Inverted Race Tube Assay for Circadian Clock Studies of the Neurospora Accessions

Although the Neurospora crassa circadian clock has been studied for forty years, population studies of natural accessions have been limited by technical difficulties associated with the conventional race tube assay (CRTA) that is used to measure asexual development (conidiation). Due to the buildup of CO2 in the CRTA that represses banding, a mutant strain band (bd) has been utilized for increased visualization of the banding phenotype. In order to study the circadian clock in natural accessions of Neurospora multiple techniques have been explored. One such technique, the rubidium chloride-supplemented race tube assay (RRTA) has been used successfully. Here we present a new technique, the Inverted Race Tube Assay (IRTA) that is a simple modification of the CRTA. We analyzed 5 natural accessions of Neurospora using CRTA, IRTA and RRTA and discuss the advantages of the IRTA in natural variation studies in Neurospora. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol51/iss1/5 12 Fungal Genetics Newsletter Inverted Race Tube Assay for Circadian Clock Studies of the Neurospora Accessions. Sohyun Park, Kwangwon Lee. Department of Plant Pathology, Cornell University, Ithaca NY 14850 Fungal Genet. Newsl. 51:12-14 Although the Neurospora crassa circadian clock has been studied for forty years, population studies of natural accessions have been limited by technical difficulties associated with the conventional race tube assay (CRTA) that is used to measure asexual development (conidiation). Due to the buildup of CO2 in the CRTA that represses banding, a mutant strain band (bd) has been utilized for increased visualization of the banding phenotype. In order to study the circadian clock in natural accessions of Neurospora multiple techniques have been explored. One such technique, the rubidium chloride-supplemented race tube assay (RRTA) has been used successfully. Here we present a new technique, the Inverted Race Tube Assay (IRTA) that is a simple modification of the CRTA. We analyzed 5 natural accessions of Neurospora using CRTA, IRTA and RRTA and discuss the advantages of the IRTA in natural variation studies in Neurospora. N. crassa has proved to be a successful model for studying the molecular bases of circadian clocks. Significant insights into the mechanism of the clock have been obtained in N. crassa including a) transcriptional/translational feedback loops in the clock that opened up the conceptual foundation for our understanding of the biological clock (Aronson et al.1994); b) the resetting mechanisms by light and temperature (Liu et al. 1998; Crosthwaite et al. 1995); c) circadian gating of cellular activities (Heintzen et al. 2001); and d) the involvement of anti-sense RNA in the circadian regulatory circuit (Kramer et al. 2003). A critical step in studying the circadian clock is the ability to easily observe and measure circadian regulated behavior. The rhythmic behavior that has been most extensively studied in N. crassa is asexual spore development using the CRTA. A race tube is a long glass tube (28 inch long) that is bent up at both ends to hold an agar medium. Mycelia or conidia are inoculated at one end of the race tube and the fungus grows toward the other end. To synchronize the cells to the same time of day, the cultures are germinated in constant light for a day and then transferred to constant darkness. Under these conditions, Neurospora initiates asexual development about every 22 hrs (Loros and Dunlap 2001). As a result of asexual development, Neurospora produces orange colored spores creating the “banding” phenotype. This easily tractable clock phenotype has been integral in dissecting the genetic structure of the Neurospora circadian oscillator. All laboratory Neurospora strains used in clock studies contain the mutation band (bd) (Sargent and Woodward 1969). In the absence of the bd mutation, the rhythmic asexual development of Neurospora in a race tube is suppressed by the accumulated CO2, a byproduct of respiration (Sargent and Kaltenborn 1972). Before the discovery of the bd mutant, researchers relied on a device to blow fresh air into the race tube to prevent the accumulation of CO2. However, blown air easily disperses the highly hydrophobic Neurospora spores inside the race tube, ruining the experiment. In the bd mutant background, the rhythmic pattern of asexual development can be visualized without the air circulation device. It was proposed that the bd mutation desensitizes the repressive role of CO2 in rhythmic asexual development in Neurospora, although its specific role is unknown (Sargent and Kaltenborn 1972). This repressive role of CO2 has been a major obstacle in studying natural variation of the circadian clock in wild-collected Neurospora accessions (Morgan and Feldman 1998). In addition to the adoption of blowing fresh air into the race tube, crossing natural accessions with the bd mutant, or the rubidium chloride-supplemented race tube assay (RRTA) (M organ and Feldman 1998) have been suggested as methods to monitor banding in Neurospora accessions. However, each of these solutions created additional issues. First, crossing the accessions with the bd strain is not desirable because it does not maintain the heterogeneity of the genomic structures between accessions. Second, the RRTA introduces another variable; the mode of rubidium chloride action is not characterized, adding an unknown environmental factor that further complicates quantitative studies (Morgan and Feldman 1998). With all these technical problems in mind, we developed a modified race tube assay, the Inverted Race Tube Assay (IRTA) (Fig. 1). We reasoned that if it is truly only the CO2 trapped in the bottom of the race tube that prevents the rhythmic asexual development [8], one could remove the heavy CO2 by simply inverting the race tube. 25 ml of media (1X Vogel, L-arginine 0.17%, D-glucose 0.1%, 1.5% agar) is poured into the race tube and autoclaved (20 min. 121 /C). As the media cools down, the media solidifies; due to the water condensation on the inside surface of the race tube, the solid media can settle on the bottom (what used to be the top) of the race tube as the race tube is turned upside down. The race tube is then placed on a flat surface overnight until the excess water inside the race tube is dried. 1 Park and Lee: Inverted Race Tube Assay for Circadian Clock Studies of the Neuro Published by New Prairie Press, 2017