The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro

Abstract

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 H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 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 H2O2 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 Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

Novel flavin-containing monooxygenase protein FMO1 interacts with CAT2 to negatively regulate drought tolerance through ROS homeostasis and ABA signaling pathway in tomato

“…1a and b ). These findings differ from those obtained for the pepper FMO gene Bs3 , which contains an NADPH binding site in the middle of amino acid residues [ 37 ].…”

“…Thus, we speculate that FMO1 negatively regulates drought resistance primarily by affecting ABA signaling then increasing ABA synthesis. Moreover, the pepper FMO gene Bs3 triggers plant cell death, impairs yeast growth, and produces H 2 O 2 , thereby promoting ABA signaling via the inactivation of the negative regulator PP2C s [ 37 , 58 ]. This finding helps explain why the FMO1 RNAi-inhibited plants in our study accumulated less H 2 O 2 and had a low expression of PP2Cs .…”

Intertwined Roles of Reactive Oxygen Species and Salicylic Acid Signaling Are Crucial for the Plant Response to Biotic Stress

“…Since pt-roGFP is targeted to the chloroplast outer plastid envelope membrane, it also allows for the imaging of stromules formation [ 73 ]. The above-mentioned sensors were used in tobacco, Arabidopsis, and potato subjected to different developmental and environmental stresses [ 73 , 76 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 ], some of them also in HR [ 63 , 100 , 101 ]. roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2 [ 93 ].…”