anion channels blockers were not able to decrease this cell death as previously described for cell death triggered by HrpW_ea alone. We also tested tetraethyl ammonium (TEA), a K⁺ channel inhibitor known to decrease AVD. Five millimolar TEA alone did not modify A. thaliana cells survival, but decreased cell death triggered by simultaneous addition of HrpN_ea and HrpW_ea 200 nM (Fig. 3D). It is noteworthy that TEA did not reduce the cell death induced by HrpN_ea alone (Fig. 3A). This result indicates that HrpN_ea induced KORC activation12,14 is not involved in cell death, mainly due to the decrease of anion current as previously proposed,12 whereas it is involved in cell death triggered by simultaneous addition of HrpN_ea and HrpW_ea cell death. This comforts the hypothesis of a second way for harpins to induce cell death through an AVD type process (Fig. 1C and D).

Further studies are needed to improve the model presented in Figure 1 concerning the role of ion channels and their place in transduction mechanisms leading to harpin-induced cell death. Studies of particular interest would be the timing and quantification of the different harpins secreted during interaction.

Figures and Tables

Figure 1 Schema presenting the putative involvements of anion and K⁺ outward rectifying channels in the control of cell death triggered in non-host Arabidopsis cells by the two harpins of Erwinia amylovora, HrpN and HrpW. (A) Effect of 200 nM HrpN alone or mixed with 0.2 nM HrpW (B). (C) Effect of 200 nM HrpW alone or mixed with 200 nM HrpN (D).

Figure 2 Simultaneous addition of 200 nM HrpW_ea and HrpN_ea triggers strong activation of anion channels and K⁺ outward rectifying channels (KORC) in suspension cell. (A) Whole cell current recordings before and after treatment with 200 nM HrpW_ea and HrpN_ea. Protocol was as indicated, V_h, holding potential; V_m, membrane potential. (B) Mean amplitude of steady state time dependent KORC at +80 mV. (C) Mean amplitudes of steady state anion currents at −200 mV. The control corresponds to the addition of an equivalent volume of the buffer used for harpin preparation. Values are given as a percentage with respect to current values before treatment. All values are mean of at least five independent experiments. Error bars correspond to standard errors.

Figure 3 Effect of 5 mM TEA, a K⁺ channel inhibitor, on cell death triggered by 200 nM HrpN_ea or 200 nM HrpN_ea and HrpW_ea. Cell death was expressed as a percentage with respect to cell death level of nontreated cells. Data are means of 4 independent experiments and error bars correspond to standard errors.

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