AtfA bZIP-type transcription factor regulates oxidative ... - Springer Link

Mol Genet Genomics (2010) 283:289–303 DOI 10.1007/s00438-010-0513-z

ORIGINAL PAPER

AtfA bZIP-type transcription factor regulates oxidative and osmotic stress responses in Aspergillus nidulans Anita Balázs · Imre Pócsi · Zsuzsanna Hamari · Éva Leiter · Tamás Emri · Márton Miskei · Judit Oláh · Viktória Tóth · Nikoletta Heged:s · Rolf A. Prade · Mónika Molnár · István Pócsi

Received: 20 March 2009 / Accepted: 18 January 2010 / Published online: 4 February 2010 © Springer-Verlag 2010

Abstract The aim of the study was to demonstrate that the bZIP-type transcription factor AtfA regulates diVerent types of stress responses in Aspergillus nidulans similarly to Atf1, the orthologous ‘all-purpose’ transcription factor of Schizosaccharomyces pombe. Heterologous expression of atfA in a S. pombe atf1 mutant restored the osmotic stress tolerance of Wssion yeast in surface cultures to the same level as recorded in complementation studies with the atf1 gene, and a partial complementation of the osmotic and oxidative-stress-sensitive phenotypes was also achieved in submerged cultures. AtfA is therefore a true functional ortholog of Wssion yeast’s Atf1. As demonstrated by RTPCR experiments, elements of both oxidative (e.g. catalase B)

and osmotic (e.g. glycerol-3-phosphate dehydrogenase B) stress defense systems were transcriptionally regulated by AtfA in a stress-type-speciWc manner. Deletion of atfA resulted in oxidative-stress-sensitive phenotypes while the high-osmolarity stress sensitivity of the fungus was not aVected signiWcantly. In A. nidulans, the glutathione/glutathione disulWde redox status of the cells as well as apoptotic cell death and autolysis seemed to be controlled by regulatory elements other than AtfA. In conclusion, the orchestrations of stress responses in the aspergilli and in Wssion yeast share several common features, but further studies are needed to answer the important question of whether a Wssion yeast-like core environmental stress response also operates in the euascomycete genus Aspergillus.

A. Balázs and Imre Pócsi contributed equally to this paper.

Keywords Stress signaling · Oxidative stress · Osmotic stress · Apoptosis · Autolysis · Auxotrophy · CESR

Communicated by J. Perez-Martin. A. Balázs · Imre Pócsi · É. Leiter · T. Emri · V. Tóth · N. Heged:s · M. Molnár · István Pócsi (&) Department of Microbial Biotechnology and Cell Biology, Faculty of Science and Technology, University of Debrecen, P.O. Box 63, Debrecen 4010, Hungary e-mail: [email protected]; [email protected] Z. Hamari (&) Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, Szeged 6726, Hungary e-mail: [email protected] M. Miskei · J. Oláh Department of Horticulture and Plant Biotechnology, Faculty of Agricultural Science, University of Debrecen, Egyetem tér 1, Debrecen 4010, Hungary R. A. Prade Department of Microbiology and Molecular Genetics, Oklahoma State University, 307 Life Sciences East, Stillwater, OK 74078, USA

Introduction In the aspergilli, SskB [mitogen activated protein kinase kinase kinase (MAPKKK)]—PbsB [mitogen activated protein kinase kinase (MAPKK)]—HogA/SakA [mitogen activated protein kinase (MAPK)] stress signaling plays a pivotal role in the regulation of diVerent types of stress responses, e.g. responses to osmotic (Han and Prade 2002; Kawasaki et al. 2002; Furukawa et al. 2005; Hagiwara et al. 2007; Vargas-Pérez et al. 2007), oxidative (Kawasaki et al. 2002; Furukawa et al. 2005; Hagiwara et al. 2007) and starvation (Xue et al. 2004) stress. Although some upstream elements of the SskB–HogA/SakA signaling pathway including the phosphotransfer protein YpdA and the response regulator SskA have been identiWed and functionally characterized (Furukawa et al. 2005; Hagiwara

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290

et al. 2007; Vargas-Pérez et al. 2007), no stress–response transcription factor downstream to HogA/SakA has been described until now. Therefore, our main goal was to identify and functionally characterize a transcriptional regulator that may transmit YpdA–SskA–SskB–PbsB–HogA/SakA stress signals to a wide array of stress response genes. The cardinal importance of MAPK signaling in the coordination of genome-wide fungal stress responses has been emphasized by several authors (Gasch et al. 2000; Chen et al. 2003, 2008; Gasch 2007; Miskei et al. 2009). To Wnd a promising candidate for acting downstream of HogA/SakA MAPK, we relied on a recent in silico reconstruction of yeast-like stress–response systems in the aspergilli (Miskei et al. 2009). The success of the application of yeast-based models to identify and annotate stress– response proteins in the genus Aspergillus led us to the conclusions that (1) the aspergilli are closer to the Wssion yeast Schizosaccharomyces pombe than to the budding yeast Saccharomyces cerevisiae when the elements and the organization of their stress–response systems are considered and compared and, as a consequence, (2) A. nidulans SskB–PbsB–HogA/SakA signaling resembles the Wssion yeast’s Wak1/Win1 (MAPKKK)–Wis1 (MAPKK)–Sty1/ Spc1 (MAPK) system, which is an ‘all-purpose’ signaling pathway transmitting various environmental stress signals to core environmental stress response (CESR) genes (Chen et al. 2003, 2008; Gasch 2007; Miskei et al. 2009). It is important to note that, downstream to Sty1/Spc1 MAPK, environmental stress signals proceed via Atf1, a bZIP-type transcriptional factor which primarily controls CESR genes in S. pombe (Shiozaki and Russell 1996; Wilkinson et al. 1996; Chen et al. 2003), and that a putative ortholog of S. pombe Atf1, called AtfA, has been annotated in A. nidulans (locus ID in Broad Institute Aspergillus nidulans database at http://www.broad.mit.edu/annotation/genome/: AN2911.3; expectation value for homology between A. nidulans AtfA and S. pombe Atf1 is E = 1 £ 10¡36, coming from BLASTP search performed in the NCBI protein database at http:// www.blast.ncbi.nlm.nih.gov/Blast.cgi; Aguirre et al. 2005; Hagiwara et al. 2008; Miskei et al. 2009). The Wrst functional analysis of AtfA was performed by Hagiwara et al. (2008), who generated and phenotypically characterized a atfA deletion mutant in A. nidulans. These authors found that AtfA—similar to SskA response regulator and HogA/SakA MAPK (Kawasaki et al. 2002; Hagiwara et al. 2007; Vargas-Pérez et al. 2007)—was required for the formation of conidia with appropriate tolerance of oxidative and heat stress. On the other hand, they did not observe any stress-sensitive phenotypes when the fungus was subjected to diVerent types of high-osmolarity and oxidative stress in surface cultures (Hagiwara et al. 2008). In this study, the atfA gene of A. nidulans was deleted, the oxidative stress (caused by tert-butylhydroperoxide,

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Mol Genet Genomics (2010) 283:289–303

H2O2 or menadione sodium bisulWte) sensitive phenotypes of a group of atfA mutants were described, and a set of oxidative and osmotic-stress-responsive genes regulated by AtfA was identiWed. Moreover, the osmotic and oxidative stress sensitivities of a atf1 mutant Wssion yeast strain (Takeda et al. 1995) were successfully complemented with the heterologous expression of AtfA. The possible roles of AtfA in the regulation of important and complex physiological processes such as environmental stress responses, apoptotic cell death and autolysis are also discussed here.

Materials and methods Strains and culture media The genotypes and origin of the A. nidulans and S. pombe strains are summarized in Table 1. For cultivation of the A. nidulans strains, standard complete medium and minimal nitrate medium (MNM) were used with appropriate nutritional supplements (Barratt et al. 1965; Cove 1966; Scazzocchio et al. 1982; http://www.gla.ac.uk/ibls/molgen/ aspergillus/supplement.html). S. pombe strains were grown on minimal medium agar (MMA; 1% glucose, 0.67% Yeast Nitrogen Base Difco™, 2% agar) and Edinburgh minimal medium 2 (EMM2, US Biological) plates or liquid cultures. When required, EMM2 was supplemented with 100 mg l¡1 leucine. Unless otherwise indicated, S. pombe and A. nidulans cultures were incubated at 30 and 37°C and were shaken at 2.5 and 3.3 Hz frequencies, respectively. Complementation of the osmotic and oxidative-stresssensitive phenotypes of atfA mutant S. pombe cells with atf1 and atfA To isolate RNA from A. nidulans, the FGSC 26 strain was cultured overnight in liquid complete medium (Cove 1966). Mycelia were washed and transferred into MNM (Barratt et al. 1965), and supplemented with 0.8 mmol l¡1 menadione sodium bisulWte to induce the transcription of oxidative-stress-responsive genes (Pócsi et al. 2005). After harvesting mycelia at 5 h incubation time, RNA was extracted with TRIZOL® Reagent (Invitrogen), atfA mRNA was reverse-transcribed with the SUPERSCRIPT™ II Kit (Invitrogen), and atfA cDNA was PCR-ampliWed with the primers shown in Table 2. The cDNA was cloned into pGEM® T Easy vector (Promega), and its sequence was veriWed by sequencing both strands. After digestions with NdeI restriction enzyme (Fermentas Ltd., Vilnius, Lithuania), the atfA cDNA was re-cloned into the S. pombe expression vectors pREP1, pREP41, and pREP81 (Maundrell 1993; Basi et al. 1993). The NT146 atf1 strain was transformed using the lithium-acetate method of Ito et al.


291

Table 1 Genotypic characterization and origin of the S. pombe and A. nidulans strains used in this study Genotypea

Strain

Origin/reference

S. pombe strains NT146 strain

h¡ leu1-32 ura4-D18 atf1::ura4

T. Toda (Cancer Research UK); Takeda et al. (1995)

HM123

h¡ leu1-32

Takeda et al. (1995)

CS 2902

pyrG89 biA1 pyroA4 riboB2

Yu et al. (2004)

FGSC 26

biA1

S. Rosén (University of Lund)

pabaA1 riboB2

Strain Collection of the Department of Microbiology, University of Szeged

A. nidulans strains

G1071

b

HZS 120

J. Clutterbuck (University of Glasgow)

HZS 145b

Strain Collection of the Department of Microbiology, University of Szeged

HZS 187

pabaA1 biA1 riboB2 atfA::riboB

This study

HZS 188

biA1 riboB2 atfA::riboB

This study

HZS 189

pabaA1 riboB2 atfA::riboB

This study

HZS 190, LE 1–24

riboB2 atfA::riboB

This study

HZS 191

pyrG89 biA1 riboB2 atfA::riboB

This study

HZS 192

pyrG89 biA1 pyroA4 riboB2 atfA::riboB

This study

HZS 193

pyrG89 pabaA1 pyroA4 riboB2 atfA::riboB

This study

HZS 194

pabaA1 pyroA4 riboB2 atfA::riboB

This study

a

All A. nidulans strains used in this study carried the veA1 mutation. leu1, ura4, pyrG89, pabaA1, biA1, pyroA1, and riboB2 are leucine, uracil, uracil, p-amino-benzoic acid (PABA), biotin, pyridoxine, and riboXavin auxotroph mutations, respectively. Chromosome localization of the above listed genes can be found at websites http://www.broadinstitute.org/annotation/genome/aspergillus_group/Home.html or http://www.gla.ac.uk/ ibls/molgen/aspergillus/index.html for A. nidulans and http://old.genedb.org/genedb/pombe/ for S. pombe b veA¡ genotype was checked by incubating the strains on MNM agar for 3 days at 37°C in complete darkness similarly to the procedure of Mooney and Yager (1990), and veA1 mutation in the initiation codon of the VeA ORF was also veriWed according to Kim et al. (2002)

