High-resolution spatiotemporal analysis of gene expression in real time: in vivo analysis of circadian rhythms in Neurospora crassa using a FREQUENCY-luciferase translational reporter.

The pacemaker of the Neurospora circadian clock is composed of a transcriptional-translational feedback loop that has been intensively studied during the last two decades. Invaluable information has been derived from measuring the expression of the central clock component frequency (frq) under liquid culture conditions. Direct analyses of frq mRNA and protein levels on solid media - where overt circadian rhythms are normally visualized - have not been trivial due to technical issues. Nevertheless, a frq promoter-luciferase reporter has recently allowed the study of frq transcription under these conditions. It is known that FRQ undergoes extensive posttranslational modifications, and changes in its levels provide important information regarding the clockworks. Here we describe a FRQ-luciferase translational fusion reporter that directly tracks FRQ levels, granting access to a better understanding and analysis of FRQ dynamics in vivo. More generally the method, which allows the investigator to follow continuous gene expression in real time in a spatially and temporally unrestricted manner, should be widely applicable to analyses of environmentally and developmentally regulated gene expression in ascomycete filamentous fungi as well as in basidiomycetes.