Table 1.
Aspergillus strains used in this study.
Figure 1.
(A∼B) cpcB mRNA levels during the life cycle of A. nidulans (A) and A. fumigatus (B). Conidia (asexual spores) are indicated as C. The numbers indicate the time (hours) after incubation in liquid MMG (Vegetative), and on solid MMG under conditions inducing conidiation (Asexual) or sexual development (Sexual). The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and each band was normalized by the amount of 18S rRNA (cpcB/18S rRNA) in the relevant lane. Ethidium bromide staining of ribosomal RNAs was used as a loading control. (C) Alignment of A. nidulans (Ani) CpcB, A. fumigatus (Afu) CpcB, A. flavus (Afl) CpcB with Gib2 of C. neoformans var. grubii (Cgr; accession: AY907679) and C. neoformans var. neoformans (Cne; accession: AY907680). ClustalW (http://align.genome.jp/) and BoxShade 3.21 (http://www.ch.embnet.org/software/BOX_form.html) were used for the alignment and presentation. (D) A phylogenetic tree of CpcB-like proteins identified in various fungal species including A. clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. oryzae, A. terreus (A = Aspergillus), C. neoformans, Neurospora crassa, Penicillium chrysogenum, Penicillium marneffei, Schizophyllum commune, Schizosaccharomyces japonicus and Talaromyces stipitatus. The putative CpcB proteins were retrieved from NCBI BlastX (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using A. nidulans CpcB. A phylogenetic tree of 14 putative CpcB homologues was generated by the TreeTop software (http://genebee.msu.su/services/phtree_reduced.html) using the alignment data from ClustalW. The phylogenetic tree is constructed based on the matrix of pairwise distances between sequences. Numbers indicate the computed distances given the residue substitution weights from the alignment data [59].
Figure 2.
Requirement of CpcB for proper growth and conidiation in A. nidulans and A. fumigatus.
(A) Photographs of the point-inoculated colonies of WT control (TNJ36), ΔcpcB (RJMP1.59-8) and complemented (C’; RJMP1.59-8C) strains of A. nidulans grown on solid MMG for 4 days (top panels) and the close-up views of the colonies (bottom panels) are shown in left panel. Photographs of the colonies of WT (AF293), ΔAfucpcB (Af293.1-7) and complemented (C’; Af293.1-7C) strains of A. fumigatus grown on solid MM+0.5%YE for 4 days (top panels), and the close-up views of the colonies (bottom panels) are shown in right panel. (B) Quantitative analyses of the diameter of colonies and conidiation levels in the designated strains. (C) Northern blot analyses for levels of brlA transcript in the WT (FGSC4) and ΔAnicpcB (RJMP1.59-8) strains of A. nidulans, and in WT (Af293) and ΔAfucpcB (Af293.1-7) strains of A. fumigatus. The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and normalized by the amounts of 18S rRNA (brlA/18S rRNA) in each sample. Ethidium bromide staining of rRNA was used as a loading control.
Figure 3.
Requirement of CpcB for sexual development in A. nidulans.
(A) Photographs of the point-inoculated colonies of WT (TNJ36), ΔcpcB (RJMP1.59-8) and complemented (C’; RJMP1.59-8C) strains of A. nidulans grown on MMG for 7 days (top panels), and the close-up views of the colonies (bottom panels) captured by a Zeiss Axioplan 2 stereomicroscope are shown. Cleistothecia are marked as CT. (B) Morphology of cleistothecia formed by the three strains. Images were captured by a Zeiss Axioplan 2 stereomicroscope. (C) Quantitative analyses of cleistothecia (CLS) per cm2 produced by the three strains grown on MMG for 7 days. (D) Northern blot analyses for levels of veA and vosA transcripts after sexual developmental induction of A. nidulans WT (FGSC4) and ΔcpcB (RJMP1.59-8) strains. The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of RNA bands using ImageJ and normalized by the amounts of 18S rRNA (veA/18S rRNA or vosA/18S rRNA) in each sample. Ethidium bromide staining of ribosomal RNAs was used as a loading control. Numbers indicate the time post sexual developmental induction.
Figure 4.
The roles of CpcB in conidial germination and hyphal growth.
(A∼B) Quantitative analyses of conidial germination and hyphal growth post conidial germination of WT (filled bar; TNJ36), ΔcpcB (blank bar; RJMP1.59-8) and complemented (C) (shaded bar; RJMP1.59-8C) A. nidulans strains on MMG (A); and of WT (AF293), ΔAfucpcB (Af293.1-7) and complemented (C’; Af293.1-7C) A. fumigates strains on MMG (B). (C) Photographs of the three A. nidulans strains grown on solid MMG for 1 day (top panels) and the close-up views (bottom panels). Note the lack of conidiophores in ΔcpcB strain. (D) Photographs of the three A. fumigates strains of grown on solid MMG for 1 day (top panels), and the close-up views (bottom panels). Note the differences in the size and number of conidiophores.
Figure 5.
CpcB is not required for ST biosynthesis.
(A) TLC of ST produced after 2 days of liquid submerged culture (MM+0.5%YE) of WT (FGSC4), ΔcpcB (RJMP1.59-8, RJMP1.59-24) and complemented (RJMP1.59-8C1, RJMP1.59-8C5) A. nidulans strains. The relative intensity (mean±SD) of ST produced by each strain was determined by ImageJ and normalized by the amount of the ST standard (spot/ST). (B) TLC of GT produced upon 2 days of liquid submerged culture (MM+0.5%YE) of WT (Af293), ΔAfucpcB (Af293.1-4, Af293.1-6, Af293.1-7) and complemented (Af293.1-7C4, Af293.1-7C5, Af293.1-7C6) A. fumigatus strains. The relative intensity (mean±SD) of GT produced by each strain was determined by ImageJ and normalized by the amount of the GT standard (spot/GT). (C) Northern blot for levels of stcU and aflR transcripts in WT (FGSC4) and ΔcpcB (RJMP1.59-8) of A. nidulans. The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and normalized by the amounts of 18S rRNA (aflR/18S rRNA or stcU/18S rRNA) in each sample. Ethidium bromide staining of ribosomal RNAs was used as a loading control.
Figure 6.
Phenotype of the ΔflbAΔcpcB and ΔrgsAΔcpcB mutants.
Photographs of colonies of WT (FGSC4), ΔcpcB (RJMP1.59-8), ΔflbA (QK1), and ΔflbAΔcpcB (QK3) strains (A); and ΔrgsA (QK2) and ΔrgsAΔcpcB (QK4) strains (B) grown on solid MMG for 3 days are shown. The two left panels show the point-inoculated strains (top and bottom), and the two right panels show close-up views of single and double mutant colonies shown in the left panels. Photomicrographs were taken by Zeiss Axioplan 2 stereomicroscope. Note the highly restricted growth of the double mutants (A and B), and the lack of conidiation in the ΔflbA and ΔflbA ΔcpcB mutant (A).
Figure 7.
A speculative model summarizing the roles of CpcB and SfaD in governing various biological processes in A. nidulans.