Chemically induced and light-independent cryptochrome photoreceptor activation.
The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP-rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.
Rosenfeldt
Gesa
G
FB Biologie-Pflanzenphysiologie, Philipps-Universität, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany.
Viana
Rafael Muñoz
RM
Mootz
Henning D
HD
von Arnim
Albrecht G
AG
Batschauer
Alfred
A
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000378:metabolism / Q000528:radiation effects; D056931:Cryptochromes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D019281:Dimerization; D004735:Energy Transfer; D017930:Genes, Reporter; D008027:Light; D008156:Luciferases / Q000378:metabolism; D049449:Luminescence; D055519:Photoreceptors, Plant / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D010944:Plants / Q000187:drug effects / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000528:radiation effects; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000378:metabolism / Q000528:radiation effects
C539629:AP 21967; D029681:Arabidopsis Proteins; D056931:Cryptochromes; D055519:Photoreceptors, Plant; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus