The bleeding canker of European horse chestnut is a recently emerged disease caused by Pseudomonas syringae pathovar aesculi (Pae). The major virulence mechanisms driven by this pathogen to infect woody hosts are scarcely investigated. However, genome comparison of the Pseudomonas syringae complex revealed conserved regions only present in E-Pae which are implicated in the metabolism of aromatic and phenolic compounds, in sucrose uptake and utilization and in fatty acid biosynthesis. The hopAB1 effector and HrpL regulon are essential for suppressing and/or triggering defences in different plant species and for symptom development, respectively. However, their contribution to the virulence of Pae is also important to analyse. Our goal was to investigate the main components contributing to Pae virulence on horse chestnut. Therefore, we conducted functional analyses through mutagenesis and complementation experiments to evaluate the genetic adaptations to infection of the woody parts of the tree. Our study revealed the important role of hopAB1 and hrpL in suppressing the plant immune response and causing disease in tobacco plants and horse chestnut. The results demonstrated that E-Pae can utilize sucrose as the sole carbon source, while deletion mutants of ScrB and scrY sucrose genes showed significant reduction in sucrose utilization when grown on sucrose as the sole carbon. Moreover, ScrB and scrY mutants showed disease symptom development on both tobacco and horse chestnut leaves. The deletion of the catB and antA WHOP related-genes encoding for the catabolism of anthranilate and catechol §have not affected the virulence in E-Pae on tobacco neither the HC leaves and their role in the woody tissue are further to be investigated. Our results highlight the role of some of the enzymatic activities encoded within E-Pae and their implication on its evolution and adaptation to woody hosts.