Abstract The understanding of the molecular mechanisms controlling plant resistance to biotrophic pathogens is an expanding and complex field of research where the identification on novel regulators is demanding. We previously reported on P69C, a gene encoding a subtilisin-like protease induced during the course of a plant-pathogen interaction. To investigate novel plant components operating in pathogen-induced signalling cascades, we screened Arabidopsis plants carrying the ?-glucoronidase reporter gene under control of the P69C promoter. Here we report the identification and characterization of a mutant, csb3 (for constitutive subtilisin3) in which the reporter construct is constitutively expressed. Healthy csb3 plants show increased accumulation of salicylic acid (SA) and express constitutively the PR-1, PR-2 and GST6 genes. Moreover, the csb3 mutant shows a strikingly enhanced resistance to the biotrophic oomycete pathogen Hyaloperonospora parasitica and to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Conversely, resistance to the necrotrophic pathogens Botrytis cinerea and Plectosphaerella cucumerina remains unaffected in csb3 plants when compared to wild-type plants. To analyze signal transduction pathways where csb3 operates, epistasis analyses between csb3 and pad4, sid2, eds5, nahG, npr1, dth9 and cpr1 were performed. These studies revealed that the enhanced resistance of csb3 plants requires intactness of synthesis and accumulation of SA and is also fully dependent on appropriate perception of SA through NPR1 and DTH9. We show that CSB3 encodes 1-hydroxy-2-methil-2-butenyl 4-diphosphate synthase (HDS), the enzyme controlling one of the terminal steps of the biosynthesis of isopentenyl diphosphate (IPP) via the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway that occurs in the chloroplast of higher plants. CSB3 is constitutively expressed in healthy plants but partially repressed in response to infection by Pst DC3000. Furthermore, through the isolation and characterization of extragenic suppressors of the csb3 mutant, along with the pharmacological complementation of the enhanced resistance phenotype of csb3 plants with fosmidomycin, and inhibitor of the MEP pathway, we proposed that CSB3 may be functioning as a negative regulator of the SA-mediated disease resistance pathway to biotrophic pathogens. Moreover, csb3 showed enhanced expression of two subtilisin-like genes named AtSBT3.3 and AtSBT3.5. The expression of these two genes is rapidly induced by Pseudomonas syringae, Plectosphaerella cucumerina and after H2O2 treatment. Conversely the expression of AtSBT3.3 and AtSBT3.5 is fully independent of SA. The loss of function of the gene AtSBT3.3 renders a phenotype of hypersusceptibility to biotrophic pathogens. These evidences point to the role of AtSBT3.3 and AtSBT3.5 in the resistance mechanism to biotrophic pathogens.