Distinct signaling pathways from the circadian clock participate in regulation of rhythmic conidiospore development in Neurospora crassa

Abstract

Several different environmental signals can induce asexual spore development (conidiation) and expression of developmentally regulated genes in Neurospora crassa. However, under constant conditions, where no environmental cues for conidiation are present, the endogenous circadian clock in N. crassa promotes daily rhythms in expression of known developmental genes and of conidiation. We anticipated that the same pathway of gene regulation would be followed during clock-controlled conidiation and environmental induction of conidiation and that the circadian clock would need only to control the initial developmental switch. Previous experiments showed that high-level developmental induction of the clock-controlled genes eas (ccg-2) and ccg-1 requires the developmental regulatory proteins FL and ACON-2, respectively, and normal developmental induction of fl mRNA expression requires ACON-2. We demonstrate that the circadian clock regulates rhythmic fl gene expression and that fl rhythmicity requires ACON-2. However, we find that clock regulation of eas (ccg-2) is normal in an fl mutant strain and ccg-1 expression is rhythmic in an acon-2 mutant strain. Together, these data point to the endogenous clock and the environment following separate pathways to regulate conidiation-specific gene expression.

FIG. 1.

The inability to complete conidiation does not affect clock control of the initiation of aerial-hypha formation. Race tube cultures of the indicated strains were grown in constant darkness for 7 days, and the growth fronts were marked every 24 h (vertical black lines). The centers of the developmental bands are indicated by the black dots. The calculated growth rates (GR) and the periods of the rhythms in hours are shown on the right. The values are means ± standard deviations determined from at least three separate experiments for each strain.

FIG. 2.

The circadian clock regulates fl gene expression. (A) The steady-state levels of fl mRNA were assayed by Northern blot analyses in frq⁺ (top) and frq⁷ (bottom) strains. The frq⁺ membrane was also probed with an eas (ccg-2)-specific probe as a control. Liquid cultures of mycelia were grown in constant darkness and harvested after the indicated number of hours in the dark (Hours DD). The approximate CTs at the times of harvest are shown below the autoradiograms. (B) Using the autoradiograms shown in panel A, fl mRNA was quantitated by densitometry and plotted as relative band intensity normalized to rRNA (not shown) versus time in the dark for both frq⁺ (solid line) and frq⁷ (dashed line). Three separate repetitions of these experiments in each strain yielded similar results.

FIG. 3.

fl mRNA accumulation is arrhythmic in the acon-2 mutant strain. (A) The levels of fl mRNA were assayed by Northern blot analyses in the acon-2 mutant strain. Liquid cultures of mycelia were grown in constant darkness at 34°C and harvested after the indicated number of hours in the dark (Hours DD). The Northern blot was probed with fl and eas (ccg-2) riboprobes. The approximate CTs at the times of harvest are indicated below the autoradiogram. (B) Following autoradiography, fl mRNA was quantitated by densitometry and plotted as relative band intensity normalized to rRNA versus time in the dark. Three separate experiments were used to determine the standard deviation of fl RNA levels.

FIG. 4.

fl mRNA is induced early in the conidiation pathway. (A) RNA was isolated from wild-type clock cells at 0, 15, 30, 60, and 120 min after induction of conidiation by desiccation. The RNA was hybridized to an fl-specific RNA probe and subsequently to an rRNA DNA probe. (B) Following autoradiography, fl mRNA was quantitated by densitometry and plotted as relative band intensity normalized to rRNA versus time after induction. Similar results were obtained with two additional repeats of this experiment.

FIG. 5.

Both eas (ccg-2) and ccg-1 are rhythmically expressed in mutants of key developmental regulators that are required for their induction by exogenous developmental cues. (A) The steady-state levels of eas (ccg-2) mRNA were assayed by Northern blot analyses in fl and acon-2 mutant strains and ccg-1 mRNA in acon-2 and acon-3 mutant strains at 24°C. Liquid cultures of mycelia were grown in constant darkness and harvested after the indicated number of hours in the dark (Hours DD). The approximate CTs at the times of harvest are indicated. (B) Following autoradiography, eas (ccg-2) and ccg-1 mRNA in each mutant background was quantitated by densitometry and plotted as relative band intensity normalized to rRNA versus time in the dark. Similar results were obtained in at least two separate repetitions of these experiments.

FIG. 6.

Model depicting direct and indirect regulation of clock output genes involved in conidiation. (See the text for a full explanation of the developmental pathway and the clock-regulated events.) Once a day, the endogenous circadian clock signals the production of aerial hyphae from vegetative hyphae (solid arrow). Independent of the time of day, exogenous environmental signals can trigger the same event (dashed arrow). Both pathways eventually lead to the production of conidia. Conidia are formed by budding of the aerial hyphae involving the formation of minor constriction chains, requiring the products of acon-2 and fld, and major constriction chains, requiring the products of fl and acon-3. The csp-1 and csp-2 genes are required for conidial separation. The genes shown to be regulated by the clock independent of the developmental pathway are linked to the clock by solid arrows. The epistatic relationships between the key developmental regulators and developmentally induced genes (6, 22, 47) are depicted by dashed arrows. Rhythmic fl expression requires its upstream developmental regulator, ACON-2 (shown as a heavy solid arrow). A solid line was not drawn to acon-2, since it is not known if acon-2 is rhythmically expressed. The fl gene is shown to act during the production of aerial hyphae and after the formation of minor constriction chains.