The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, though little information exists regarding biological roles. Here we show that the cysteine (Cys-) and glutamine (Gln)-specific amino-terminal (Nt)-amidase NTAQ1 branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. Both ntaq1 and prt6 mutants of Arabidopsis thaliana showed enhanced basal resistance to the hemibiotroph pathogen Pseudomonas syringae, associated with constitutive expression of defence-response genes and activation of synthesis pathway of the phytoalexin camalexin. We observed a role in stomatal defence for the Nt-Cys substrates ETHYLENE RESPONSE FACTOR VII transcription factors. Transgenic barley lines with reduced HvPRT6 expression showed enhanced resistance against Ps japonica and Blumeria graminis f. sp. Hordei, indicating a conserved role of the pathway in immunity. These data demonstrate that different branches of the N-end rule pathway act at distinct levels of the plant immune response.