A circadian oscillator in Aspergillus spp. regulates daily development and gene expression

Abstract

We have established the presence of a circadian clock in Aspergillus flavus and Aspergillus nidulans by morphological and molecular assays, respectively. In A. flavus, the clock regulates an easily assayable rhythm in the development of sclerotia, which are large survival structures produced by many fungi. This developmental rhythm exhibits all of the principal clock properties. The rhythm is maintained in constant environmental conditions with a period of 33 h at 30 degrees C, it can be entrained by environmental signals, and it is temperature compensated. This endogenous 33-h period is one of the longest natural circadian rhythms reported for any organism, and this likely contributes to some unique responses of the clock to environmental signals. In A. nidulans, no obvious rhythms in development are apparent. However, a free running and entrainable rhythm in the accumulation of gpdA mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) is observed, suggesting the presence of a circadian clock in this species. We are unable to identify an Aspergillus ortholog of frequency, a gene required for normal circadian rhythmicity in Neurospora crassa. Together, our data indicate the existence of an Aspergillus circadian clock, which has properties that differ from that of the well-described clock of N. crassa.

FIG. 1.

A. flavus forms bands of sclerotia in constant darkness. A. flavus strain 12S was inoculated onto race tubes containing CM and grown in 12:12 LD cycles for 5 days at 30°C before transfer into DD (A) or LL (B). The time of transfer is marked with a heavy black line. Bars below the race tubes represent the times that the cultures were in the light (white bars) or dark (black bars). Representative race tubes are shown. The direction of growth is from left to right. The mycelial growth front was marked at the same time each day (black lines) for reference. (C) A. flavus strain 12S was inoculated onto race tubes containing PDA medium and grown in 12:12 LD cycles for 7 days at 30°C before transfer into DD. In each panel, an intensity tracing of the race tubes is shown below, with the lines on the x axis representing the 24-h growth front marks.

FIG. 2.

A. flavus 12S entrains to different light and temperature cycles. (A) A. flavus 12S was inoculated onto CM race tubes and allowed to grow for 3 days in LL at 30°C (not shown). Race tubes were then transferred into 20-, 24-, or 36-h LD cycles at 30°C. Representative race tubes are shown on the left, with bars and intensity tracings as in Fig. 1. The mycelial growth fronts were marked daily at the dark-to-light transition (black lines). The direction of growth is from left to right. The phase of the center of the sclerotial band relative to the dark-to-light transition was measured in hours and plotted (top right side of figure). Positive numbers indicate that the band occurred prior to the dark-to-light transition, and negative numbers indicate that the band occurred after the dark-to-light transition. Values are means ± the SEM (n ≥ 23). (B) A. flavus strain 12S was inoculated onto CM race tubes and grown for 3 days in 12:12 LD at 30°C (not shown) and then transferred to DD with 24-h (12:12) cycles of 30 and 38°C. The mycelial growth front was marked at the transition from low to high temperature. The heavy line indicates when the cultures were transferred to constant 30°C. The bar below the race tubes indicates the time at 30°C (white) and 38°C (gray shaded). Tracings of the image are shown below the race tube.

FIG. 3.

The sclerotial rhythm in A. flavus is reset by environmental signals. A PRC was generated as described in Materials and Methods. The phase response was calculated as the difference in position of the sclerotial band after light treatment compared to the untreated controls. The x axis indicates the circadian time (CT) as calculated from the 33-h FRP of A. flavus 12S on CM medium at 30°C; the y axis shows the phase shift measured in hours of advance (positive numbers) or delay (negative numbers). Values are means ± the SEM (n = 5).

FIG. 4.

The sclerotial rhythm in A. flavus is temperature compensated. (A) A. flavus strain 12S was inoculated onto CM race tubes and allowed to grow at 30°C in 12:12 LD cycles for 3 days. Cultures were then transferred to the indicated temperatures in DD for 7 days. The data are plotted as the average FRP versus temperature. (B) The growth rate of A. flavus is plotted versus the temperature. In panels A and B, values are means ± the standard deviation (n ≥ 14).

FIG. 5.

gpdA mRNA accumulates rhythmically in A. nidulans strain A4. (A) Total RNA was isolated from A. nidulans strain A4 grown in DD (black bar) and harvested every 4 h for 2 consecutive days. Two independent experiments are shown. The Northern blots were probed with a PCR product derived from the A. nidulans gpdA gene to identify the 1-kb gpdA mRNA. Ethidium bromide-stained 25S rRNA is shown as an RNA loading control. A plot of the relative band intensities from the Northern blot is shown below. This experiment was repeated four times with similar results; however, as revealed in a comparison of the two experiments shown, the phasing of the peak of expression varied over eight circadian hours. (B) Total RNA was isolated from A. nidulans strain A4 grown in cycles of 12 h of light at 38°C (white bar) and 12 h of dark at 30°C (black bar) and then harvested every 4 h for 48 h. Northern blots were probed as in A. The zeitgeber time (ZT) is the time in hours from the start of a 12:12 LD cycle. The lights were turned on at ZT0 and turned off at ZT12. The relative band intensities are plotted below.