The pkaB gene encoding the secondary protein kinase A catalytic subunit has a synthetic lethal interaction with pkaA and plays overlapping and opposite roles in Aspergillus nidulans

Abstract

Filamentous fungal genomes contain two distantly related cyclic AMP-dependent protein kinase A catalytic subunits (PKAs), but only one PKA is found to play a principal role. In Aspergillus nidulans, PkaA is the primary PKA that positively functions in vegetative growth and spore germination but negatively controls asexual sporulation and production of the mycotoxin sterigmatocystin. In this report, we present the identification and characterization of pkaB, encoding the secondary PKA in A. nidulans. Although deletion of pkaB alone does not cause any apparent phenotypic changes, the absence of both pkaB and pkaA is lethal, indicating that PkaB and PkaA are essential for viability of A. nidulans. Overexpression of pkaB enhances hyphal proliferation and rescues the growth defects caused by DeltapkaA, indicating that PkaB plays a role in vegetative growth signaling. However, unlike DeltapkaA, deletion of pkaB does not suppress the fluffy-autolytic phenotype resulting from DeltaflbA. While upregulation of pkaB rescues the defects of spore germination resulting from DeltapkaA in the presence of glucose, overexpression of pkaB delays spore germination. Furthermore, upregulation of pkaB completely abolishes spore germination on medium lacking a carbon source. In addition, upregulation of pkaB enhances the level of submerged sporulation caused by DeltapkaA and reduces hyphal tolerance to oxidative stress. In conclusion, PkaB is the secondary PKA that has a synthetic lethal interaction with PkaA, and it plays an overlapping role in vegetative growth and spore germination in the presence of glucose but an opposite role in regulating asexual sporulation, germination in the absence of a carbon source, and oxidative stress responses in A. nidulans.

FIG.1.

Summary of pkaB, encoding the secondary PKA catalytic subunit. A. A partial restriction map of the pkaB gene region is shown. The pkaB ORF (filled box), the mRNA coding region (arrow), and the three introns (marked by discontinuities on the arrow) were determined by reverse transcription-PCR and random amplification of cDNA ends followed by sequence analyses. B. Steady-state mRNA levels of pkaB in various growth and developmental stages. Numbers indicate the times of incubation in liquid MMG (Veg) or solid MMG under the conditions inducing asexual development or sexual development. The pkaB mRNA levels were low at 24 and 96 h post-asexual and -sexual developmental induction. C. Amino acid sequence alignment of A. nidulans PkaB (An_PkaB), S. cerevisiae Tpk1P (Sc_Tpk1), A. fumigatus PkaC2 (Af_PkaC2), and U. maydis Uka1 (Um_Uka1), showing identities (black boxes) and similarities (gray boxes). The proteins were aligned with ClustalW (5) and displayed by BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). D. Phylogenetic tree of PkaA, PkaB, and SchA of A. nidulans, PkaC1 and PkaC2 of A. fumigatus, CpkA and Cpk2 of M. grisea, XP324862.1 and XP326095.1 of N. crassa, Tpk1p, Tpk2p, and Tpk3p of S. cerevisiae, Adr1, and Uka1 and UM00484.1 of U. maydis. The ClustalW alignment (5) of the fungal proteins was presented by TREEVIEW. The length of the bar represents an evolutionary distance of 0.1 amino acid substitutions per site.

FIG. 2.

Overexpression of pkaB enhances vegetative growth. Photographs of the colonies of wild-type (WT; FGSC26), ΔpkaB (RSA53.143), alcA(p)::pkaB (RNI5.3), ΔpkaA (RSA53.130), ΔpkaA alcA(p)::pkaB (RNI6), and ΔpkaA ΔpkaB alcA(p)::pkaB (RNI7) strains grown on MMG (noninducing) or MMT plus YE (threonine plus 5 g/liter YE) for 3 days or on MMT for 6 days at 37°C are shown. Yeast extract was added to achieve both alcA induction and enhanced growth. While ΔpkaB caused no apparent phenotypic changes, overexpression of pkaB resulted in elevated hyphal proliferation and reduced asexual sporulation on both MMT plus YE and MMT (upper panel). Furthermore, overexpression of pkaB rescued the growth defects caused by ΔpkaA (lower panel).

FIG. 3.

Upregulation of pkaB rescues germination defects caused by deletion of pkaA. A. The conidia of wild-type (WT; FGSC26), ΔpkaB (RSA53.143), ΔpkaA (RSA53.130), alcA(p)::pkaB (RNI5.3), ΔpkaA alcA(p)::pkaB (RNI6), and ΔpkaA ΔpkaB alcA(p)::pkaB (RNI7) strains were inoculated (10⁸ spores/100 ml) into liquid MMG and incubated at 37°C, 250 rpm, for 24 h. Total RNA was isolated from each sample, 6 μg of total RNA was loaded per lane, and equal loading was confirmed by ethidium bromide staining of rRNA. Note that the presence of the alcA(p)::pkaB construct resulted in elevated pkaB mRNA levels (∼10-fold). While deletion of pkaA did not affect the pkaB mRNA levels in the absence of alcA(p)::pkaB, somehow ΔpkaA and ΔpkaA ΔpkaB caused reduced accumulation of the pkaB mRNA derived from the alcA promoter. Due to the differences in transcriptional termination, the pkaB mRNA derived from the alcA promoter is about 0.5 kb bigger than the endogenous pkaB transcript shown in wild-type (FGSC26) and ΔpkaA (RSA53.130) strains. Because wild-type and ΔpkaA strains showed extremely low pkaB mRNA levels compared to the strains containing alcA(p)::pkaB, different exposure conditions were employed to make the pkaB transcripts clearly visible in all the strains tested. It is important to note that the pkaB transcripts shown in alcA(p)::pkaB, ΔpkaA alcA(p)::pkaB, and ΔpkaA ΔpkaB alcA(p)::pkaB strains resulted from a 20-h exposure with one intensifying screen, whereas the pkaB transcripts shown in wild-type and ΔpkaA strains were visualized by a 24-h exposure with two intensifying screens. Therefore, the actual relative levels of the pkaB mRNA in wild-type and ΔpkaA strains must be reduced by twofold. B. The conidia of wild-type (FGSC26), alcA(p)::pkaB (RNI5.3), ΔpkaA (RSA53.130), and ΔpkaA alcA(p)::pkaB (RNI6) strains were inoculated on solid MMG and incubated at 37°C for 10 h. Note that the presence of an ectopic copy of alcA(p)::pkaB could rescue the germination defects caused by ΔpkaA.

