The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H(2)O(2) in vitro

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H(2)O(2). Since H(2)O(2) is a hallmark metabolite in plant immunity, we analyzed the role of H(2)O(2) during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H(2)O(2) production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3(S211A) protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3(S211A) also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3(S211A) had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H(2)O(2) produced by Bs3 on its own is not sufficient to trigger HR.