Ca²⁺‐dependent lipid binding and membrane integration of PopA, a harpin‐like elicitor of the hypersensitive response in tobacco

Abstract

SummaryPopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipidbinding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca 2+ + + + for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion-conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier.

Phytotoxicity and Innate Immune Responses Induced by Nep1-Like Proteins

“…Importantly, the bacterial effector protein HrpZ Psph from P. syringae pv phaseolicola mediated ion pore formation as shown previously (Lee et al, 2001). HrpZ Psph and related bacterial effectors form ion-conducting pores not only in artificial membranes but also in biological membranes, such as the plasma membrane of Xenopus laevis oocytes (our unpublished data; Racape et al, 2005). However, NLP Pya proved insufficient to generate any ion currents in this system ( Figure 8C), suggesting that it did not directly affect membrane integrity through intrinsic ionophore activity.…”

“…Liposomes filled with the cation-sensitive fluorescent dye Sodium Green (Invitrogen) were obtained from Novosom. Ion pore formation experiments and patch-clamp analyses using Xenopus laevis oocytes were performed as described (Racape et al, 2005).…”

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

“…This hypothesis was confirmed when 0.2 nM HrpW ea was found to inhibit HrpN ea -induced cell death. Although the PopA rs harpin was recently found to form oligomers through the conserved GxxxG amino acid motif (Racape et al, 2005), this inhibition is unlikely to be the result of the direct interaction between HrpW ea and HrpN ea because GST pull-down technology did not show any interaction between HrpW ea and HrpN ea (data not shown). In addition, the fact that the most effective concentration of HrpW ea to inhibit HrpN ea -induced cell death was onethousandth of that of HrpN ea , also constitutes evidence against a direct protein-protein interaction.…”

Identification of Harpins in Pseudomonas syringae pv. tomato DC3000, Which Are Functionally Similar to HrpK1 in Promoting Translocation of Type III Secretion System Effectors

“…R. solanacearum GMI1000 also expresses the single-domain harpin PopA, which has been shown to form ion-conducting pores in artificial and biological membranes. However, popA mutants have no detectable phenotype (4,57). It would be interesting to determine if PopA overexpression could complement any of the ⌬popF1 phenotypes.…”