Bacterial Assay to Study Plant Sensor Histidine Kinases
Cytokinins, like other phytohormones, act in plants as signaling molecules at very low concentrations. The system that mediates between their chemical recognition and the responses that they induce requires a hormone receptor that, together with down-stre
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. Introduction In 2001 receptors of cytokinins (an important group of plant hormones) were identified as members of the family of histidine kinases (1, 2). Histidine kinases form the sensor part of two-component signal transduction pathways that are known to respond to various external stimuli in prokaryotes and lower eukaryotes. Their function in perception of cytokinins in higher plants was confirmed using heterologous complementation of yeast and bacterial twocomponent regulatory pathways ((1, 2), reviewed in ref. 3). In the latter case, expression of histidine kinases CRE1/AHK4 and AHK3 in the eubacterium E. coli resulted in the first demonstration of
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_7, © Springer Science+Business Media, LLC 2011
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functional complementation of an E. coli sensor mutant by a plant receptor gene (2, 4). AHK histidine kinases share some structural features with the RcsC receptor, the part of the two-component signaling pathway that leads to activation of the cps (capsular polysaccharide synthesis) operon through phosphorylation of the Hpt element YojN and the response regulator RscB (5). When CRE1/ AHK4 or AHK3 are expressed in the DrcsC and cps::lacZ mutant strain, b-galactosidase, which can easily be determined quantitatively, is produced in the presence of cytokinin (Fig. 1). This bacterial assay has been used to confirm the ability of cytokinin receptors
Fig. 1. Bacterial assay based on expression of histidine kinases CRE1/AHK4 and/or AHK3 in E. coli sensor mutant. AHK histidine kinases share some structural features with the RcsC receptor, the part of the two-component signaling pathway leading to activation of the cps operon through the phosphorylation of the Hpt element YojN and the response regulator RscB (5). Activation of the cytokinin receptor in the DrcsC and cps::lacZ mutant strain can easily be scored by quantitative fluorometric assay using the fluorogenic substrate of b-galactosidase, MUG and/or by the formation of blue colonies on X-Gal-containing medium. Abbreviations: D receiver domain, Da and Db receiver domains of a and b, respectively, H histidine kinase, MUG 4-methyl umbelliferyl galactoside, X-Gal 5-bromo-4-chloro-3-indolyl-b-d-galactoside. This figure is displayed in greyscale in the print edition but available in color in the electronic version.
7 Bacterial Assay to Study Plant Sensor Histidine Kinases
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of Arabidopsis thaliana and Zea mays to perceive cytokinins in vitro and for their molecular and biochemical characterization (1–3, 6, 7). This works revealed that the receptors recognize naturally occurring isoprenoid and aromatic cytokinins and also, albeit to varying extents, synthetic cytokinins derived from phenylurea. Furthermore, they allowed the demonstration that individual receptors of Arabidopsis and maize differ in ligand specificity, being most sensitive to the bases of the isoprenoid-type cytokinins trans-zeatin and i
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