Abstract
Neurospora crassa has been utilized as a model organism for studying biological, regulatory, and circadian rhythms for over 50 years. These circadian cycles are driven at the molecular level by gene transcription events to prepare for environmental changes. N. crassa is typically found on woody biomass and is commonly studied on agar-containing medium which mimics its natural environment. We report a novel method for disrupting circadian gene transcription while maintaining light responsiveness in N. crassa when held in a steady metabolic state using bioreactors. The arrhythmic transcription of core circadian genes and downstream clock-controlled genes was observed in constant darkness (DD) as determined by reverse transcription-quantitative PCR (RT-qPCR). Nearly all core circadian clock genes were up-regulated upon exposure to light during 11hr light/dark cycle experiments under identical conditions. Our results demonstrate that the natural timing of the robust circadian clock in N. crassa can be disrupted in the dark when maintained in a consistent metabolic state. Thus, these data lead to a path for the production of industrial scale enzymes in the model system, N. crassa, by removing the endogenous negative feedback regulation by the circadian oscillator.
Highlights
Our results demonstrate that circadian gene regulation was suppressed in DD cultures while genes linked to the circadian clock were up regulated during light-on cycles in light or dark exposure (LD) experiments
For LD experiments, after the initial 24 hr light exposure the cultures were exposed to 11 hr light/dark cycles during steady state starting with a dark cycle
The successful growth of N. crassa in an all-dark (DD) continuous culture reactor was reported by Crosthwaite and coworkers in 2007 with no data presented for LD entrainment[26]
Summary
The period of the circadian clock can be determined from the simultaneous formation of rhythmic conidial “bands” (the physical manifestation of the asexual sporulation state in N. crassa) in conjunction with the development of branching hyphae from the initial inoculation point The design of these assays are ideal to simulate how N. crassa survives in nature where the results of cellular metabolism (for example: changes in local pH, nutrient availability and waste accumulation) are not controlled but instead are responded to by the metabolism of the fungal cells. A mycelium disc is transferred to a nutrient-deficient medium and the cells are harvested periodically to analyze gene transcription and protein expression levels While these assays are designed for understanding the fungus in a liquid culture, some factors such as nutrient diffusion, localized pH gradients, and oxygen tension, are not well-controlled and the resulting environmental stressors could impact the circadian clock independently. These genes displayed arrhythmic expression patterns under DD conditions and were up regulated with light exposure
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