HrpW interacts with NADH dehydrogenase of apple

During the infection, Erwinia amylovora secretes in the host cell at least twelve different effectors, which are involved in the establishment of the disease. Harpins are part of the proteins delivered in the plant cell via the type three secretion system. These proteins are known to be heat stable, glycine rich, without cysteine residues, and trigger a HR in non-host plant. In particular, the mechanism of action of harpin N has been well studied. HrpW, one of the other harpin members, is present in many phytopathogenic bacteria such as E. amylovora and Pseudomonas syringae pv tomato, and possesses a class III pectate lyase domain, but nobody so far has reported a pectate lyase activity. Also hrpW mutants induce symptoms as strong, or even stronger, than wild-type strains, suggesting that this protein, contrarily to HrpN, is not required for a full virulence of the bacteria. However, recently HrpW has been described as a HR negative modulator in non-host plants such as Nicotiana tabacum and Arabidopsis thaliana. On Arabidopsis cell cultures, depending on its concentration, it could trigger, like HrpN, a defense response, or act antagonistically to HrpN, inhibiting cell death and ROS production. At present nobody has deeply investigated the effect of HrpW in host plants. We undertook this project to identify apple proteins interacting with this effector by screening a yeast two hybrid apple library. We identified several potential protein interactors that have been further tested by Bimolecular Fluorescent Complementation (BiFC). This test in tobacco confirmed that HrpW is able to interact with the N domain of the apple NADH dehydrogenase. Indeed when HrpW and the apple NADH dehydrogenase are co-infiltrated in tobacco an HR response can be observed after 2 days. The Interaction between HrpW and the apple NADH dehydrogenase located in the mitochondria can the ROS production and induce the HR reaction observed in non-host plant. The effect of this interaction in apple is under investigation