FIG. 4.

Inhibitory roles of pkaB in germination. A. The conidia of wild-type (WT; FGSC26), alcA(p)::pkaB (RNI5.2), ΔpkaA (RSA53.130), and ΔpkaA alcA(p)::pkaB (RNI6) strains were inoculated on solid MMT (inducing medium) and incubated at 37°C for 10 h. Note that while most of the wild-type conidia germinated, only a few mutant conidia germinated. The arrows show the alcA(p)::pkaB mutant conidia forming a germ tube. Although culture conditions were not identical, the pkaB mRNA levels in alcA(p)::pkaB (RNI5.2) and ΔpkaA alcA(p)::pkaB (RNI6) strains grown in liquid MMT for 24 h (see Materials and Methods) were about 20-fold higher than those of the wild type (not shown). B. The conidia of wild-type (WT; FGSC26), alcA(p)::pkaB (RNI5.2), ΔpkaA (RSA53.130), and ΔpkaA alcA(p)::pkaB (RNI6) strains were inoculated on the solid medium lacking a carbon source and incubated at 37°C for 31 h. An electrophoresis-grade agarose was used as a solidifying agent to avoid the introduction of unwanted energy sources. At 31 h, most of the wild-type conidia were able to germinate and about 20% of the ΔpkaA mutant conidia could form germ tubes (arrows). However, upregulation of pkaB caused by alcA(p)::pkaB completely blocked germination of spores regardless of the presence or absence of pkaA. Although culture conditions were different, the pkaB mRNA levels in alcA(p)::pkaB (RNI5.2) and ΔpkaA alcA(p)::pkaB (RNI6) strains incubated in the liquid medium lacking a carbon source at 37°C for 24 h (see Materials and Methods) were about fivefold higher than those of the wild type (data not shown).

FIG. 5.

Upregulation of pkaB enhances submerged sporulation caused by ΔpkaA. The conidia (10⁸/100 ml) of wild-type (WT; FGSC26), alcA(p)::pkaB (RNI5.2), ΔpkaA (RSA53.130), and ΔpkaA alcA(p)::pkaB (RNI6) strains were inoculated in liquid MMG and incubated at 37°C, 250 rpm, for 20 h. The arrows indicate conidiophore-like structures formed in ΔpkaA and ΔpkaA alcA(p)::pkaB strains. The steady-state mRNA levels of brlA were examined at 26 h of incubation. Transcripts of brlA were detectable in the ΔpkaA and ΔpkaA alcA(p)::pkaB mutants. Note that brlA is highly accumulated by upregulation of pkaB in the absence of pkaA. For the pkaB mRNA levels in individual strains, see Fig. 3A.

FIG. 6.

Upregulation of pkaB or ΔpkaA reduces hyphal tolerance to oxidative stress. The hyphae of wild-type (WT; FGSC26), ΔpkaB (RSA53.111), alcA(p)::pkaB (RNI5.2), ΔpkaA (RSA53.126), and alcA(p)::pkaA (RNI8) strains grown on solid MMG at 37°C for 26 h were treated with 0, 10, 50, 150, and 300 mM H₂O₂ for 10 min in triplicate. The plates were then incubated at 37°C for an additional 24 h. A. The colonies that survived through 0 mM (control) or 50 mM H₂O₂ treatment are shown. Note that no alcA(p)::pkaB or ΔpkaA mutants survived after treatment, whereas the alcA(p)::pkaA and ΔpkaB mutants did not exhibit differences in survival rates compared to the wild type. B. The bar graph shows the relative survival rates of the hyphae of wild-type, alcA(p)::pkaB, and ΔpkaA strains.

FIG. 7.

Proposed model for PKA-mediated signaling in A. nidulans. We propose that the two PKAs are differently regulated by G protein signaling and that overlapping and opposite roles of PkaA (primary PKA) and PkaB (secondary PKA) control various biological processes. FlbA-FadA (Gα)-controlled vegetative growth signaling is in part transduced by PkaA, which in turn represses conidiation and ST production (34). The demonstration that GanB and SfaD::GpgA, as well as PkaA, are required for proper germination of spores (10, 19, 30, 33) indicates that PkaA may be activated by both GanB and FadA. RgsA-controlled GanB signaling is also associated with activation of the stress response (13). The fact that ΔpkaA delayed conidial germination and reduced hyphal tolerance to oxidative stress further supports the idea that PkaA may be activated by GanB. While PkaB can stimulate vegetative growth and activate conidial germination in the presence of glucose, upregulation of pkaB blocks germination of spores in the absence of a carbon source and reduces hyphal tolerance to oxidative stress. This suggests that the resulting cellular responses for PkaB and GanB-PkaA signaling may be opposite. Thus, if PkaB functions downstream of GanB, the activity of PkaB may be negatively regulated by GanB. GanB-mediated signaling may activate other stress responses via a different (possibly a mitogen-activated protein kinase) downstream signaling cascade.