Elucidating the molecular mechanism of how the executor protein Bs3 from pepper triggers plant cell death

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The pepper resistance (R) gene Bs3 is transcriptionally activated by the Xanthomonas transcription activator-like effector protein AvrBs3. Bs3 is a structurally unique plant R protein that triggers cell death by unknown means. Bs3 is most related to YUCCAs, a plant-specific family of flavin-dependent monooxygenases (FMOs) that induce cell proliferation but not cell death. YUCCAs bind NADPH2 and O2 to convert indole-3-pyruvate (IPA) into auxin (Indole-3-acetic acid; IAA). Alternatively YUCCAs transfer reduction equivalents directly to O2 resulting in H2O2 production. In planta expression of Bs3 causes increased H2O2- but not increased IAA-levels, suggesting that Bs3 triggers cell death via H2O2 production. Indeed, recombinant Bs3 protein produces 5-fold more H2O2 than YUCCA6, a representative of the Arabidopsis YUCCA family. Photometric analysis suggests that O2-charged Bs3 is less stable than the O2-charged YUCCA6, and rationalizes why Bs3 produces more H2O2 than YUCCA6. Domain-swaps indicate that polymorphisms adjacent to a conserved cysteine determine functional differences between Bs3 and YUCCAs. Yeast two-hybrid (Y2H) studies identified the Arabidopsis transcription factor TCP9 as a putative Bs3 interactor. DNA binding of TCP proteins is redox-regulated and TCP9 activates expression of isochorismate synthase, a key enzyme of salicylic acid (SA) synthesis. Notably, Bs3-triggered cell death correlates with an increase in SA, which is in agreement with a model where Bs3 activates TCP9 via production of H2O2.Two distinct aspects of Bs3-triggered cell death will be studied within this proposal: Firstly, we want to elucidate functional differences between Bs3 and YUCCA proteins and to define the causal polymorphic residues. Secondly, we aim to identify and study the signal components that Bs3 employs to trigger cell death. To study Bs3- and YUCCA6-dependent H2O2 production in vivo both proteins will be fused to visual and functional reporters. In a complementary approach high-resolution microscopy will spatially correlate the presence of Bs3/YUCCA6 and H2O2. Thiol-specific, epitope-tagged probes will be used in conjunction with mass spectrometry (MS) to identify redox-sensitive cysteine residues in Bs3/YUCCA6. Subsequently we will elucidate the exact nature of given thiol modifications and their functional relevance. Coimmunoprecipitation (CoIP) linked to MS will be used to identify putative Bs3/YUCCA6 interactors. H2O2 produced by Bs3 is likely to induce sulfenylation of redox-regulated signal components. We will use sulfenic acid-specific, epitope-tagged probes in CoIP-MS to identify redox-regulated proteins. Subsequently functional relevance of putative Bs3 signalling components identified via CoIP-MS will be clarified by analysis of corresponding Arabidopsis and/or pepper mutant plants. In sum these studies will provide the first insights into how Bs3 triggers a plant defense reaction.