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Plants harbor various beneficial microbes that modulate their innate immunity, resulting in induced systemic resistance (ISR) against a broad range of pathogens. Camalexin is an integral part of Arabidopsis innate immunity, but the contribution of its biosynthesis in ISR is poorly investigated. We focused on camalexin accumulation primed by two beneficial bacteria, Pseudomonas fluorescens and Bacillus subtilis , and its role in ISR against Botrytis cinerea and Pseudomonas syringae Pst DC3000. Our data show that colonization of Arabidopsis thaliana roots by beneficial bacteria triggers ISR against both pathogens and primes plants for enhanced accumulation of camalexin and CYP71A12 transcript in leaf tissues. Pseudomonas fluorescens induced the most efficient ISR response against B. cinerea , while B. subtilis was more efficient against Pst DC3000. Analysis of cyp71a12 and pad3 mutants revealed that loss of camalexin synthesis affected ISR mediated by both bacteria against B. cinerea. CYP71A12 and PAD3 contributed significantly to the pathogen-triggered accumulation of camalexin, but PAD3 does not seem to contribute to ISR against Pst DC3000. This indicated a significant contribution of camalexin in ISR against B. cinerea , but not always against Pst DC3000. Experiments with Arabidopsis mutants compromised in different hormonal signaling pathways highlighted that B. subtilis stimulates similar signaling pathways upon infection with both pathogens, since salicylic acid (SA), but not jasmonic acid (JA) or ethylene, is required for ISR camalexin accumulation. However, P. fluorescens -induced ISR differs depending on the pathogen; both SA and JA are required for camalexin accumulation upon B. cinerea infection, while camalexin is not necessary for priming against Pst DC3000. [ABSTRACT FROM AUTHOR]