Basic-zipper-type transcription factor FlbB controls asexual development in Aspergillus nidulans

Abstract

The fungal colony is a complex multicellular unit consisting of various cell types and functions. Asexual spore formation (conidiation) is integrated through sensory and regulatory elements into the general morphogenetic plan, in which the activation of the transcription factor BrlA is the first determining step. A number of early regulatory elements acting upstream of BrlA (fluG and flbA-E) have been identified, but their functional relations remain to be further investigated. In this report we describe FlbB as a putative basic-zipper-type transcription factor restricted to filamentous fungi. FlbB accumulates at the hyphal apex during early vegetative growth but is later found in apical nuclei, suggesting that an activating modification triggers nuclear import. Moreover, proper temporal and quantitative expression of FlbB is a prerequisite for brlA transcription, and misscheduled overexpression inhibits conidiation. We also present evidence that FlbB activation results in the production of a second diffusible signal, acting downstream from the FluG factor, to induce conidiation.

FIG. 1.

General description of flbB. (A) Genome localization and general description of flbB. Black segments represent exons, and black lines designate introns. (B) Sequence alignment of FlbB homologues. Arrows indicate mutations which define changes in the amino acid sequence. Regions with low similarity are excluded. Genedoc software was used (version 2.6.003; www.psc.edu/biomed/genedoc). (C) Phylogenetic tree of bZIP domains identified in various fungal and yeast species. The bZIP branch including FlbB bZIP is framed. Abbreviations: Ater, A. terreus; Bcin, B. cinerea; Sscl, S. sclerotiorum; Aory, A. oryzae; Afum, A. fumigatus; Cimm, C. immitis; Snod, S. nodorum; Ncra, N. crassa; Fgra, F. graminearum; Mgri, M. grisea; Spom, S. pombe; Scer, S. cerevisiae. Phylogenetic and molecular evolutionary analyses were conducted using MEGA software, version 3.1 (neighbor-joining method, with a bootstrap of 50,000 replicates and amino p-distance substitution model).

FIG. 2.

Mutant characterization in solid medium. Phenotypes of wild-type (WT; TN02A3), ΔflbB (BD143), flbB100, flbB101, and flbB102 strains grown on solid MMA, MMA with reduced glucose, MMA with reduced nitrate, and MMA with KCl (see Materials and Methods) for 72 h are shown. Bar = 1 cm.

FIG. 3.

Mutant characterization in liquid media. Phenotypes of wild-type (WT; TN02A3), ΔflbB (BD143), flbB100, flbB101, and flbB102 strains after 18 h of culture in MMA, followed by transfer to MMA-glucose, MMA-nitrate, or MMA plus KCl and MES for a further 20 h (see Materials and Methods), are shown. Arrowheads indicate conidium-like structures. Bar = 30 μm.

FIG. 4.

Extracellular complementation. The ΔflbB (BD143) mutant acts as a donor of a signaling molecule to a ΔfluG (TTA127.4) strain at zones of contact between colonies. In turn, ΔflbB (BD143) conidiates when in contact with a sporulating strain (MAD782). The arrows indicate the complemented strains. There is no complementation between two flbB deletion strains (BD143 and BD164). All other flbB mutants described in this study display similar behavior. Right panels show close-up views (∼×10 magnification of left panel) of the framed zones. Bar = 0.6 cm.

FIG. 5.

flbB and brlA mRNA expression during the A. nidulans life cycle. (A) Steady-state mRNA levels of flbB and brlA during the life cycle of strain FGSC4. C and Ac represent conidia (asexual spores) and ascospores (sexual spores), respectively. Numbers indicate the time (hours) of incubation in liquid MMA with glucose (Veg) or solid MMA with glucose under conditions inducing asexual development (Asexual) or sexual development (Sexual). Note that flbB is expressed during vegetative growth and early phase of asexual development as well as in ascospores, while brlA is expressed as expected, during the early and medium stages of asexual development. (B) flbB and brlA mRNA expression levels, during asexual development, in the null mutant compared to the wild type (WT).

FIG. 6.

Intracellular localization of FlbB. (A) The FlbB-GFP protein accumulates at the hyphal apex after 16 h of static culture in liquid MMA. Fluorescence at the cytoplasm is also visible, although in these images the contrast has been intentionally modified to show that no nuclei are fluorescent at 16 h. After 24 h and 36 h, fluorescence is also present in the proximal nuclei, as determined by DAPI staining. Arrowheads indicate fluorescent nuclei. (B) Proximity of FlbB to the tip of the cell. Arrowheads indicate the localization of FlbB at the apex and in the proximal nucleus. Bar = 10 μm.

FIG. 7.

FlbB is a potential transcription factor. Yeast strains expressing the Gal4_DBD::AflR (positive control) (28), pTLexA alone (negative control), LexA_DBD::FlbB-F (full length; 1 to 427 aa), LexA_DBD::FlbB-N (N-terminal region including the bZIP domain; aa 1 to 172) and LexA_DBD::FlbB-C (C-terminal region without the bZIP domain; aa 166 to 427) fusion proteins were spotted on the medium without uracil and histidine (−UH) in the presence of various concentrations (0, 5, and 10 mM) of 3-AT in serial dilutions. Plates were incubated at 30°C for 3 days. Yeast strains expressing LexA_DBD::FlbB-F and LexA_DBD::FlbB-C grew in the presence of 5 mM and 10 mM 3-AT, respectively, whereas those expressing LexA_DBD::FlbB-N or vector alone did not grow on the same medium. Five individual transformants for each construct were then tested for their β-galactosidase activities; mean values ± standard deviations are presented on the right. Rapid exhibition of blue color and high levels of β-galactosidase activity (489 ± 12) by the strains expressing LexA_DBD::FlbB-C confirms that the C-terminal region of FlbB contains a potential transactivation domain.

FIG. 8.

Overexpression of flbB inhibits conidiation. (A) Photographs of the colonies of control (TNI22.1) and alcA(p)::flbB (RNI24.5) strains grown on MMA plus glucose (MMG; noninducing) and MMA plus yeast extract (MMT+5Y; threonine-inducing alcA promoter, with 5 g/liter yeast extract enhancing growth) for 5 days are shown. Overexpression of flbB resulted in a blockage in conidiation. (B) Control (TNI22.1) and alcA(p)::flbB (RNI24.5) strains were grown in liquid glucose medium at 37°C and 250 rpm for 18 h and then transferred onto solid medium with threonine (MMT; inducing) as the sole carbon source. Samples were collected at 18 h of vegetative growth (Veg) and 6, 12, and 24 h after asexual developmental induction (Asex) on MMT. Total RNA was isolated and subjected to Northern blot analyses for the levels of brlA and flbB mRNAs. Equal loading of total RNA was evaluated by ethidium bromide staining of rRNA. (C) Close-up views of control (TNI22.1) and alcA(p)::flbB (RNI24.5) strains grown on MMT for 48 h after transfer onto solid MMT. The control strain produced conidiophores abundantly (arrows), whereas alcA(p)::flbB strain (RNI24.5) formed elongated aerial hyphae without conidiophores.