Many bacterial plant pathogens have a gene-for-gene relationship that determines host specificity. However, there are pathogens such as the xylem-limited bacteriumXylella fastidiosathat do not carry genes considered essential for the gene-for-gene model, such as those coding for a type III secretion system and effector molecules. Nevertheless,X. fastidiosasubspecies are host specific. A comparison of symptom development and host colonization after infection of plants with several mutant strains in two hosts, grapevines and almonds, indicated thatX. fastidiosavirulence mechanisms are similar in those plants. Thus, we tested if modification of gene regulation patterns, by affecting the production of a cell–cell signalling molecule (DSF), impacted host specificity inX. fastidiosa. Results show that disruption of therpfFlocus, required for DSF synthesis, in a strain incapable of causing disease in grapevines, leads to symptom development in that host. These data are indicative that the core machinery required for the colonization of grapevines is present in that strain, and that changes in gene regulation alone can leadX. fastidiosato exploit a novel host. The study of the evolution and mechanisms of host specificity mediated by gene regulation at the genome level could lead to important insights on the emergence of new diseases.