(1983) with the following plasmids: pREP41-atf1 (positive control carrying the Wssion yeast atf1 gene; a kind gift from Dr. T. Toda, Cancer Research, UK), pREP41 (negative control), and the plasmids carrying the A. nidulans atfA gene (pREP1-atfA, pREP41-atfA, and pREP81-atfA). Transformed cells were plated on MMA. To examine the osmotic stress sensitivity on plates, serial dilutions of the transformed and control cultures pregrown in liquid EMM2 overnight were pipetted onto EMM2 agar plates supplemented with 1.1 mol l¡1 KCl or 0.4 mol l¡1 NaCl, and were grown for 5 days. When the osmotic stress sensitivity was tested in liquid culture, EMM2 containing either 1.0 mol l¡1 KCl or 0.5 mol l¡1 NaCl was inoculated with S. pombe cells at a cell density of OD630 t 0.10. Propagation of the cells was monitored spectrophotometrically at = 630 nm with an EL 340 Microplate Bio Kinetics Reader (Bio-Tek™ Instruments) at 2 h intervals. EMM2-cultured S. pombe cells (OD630 = 0.10– 0.15) were also exposed to oxidative stress by supplementing the liquid culture media with 30% (v/v) H2O2 at a Wnal concentration of 2.0 mmol l¡1. After 1 h incubation, Wssion yeast cells were washed with water, and were re-suspended in H2O2-free EMM2. When cells recovered from the oxidative

stress (t13 h), their propagation was monitored by measuring OD630 at 2 h intervals. Construction and stress sensitivity of A. nidulans atfA deletion mutants The atfA gene was replaced with the riboB marker gene in the HZS 120 atfA+ strain. The substitution cassette was constructed by double-joint PCR (Yu et al. 2004; primers for the deletion construct are summarized in Table 2; the targeting sequences for homologous recombination were 1,325 and 2,451-bp-long upstream and downstream to the atfA gene, respectively), and was transformed directly in the HZS 120 recipient strain (Tilburn et al. 1983). Selected transformants were pre-screened for the absence of the atfA gene product by PCR (Table 2), and transformants without amplicon were also checked by genomic DNA Southern blot analysis. Three single-copy transformants (genotype pabaA1 riboB2 atfA::riboB) characterized with light green conidia were crossed with the CS 2902 strain, and a set of atfA progenies with diVerent auxotrophy markers was collected. All riboB+ progenies of the independent crosses proved to be atfA deleted mutants by Southern analyses.

123

292 Table 2 PCR primers used in the genetic manipulations


Primers

Sequences

Reverse transcription of atfA mRNA atfA RT

5⬘-cgccagttagtcaagtgatattattcc-3⬘

AmpliWcation of atfA cDNAa atfA amp frw

5⬘-cgcatatgtctgccgccgtggcttc-3⬘

atfA amp rev

5⬘-ggcatatgtcaagtgtatggaggatt-3⬘

Double-joint PCR atfA upstream frw

5⬘-gatagaaacccgcagaacgaagagg-3⬘

atfA upstream nested frw

5⬘-gagtattggctaaactgcagtgcggcaag-3⬘

atfA upstream rev

5⬘-ccggtcgataaatcagctgaataatagag-3⬘

atfA riboB chimera frw

5⬘-ctctattattcagctgatttatcgaccggacgtagtgtagattcaggcacattgaagcg-3⬘

atfA riboB chimera rev

5⬘-acagaaaccgttgcgccagttagtcaagtggccatgactactaggtggtgctatc-3⬘

atfA downstream frw

5⬘-cacttgactaactggcgcaacggtttctgt-3⬘

atfA downstream nested rev

5⬘-cctatgtagaatctcagaaactgtcttc-3⬘

atfA downstream rev

5⬘-agctggacatagtgtttgacctgcacg-3⬘

Screening the absence of atfA in transformants a

The primers included NdeI restriction sites (italics)

