MpkA-Dependent and -independent cell wall integrity signaling in Aspergillus nidulans

Abstract

Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Delta and mpk1Delta mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Delta and swi6Delta mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmADelta, Answi4Delta Answi6Delta, and mpkADelta strains) and analyzed mpkA and CWG transcripts after treatment with a beta-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The beta-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkADelta strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except alpha-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae.

FIG. 1.

In silico reconstruction of the Aspergillus CWIS pathway based on Aspergillus and S. cerevisiae genome information. The putative orthologous proteins involved in CWIS in Aspergillus and S. cerevisiae are represented; An, Ao, and Af indicate the number of BLAST hits for A. nidulans, A. oryzae, and A. fumigatus, respectively. Most genes for CWIS are nonredundant and are well conserved among the Aspergillus fungi and S. cerevisiae. Shaded circles represent transcription factors. Rectangles with rounded edges represent components of the MAPK signaling pathway. Ellipses represent signals that are not part of the MAPK signaling pathway. MAPKK, MAPK kinase; MAPKKK, MAPK kinase kinase.

FIG. 2.

A. nidulans rlmA complements an S. cerevisiae rlm1Δ mutant, while rlmA derivatives and AN8676.3 do not. (A) The mutation of residues L432 and V434 into Ala yielded mutant M1, and the mutation of residues I436 and P437 into Ala yielded mutant M2. (B and C) Serial fivefold dilutions of rlm1 mutants harboring the following plasmids were spotted onto plates supplemented with 7 mM caffeine and 2% glucose or 2% galactose at 30°C for 3 days (Glc or Gal alone) or 6 days (Glc-caffeine or Gal-caffeine): pYES2 (vector), YEp195-RLM1 (RLM1 wild type; Rlm1p), pYESrlmA (rlmA wild type; RlmA), pYESrlmA-M1 (rlmA mutant M1; RlmA-M1), pYESrlmA-M2 (rlmA mutant M2; RlmA-M2), and pYES-AN8676.3 (AN8676.3). A. nidulans rlmA, its derivatives, and AN8676.3 were transcribed by the yeast GAL1 promoter, which is inducible and repressible in the presence of galactose and glucose, respectively. The yeast rlm1 mutant is sensitive to caffeine, and the expression of the wild-type rlmA or yeast RLM1 gene, but not that of rlmA derivatives and AN8676.3, suppressed the caffeine sensitivity of the yeast rlm1 mutant.

FIG. 3.

Complementation of the mpk1 mutant by expression of the mpkA cDNA. Serial fivefold dilutions of mpk1 mutants harboring the following plasmids were spotted onto YPD plates at 23 and 37°C for 3 days: YEpGAP (vector), YCplac33-MPK1 (Mpk1p), and YEpGAPmpkA (MpkA). A. nidulans mpkA was transcribed by the constitutive GAPDH promoter. The yeast mpk1 mutant displays an autolytic lethal phenotype at 37°C, and the expression of the mpkA or yeast MPK1 gene suppressed the thermosensitivity of the yeast mpk1 mutant.

FIG. 4.

The rlmAΔ strain exhibited sensitivity to CFW. A wild-type strain and an rlmAΔ strain were inoculated (as a suspension of ca. 10⁴ conidiospores) onto minimal-medium plates containing CFW, a potent inhibitor of chitin synthesis, and were cultured at 30°C for 6 days.

FIG. 5.

