The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2in vitro

ABSTRACT The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding transcription activator-like effector AvrBs3 from the bacterial pathogen Xanthomonas . Bs3 is homologous to flavin monooxygenases (FMOs), an enzyme class that has NADPH oxidase activity and can produce H 2 O 2 , a hallmark metabolite in plant immune reactions. Histochemical staining of infected pepper leaves and a translational fusion of Bs3 to the redox reporter roGFP2 both indicated that the Bs3-dependent HR induces a local increase in H 2 O 2 levels in planta . Moreover, our in vitro studies with recombinant Bs3 protein confirmed its NADPH oxidase activity. To test if the NADPH oxidation of Bs3 induces HR, we adapted previous studies which have uncovered mutations in fungal FMOs that result in higher NADPH oxidase activity. We replicated one of these mutations and demonstrated that the generated recombinant Bs3 S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3 in vitro . Translational fusions to roGFP2 showed that Bs3 S211A also had increased NADPH oxidase activity in planta . Interestingly, while the mutant derivative Bs3 S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta . Ultimately, this reveals that Bs3 produces H 2 O 2 in planta , but that the H 2 O 2 produced by Bs3 on its own is not sufficient to trigger HR. We also demonstrated that expression of Bs3 not only triggered HR in plants, but also inhibited proliferation of yeast, which lends this model system to be utilized for the genetic dissection of Bs3 function in future studies. One sentence summary The executor-type resistance protein Bs3 from pepper ( Capsicum annuum ) acts as an NADPH oxidase but reactive oxygen species produced by Bs3 are not sufficient to trigger plant cell death