frw

5⬘-gtactgacttgatgatcttccagaggc-3⬘

rev

5⬘-gtctgttagtatactgataaattagagg-3⬘

In stress sensitivity studies, the agar plate assays of Hagiwara et al. (2007, 2008) were used with minor modiWcations. Freshly grown (7 days) conidia (105 suspended in 5 l aliquots of PBS–0.1% Tween 20) were spotted on MNM plates (Barratt et al. 1965) containing the following stressgenerating agents: oxidative stress: 0.6–0.8 mmol l¡1 tertbutylhydroperoxide (tBOOH; stimulates lipid peroxidation), 6.0 mmol l¡1 H2O2 (increases the peroxide content of the cells), 0.12 mmol l¡1 menadione sodium bisulphite (MSB; elevates intracellular superoxide concentrations), 2.0 mmol l¡1 diamide [disturbs the glutathione status of the cells via instantaneous and stoichiometrical oxidation of glutathione (GSH) to glutathione disulphide (GSSG)], osmotic stress: 1.5 mol l¡1 KCl, 1.5 mol l¡1 NaCl, 2.0 mol l¡1 sorbitol. Stress plates were incubated for 5 days, and the osmotic stress tolerances of selected prototroph strains were tested at both 30 and 37°C temperatures (Han and Prade 2002). To test the tBOOH-sensitivity of A. nidulans mycelia on agar plates, freshly-harvested conidia were pipetted onto circle-shaped, 2-cm-diameter cellophane sheets laid on the surfaces of MNM agar plates and were incubated for 24 h. Mycelial mats were transferred with the sheets onto freshly prepared MNM agar also supplemented with 0–3.0 mmol l¡1 tBOOH. The oxidative stress sensitivity of atfA mutants was also studied in submerged cultures. Late exponential growth phase vegetative tissues of selected mutant and control strains were transferred into fresh MNM and were cultivated further for 30 h according to the protocol of Pócsi et al. (2005). The starting dry cell mass (DCM) was set to approximately 4 mg ml¡1 for all strains, and culture media were either supplemented with 75–300 mmol l¡1 H2O2,

123

0.38–2.0 mmol l¡1 tBOOH, 0.4–0.8 mmol l¡1 MSB or 3.0 mmol l¡1 diamide (stress-exposed cultures) or left untreated (controls). The oxidative, heat, and cold stress sensitivities of A. nidulans conidia were estimated according to Hagiwara et al. (2007, 2008). Freshly grown conidia (105 ml¡1) were treated with 100 and 200 mmol l¡1 H2O2 at 37°C for 20 min, were subjected to heat stress at 50°C for 30 and 60 min or were kept at 4°C for 7 days in glucose-free MNM. Stress-exposed conidia were spread on nutrient agar plates, and the numbers of colonies were counted after 2 days incubation at 37°C. The conidiospore forming capabilities of A. nidulans strains were determined as published previously by Vargas-Pérez et al. (2007). Cell death and autolysis of the HZS 190 atfA mutant The small molecular mass antifungal protein produced by Penicillium chrysogenum (PAF) causes apoptosis-like cell death in sensitive Wlamentous fungi like A. nidulans (Leiter et al. 2005; Marx et al. 2008). The PAF sensitivity of the A. nidulans strains was examined by the microplate assay of Kaiserer et al. (2003) and the agar diVusion test of Binder et al. (2010). To monitor progressing apoptosis in carbon-depleted submerged cultures, phosphatidylserine externalization on protoplasts was determined by Annexin V assay at 48 and 72 h incubation times as described elsewhere (Leiter et al. 2005; Pócsi et al. 2009). Concomitant autolysis was characterized with DCM and cell vitality declinations as well as with extracellular chitinase and proteinase productions (Emri et al. 2004, 2005 and Pócsi et al. 2009).