Micafungin-activated CWIS of S. cerevisiae and A. nidulans. (A) The S. cerevisiae wild-type strain was grown to an A₆₀₀ of 0.7 in YPD medium at 30°C and then treated with 1 μg of micafungin/ml (final concentration). The upper panel shows the results of immunoblotting with anti-phospho-p44/42 MAPK antibodies (anti-p44/42). The lower panel shows the results of immunoblotting with anti-Mpk1p antibodies (anti-Mpk1) as a loading control. (B) The S. cerevisiae wild-type and rlm1Δ strains were grown to an A₆₀₀ of 0.7 in YPD medium at 30°C and then treated with 1 μg of micafungin/ml (final concentration). Levels of transcription of MLP1 were determined by quantitative RT-PCR using specific primers (Table 1). As an internal control, the RT-PCR mixture contained a primer pair specific for the RPL28 gene. The y axis displays relative mRNA levels. t, time. (C) The A. nidulans wild-type strain was cultured at 30°C for 24 h in CD liquid medium and treated with 0.01 μg of micafungin/ml (final concentration). The upper panel shows the results of immunoblotting with anti-phospho-p44/42 MAPK antibodies. The lower panel shows the results of immunoblotting with anti-extracellular signal-regulated kinase 2 antibodies (anti-ERK-2) as a loading control.

FIG. 6.

Analysis of expression of cell wall-related genes and mpkA in rlmAΔ and mpkAΔ strains. Levels of transcription of the indicated genes were determined by means of quantitative RT-PCR using specific primers (Table 1) for each gene. As an internal control, each RT-PCR mixture also contained a primer pair specific for a histone H2B gene. The y axes display relative mRNA levels. Cells of the wild-type, rlmAΔ, and mpkAΔ strains were precultured to the logarithmic growth phase at 30°C and further incubated with 0.01 μg of micafungin/ml (final concentration) at 30°C for 0, 30, 60, or 120 min. Total RNAs were extracted from the cells. t, time.

FIG. 7.

The mpkAΔ strain exhibited sensitivity to CFW and micafungin. A wild-type strain and an mpkAΔ strain were inoculated (as a suspension of ca. 10⁴ conidiospores) onto minimal-medium plates containing CFW, a potent inhibitor of chitin synthesis (A), and a β-1,3-glucan synthase inhibitor, micafungin (B), and were cultured at 30°C for 6 days.

FIG. 8.

Assay of GUS activity with mpkA-GUS gene fusion in the mpkAΔ strain. (A) Construction of the plasmid used for the reporter assay. uidA, T-agdA, aurA^R, and AMA1 represent (respectively) the E. coli GUS gene as the reporter, the terminator region of the A. oryzae agdA gene, the A. nidulans aureobasidin A resistance gene as a selectable marker, and the A. nidulans replication origin. The plasmid containing the mpkA promoter (up to 995 bp upstream of the translation initiation codon) was designated pAURGUSmpkA. (B) GUS activities of the transformants with the mpkA promoter (P-mpkA)-GUS gene fusion. GUS activity was measured as described in Materials and Methods.

FIG. 9.

Expression of mpkA in response to micafungin in the Answi4Δ and Answi6Δ disruption mutants. The expression of the mpkA gene in the Answi4Δ strain (A) and the Answi6Δ strain (B) was analyzed by Northern blotting and compared with that in the wild type. A histone H2B gene was used as a control. Cells of all three strains were precultured to the logarithmic growth phase at 30°C and further incubated with 0.01 μg of micafungin/ml (final concentration) at 30°C for 0, 30, 60, or 120 min. Total RNAs were extracted from the cells. Probes were prepared as described in Materials and Methods. t, time.

FIG. 10.

Schematic model of transcriptional regulation of mpkA and of cell wall-related genes via cell wall stress signaling in A. nidulans. Based on the study results, we hypothesize that A. nidulans has the following transcriptional regulation system: (i) mpkA, MpkA dependent but not RlmA dependent; (ii) agsA and agsB, MpkA dependent and partly dependent on RlmA; (iii) gfaA, dependent on both MpkA-RlmA and an unidentified non-MpkA system; and (iv) fksA, gelA, gelB, chsA, chsB, chsC, chsD, csmA, and csmB, independent of the MpkA-RlmA system. The cell wall integrity pathway regulates mainly the transcription of α-1,3-glucan biogenesis-related genes. The transcripts of β-1,3-glucan and chitin biogenesis-related genes are regulated mainly by other, unknown signaling that may be activated by a cell wall stress such as micafungin treatment. X, unknown factor; −, downregulated; +, upregulated.