Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Tuning Pectin Methylesterification in Plant Immunity: Post-Transcriptional Regulation of Pectin Methylesterases by Subtilases

The plant cell wall (CW) is the foremost barrier where plant-pathogen interactions occur. The CW is a complex and dynamic compartment composed of cellulose micro-fibrils embedded in a dense network of hemicelluloses and pectins. Pectins are synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by plant Pectin Methylesterases (PMEs). In Arabidopsis thaliana there are 66 PME isoforms involved in important development processes and in plant immunity during pathogenesis. 45 isoforms are ProPMEs and possess, in addition to the PME domain, an N-terminal Pro-region that shows sequence similarities with the PME Inhibitors. A. thaliana induces the expression of specific PME isoforms during the attack of the necrotrophic fungus Botrytis cinerea. The amino acidic sequences alignment of these isoforms shows that they all are ProPMEs. Among them, AtProPME17 is particularly interesting since it is induced after the infection of a wide-range of plant pathogens and can be considered a general biomarker of pathogenesis. ProPME activity can be post-transcriptionally regulated by specific Subtilases (SBTs), serine-like proteases that process conserved recognition sites releasing the PME domain from the Pro-region. AtSBT3.3, AtSBT3.4 and AtSBT3.5 were found to be temporally co-expressed with AtProPME17 in A. thaliana plants during B. cinerea infection and selected for further analysis. Homozygous A. thaliana T-DNA insertional mutants have been isolated and characterized. During the infection process, the decreased expression of AtSBT3.3 and AtProPME17 in their respective mutants determines a lower increase of PME activity and an increased susceptibility to B. cinerea respect to the control plants. By exploiting heterologous expression in Pichia pastoris and Escherichia coli, it has been observed that AtProPME17 is a functional PME performing a blockwise pattern of pectin de-methylesterification and showing an optimal activity at alkaline pH. Moreover, we demonstrated that the Pro-region acts as an intramolecular inhibitor of AtProPME17 activity.