title: A Universal Stress Protein Involved in Oxidative Stress Is a Phosphorylation Target for Protein Kinase CIPK6.
abstract: Calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins into phosphorylation events that result into adaptation to environmental stresses. Few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible Programmed cell death in immunity transforming Ca2+signals into reactive oxygen species (ROS) signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two-hybrid approach was carried on and a protein was identified that contained a Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named "SlRd2". SlRd2 was an ATP-binding protein that formed homodimers in planta. SlCipk6 and SlRd2 interacted using coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves and the complex localized in the cytosol. SlCipk6 phosphorylated SlRd2 in vitro, thus defining, to our knowledge, a novel target for CIPKs. Heterologous SlRd2 overexpression in yeast conferred resistance to highly toxic LiCl, whereas SlRd2 expression in Escherichia coli UspA mutant restored bacterial viability in response to H2O2treatment. Finally, transient expression of SlCipk6 in transgenic N benthamiana SlRd2 overexpressors resulted in reduced ROS accumulation as compared to wild-type plants. Taken together, our results establish that SlRd2, a tomato UspA, is, to our knowledge, a novel interactor and phosphorylation target of a member of the CIPK family, SlCipk6, and functionally regulates SlCipk6-mediated ROS generation.
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, 41092 Sevilla, Spain.
authors.forename: Emilio
authors.identifier: 0000-0001-7978-3164
authors.initials: E
authors.lastname: Gutiérrez-Beltrán
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, 41092 Sevilla, Spain.
authors.forename: José María
authors.identifier: 0000-0003-2273-5000
authors.initials: JM
authors.lastname: Personat
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, 41092 Sevilla, Spain.
authors.forename: Fernando
authors.identifier: 0000-0002-5143-3709
authors.initials: F
authors.lastname: de la Torre
authors.affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla/Consejo Superior de Investigaciones Científicas, 41092 Sevilla, Spain olga_delpozo@ibvf.csic.es.
authors.forename: Olga
authors.identifier: 0000-0001-9852-0431
authors.initials: O
authors.lastname: Del Pozo
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018021:Lithium Chloride / Q000494:pharmacology; D018551:Solanum lycopersicum / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D010940:Plant Proteins; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D018021:Lithium Chloride