More than a repair enzyme: Aspergillus nidulans photolyase-like CryA is a regulator of sexual development

Abstract

Cryptochromes are blue-light receptors that have presumably evolved from the DNA photolyase protein family, and the genomes of many organisms contain genes for both types of molecules. Both protein structures resemble each other, which suggests that light control and light protection share a common ancient origin. In the genome of the filamentous fungus Aspergillus nidulans, however, only one cryptochrome/photolyase-encoding gene, termed cryA, was identified. Deletion of the cryA gene triggers sexual differentiation under inappropriate culture conditions and results in up-regulation of transcripts encoding regulators of fruiting body formation. CryA is a protein whose N- and C-terminal synthetic green fluorescent protein fusions localize to the nucleus. CryA represses sexual development under UVA (350-370 nm) light both on plates and in submerged culture. Strikingly, CryA exhibits photorepair activity as demonstrated by heterologous complementation of a DNA repair-deficient Escherichia coli strain as well as overexpression in an A. nidulans uvsBDelta genetic background. This is in contrast to the single deletion cryADelta strain, which does not show increased sensitivity toward UV-induced damage. In A. nidulans, cryA encodes a novel type of cryptochrome/photolyase that exhibits a regulatory function during light-dependent development and DNA repair activity. This represents a paradigm for the evolutionary transition between photolyases and cryptochromes.

Figure 1.

CryA classification. CryA amino acid sequence was aligned to six (6-4)-photolyases, 17 animal cryptochromes, 10 CRY-DASH proteins, 12 class I CPD-photolyases, and 11 plant cryptochromes (Daiyasu et al., 2004). (A) Multiple sequence alignment (T-COFFEE; position 601–675) illustrates sequence conservation of CryA and two sequences from each cryptochrome/photolyase subfamily in FAD binding domain. Degree of sequence conservation: black (100%), dark gray (80%), light gray (60%), and white (<60%); amino acids of the same similarity group were treated as identical. (B) Radial cladogram of phylogenetic relationships of cryptochromes/photolyases including CryA. The cladogram represents a consensus tree combining maximum parsimony, neighbor-joining, and maximum-likelihood calculations, based on the multiple-sequence alignment generated with T-COFFEE. Bootsrap probabilities are given for the nodes separating animal CRYs/6-4 photolyases from plant CRYs/class I CPD photolyases (1.00) and plant CRYs from class I CPD photolyases (0.99). (C) Pseudo three-dimensional representation of the PCA from the ClustalW alignment. Shown are the first three principal components of a vector space, which represent the subspace with greatest variance. (D) cryA expression was monitored in an A. nidulans wild-type strain by RT-PCR (top) and Northern hybridization experiments (bottom). Strain FGSC A4 was vegetatively grown in submerged culture for 24 h (Veg), transferred onto plates, and kept in the light to induce the asexual sporulation (Asex) or incubated in the darkness to induce sexual development (Sex). Samples were prepared at the given time points of 12 and 24 h of asexual and 12, 24, 48, and 72 h of sexual development. Ethidium bromide-stained rRNA was used as loading control in Northern blots. Signals from RT-PCRs and Northern hybridizations are compatible with each other, indicating that cryA expression is low during vegetative growth and early asexual and sexual development.

Figure 2.

Deletion of fungal cryA results in Hülle cell formation in submerged culture. Phenotypical characterization of the cryAΔ strain. Light microscopy illustrates wild-type growth resulting in hyphal mats (left), whereas a cryAΔ strain produces Hülle cells with circular, banana-like cell wall shape (middle); the deficiency is complemented by cryA homologous gene replacement (right).

Figure 3.

Nuclear CryA represses the transcripts of regulatory factors of sexual development to reveal a negative feedback loop. (A) N- and C-terminal fusions of CryA to sGFP were expressed to monitor subcellular location of the fusion proteins; to visualize the nuclei in vivo, a red fluorescence mRFP::H2A fusion was used. (B) Comparative Northern hybridization of sexual development regulators in the wild-type strain A4 and the cryAΔ strain: RNA levels for the light-dependent regulator veA increase in cryAΔ after 48 h after induction, corresponding to the appearance of Hülle cells, and increase further up to 72 h. nsdD and rosA regulator mRNAs are elevated in cryAΔ, but almost undetectable in A4. gpdA (glycolytic gene) served as internal control. (C) rosA expression is dependent on expression of the nsdD gene as demonstrated by overexpression of nsdD. (D) Model of cryA-regulated negative feedback loop of fungal development. Northern blot experiments were repeated three times.

Figure 4.

Photon fluence-rate response curves for the photoinhibition of cleistothecia formation. (A) After inoculating Petri dishes with 10⁴ spores per plate, the materials were irradiated for 100 h with monochromatic overhead light at the indicated photon-fluence rates. Blue circles, wild-type; red squares, cryA mutant; green triangles, complementation strain (comp+). Vertical bars indicate the standard deviations. (B) Formation of cleistothecia (spherical fruiting bodies) of A. nidulans growing on solid medium. The photographs show the densities of the cleistothecia of the wild-type, mutant, and complementation strains that were raised under 350-, 370-, 454-, and 680-nm light sources. White bar, 200 μm.

Figure 5.

Dose–response curves for photokilling and photoreactivation in E. coli and Aspergillus nidulans strains. (A) Photoreactivation experiments in the repair-defective E. coli strain SY2 (uvrA⁻, recA⁻, phr⁻) transformed with plasmid pGEX-4T-2 as control, pGEX-cryA, or pMS969 as positive control. UV-irradiated E. coli cells were either kept in darkness (black symbols) or illuminated with UVA_{366 nm} (light symbols). E. coli Phr and A. nidulans CryA show photoreversal activity after UVA_{366 nm} treatment. (B) A. nidulans UV sensitivity experiment; spores of A. nidulans strains A4 (wild-type), cryAΔ, uvsBΔ, and cryA-OE were exposed to UV_{254 nm} radiation followed by 1 h UVA_{366 nm} treatment; the extent of surviving spores was quantified as colony forming units after 48 h. Whereas there is no difference between A4 and cryAΔ, the UV resistance of uvsBΔ is increased by overexpression of cryA (∼6-fold). Vertical bars indicate the standard deviations.