Rhythmicity of TOR (Target of Rapamycin) Activity Supports Circadian Function in <i>Neurospora crassa</i>

Circadian (24 h) rhythmicity is a nearly-ubiquitous property of eukaryotic cells, and the mechanisms that generate this rhythmicity have been studied in a number of organisms for many years. However, there are still gaps in our understanding of the generation and regulation of rhythms. Although transcription/translation feedback loops (TTFLs) are said to be essential to generating rhythmicity in eukaryotes, there are many examples of rhythmicity seen in organisms without functioning TTFLs. Our lab previously found that two genes that function in the TOR (Target of Rapamycin) pathway, <i>vta</i> and <i>gtr2</i>, are essential for non-TTFL rhythmicity in the fungus <i>Neurospora crassa.</i> These two mutants were also shown to dampen the output rhythm of spore-formation (conidiation) and the rhythm of the TTFL component protein FRQ, and dampen the amplitude of the underlying oscillator in strains with functioning TTFLs. Therefore, we are interested in the role of TOR in generating and/or sustaining rhythmicity in both the presence and absence of a functioning TTFL. Here we report the development and validation of an improved assay for TOR activity in <i>Neurospora</i>, and using this assay we demonstrate that TOR activity displays circadian rhythmicity in strains with functioning TTFLs. The period of TOR rhythmicity is affected by a long-period mutation in a TTFL component (<i>frq⁷</i>), and the TOR rhythm is dampened in the <i>vta</i> and <i>gtr2</i> knockouts. The mean level of TOR activity (mesor) in the <i>vta</i> and <i>gtr2</i> knockout mutants is within the range of the TOR rhythm in wild type, indicating that it is rhythmicity of TOR, not a constant level of activity, that is required to sustain output rhythmicity. These results establish <i>Neurospora</i> as a valuable model for investigating the role of TOR in circadian rhythmicity and implicate TOR activity as a rhythmic state variable of the circadian system.