Modulazione perossinitrito-dipendente delle vie di trasduzione MAP chinasiche nell’interazione pianta-patogeno

During an incompatible plant-pathogen interaction, the early events which play a key role in the activation of disease resistance are the accumulation of nitric oxide (NO) and the generation of the reactive oxygen species (ROS), such as the superoxide anion (O2-) and hydrogen peroxide (H2O2). NO can react with O2- to generate peroxynitrite (ONOO-), a potent oxidant able to react directly with proteins leading to the nitration of protein tyrosine residues. Tyrosine-nitration is a post-translational modification, which may alter conformation, structure and catalytic activity of nitrated proteins. An increase of Tyr-nitrated protein level and the identification of some of these targets have been previously reported in Arabidopsis thaliana plants challenged with an avirulent pathogen, suggesting a possible physiologically relevant role of nitration during the Hypersensitive Response (HR). The MAP kinase (MAPK) cascade, a signalling pathway able to transduce environmental changes in cellular responses, is other key component of plant defense response. Via a phosphorelay mechanism, which is fine regulated to guarantee the specificity of the cellular response, the MAPK cascade, minimally composed of a MAPKKK, a MAPKK and a MAPK, link upstream receptors to downstream targets. Since, in animals, Tyr-nitration has been reported to be relevant for MAPK cascade regulation, and previous data obtained in Prof. Delledonne laboratory, showed an effect of nitration on the activity of the tobacco MAPKK, NtMEK2, in this work, to understand if target of nitration in plant could be the MAPKK component, we analyzed the effect of ONOO- on six different MAPKK of A. thaliana (AtMKK). We demonstrated that (i) the Tyr nitration is a selective process since, among six MAPKK, only two, AtMKK4 and AtMKK9, were nitrated in vitro by ONOO-; (ii) the Tyr-nitration doesnʼt depend on the Tyr residue content in a protein, since the AtMKK with the highest number of Tyr residues were not nitrated; and (iii) the conformation could influence the accessibility of the Tyr residues to nitration, since the AtMKK5, which shows high homology with AtMKK4, was not nitrated. Since AtMKK4 is involved in plant defence, we also investigated in vitro the effect of ONOO- on the activity of the constitutively active form AtMKK4DE, and we demonstrated that it is inhibited. Moreover, the identification by mass spectrometry of two Tyr residues nitrated, located close and inside the ATP-binding site, suggest that this inhibition is correlated with the incapacity of nitrated AtMKK4DE to bind ATP. Then, to confirm the AtMKK4 inhibition by nitration in vivo, we showed that ONOO--treatment strongly delays AtMKK4DE-induced hypersensitive cell death in tobacco plants. Interestingly, AtMKK5, which shares high homology with AtMKK4, was not nitrated and its activity was not affected by ONOO--treatment, suggesting that AtMKK4 and AtMKK5 redundancy could be compromised by Tyr- nitration during the HR.