IdentiWcation of stress-responsive AtfA-target genes using real-time polymerase chain reaction (RT-PCR) assays Late exponential growth phase mutant and control strain mycelia (Pócsi et al. 2005) were exposed to stress-generating agents for 0.5 h according to Asano et al. (2007) and Hagiwara et al. (2008). The selected agents and concentrations were 75 mmol l¡1 H2O2, 1.0 mmol l¡1 tBOOH and 0.6 mol l¡1 NaCl. In a separate set of experiments, oxidative stress was also generated by treating mycelia with 5.0 mmol l¡1 H2O2, 0.8 mmol l¡1 tBOOH or 60 mol l¡1 MSB (Asano et al. 2007). Total RNA was extracted as described elsewhere (Pócsi et al. 2005), and RT-PCR assays were optimized and performed according to Pusztahelyi et al. (2006) using QuantiTect™SYBR®Green RT-PCR Kit (Qiagen, Germany). The putative stressresponsive genes tested in these assays are summarized in Table 3 together with the primer pairs used to amplify speciWc gene transcripts. In each RNA sample, 18S rRNA transcripts were also quantiWed as reference gene transcripts using the 5⬘-ttctgccctatcaact-3⬘ (frw) and 5⬘-ggctgaaacttaaaggaattg-3⬘ (rev) primers (Kato et al. 2003). Normalized relative transcript levels were estimated by the ‘delta–delta method’ and quantiWed with ¡CP = ¡[CPsample(target ¡ reference) ¡ CPcontrol(target ¡ reference)] = CPtarget(con trol ¡ sample) ¡ CPreference(control ¡ sample) RT-PCR cycle number of crossing point diVerences, where CP values stand for the cycle numbers of crossing points recorded for the tested target gene (CPtarget) and for the reference 18S rRNA (CPreference), respectively, either in stress-treated samples (CPsample) or in non-stressed controls (CPcontrol) (PfaZ 2001). This model presumes that RT ampliWcation eYciencies (E values) are optimal and identical (Etarget = Ereference = 2) for both the target and the reference (18S rRNA) genes and the gene expression ratio of the target gene in the sample and the control is 2¡CP (PfaZ 2001). Data statistics The variations between experiments were estimated by standard deviations (SDs) and the statistical signiWcance of changes in the parameters studied was estimated by the Student’s t-test. Only changes with probability levels of P · 5% were regarded as statistically signiWcant (Pócsi et al. 2005; Pusztahelyi et al. 2006). In RT-PCR measurements, the SD values of CPs determined for each gene and culture conditions tested were always within the range of 0.6–1.5 cycles, which were comparable to those found previously for other A. nidulans genes (Emri et al. 2006; Molnár et al. 2006; Pusztahelyi et al. 2006). SDs for ¡CP values were calculated following the rules for the linear combination of measurements (Barford 1967). In statistical

293

signiWcance calculations, ¡CP (mean § SD) values were compared to ¡CP values calculated for unstressed control cultures alone with the simpliWcations CPtarget = 0 § SD and CPreference = 0 § SD (Pusztahelyi et al. 2006).

Results The bZIP-type transcription factor AtfA of A. nidulans is considered as a putative ortholog of Wssion yeast’s ‘all-purpose’ transcription factor Atf1 and, hence, may regulate a broad spectrum of stress responses (Miskei et al. 2009). To test this hypothesis, numerous genetic, physiological, and gene expression experiments were carried out and some results heavily supported the hypothesized stress response regulatory function of AtfA. Aspergillus nidulans’s AtfA is a true functional ortholog of S. pombe’s Atf1 Functional characterization of AtfA was Wrst performed in Wssion yeast by heterologously expressing AtfA in the S. pombe NT146 atf1 deletion strain. A. nidulans atfA cDNA was isolated with primers designed to the predicted cDNA sequence of XM_655423 (http://www.ncbi.nlm.nih. gov/entrez/viewer.fcgi?db=nuccore&id=67524906; Galagan et al. 2005). The sequence of the cDNA fully matched the XM_655423 sequence and was submitted to the NCBI (accession number: EU877709; http://www.ncbi.nlm.nih. gov/). The isolated atfA cDNA was cloned subsequently into pREP expression vectors, which provide diVerent expression levels in Wssion yeast (Maundrell 1993; Basi et al. 1993). Overexpression of Atf1 under the wild-type nmt1 promoter (pREP1 plasmid) in S. pombe is lethal (Takeda et al. 1995). Similarly, expression of the pREP1atfA construct in Wssion yeast proved to be deleterious (data not shown). Therefore, moderate expression of AtfA was achieved by transforming the atf1 S. pombe strain with the pREP41-atfA construct. Moderate expression of the native protein (pREP41-atf1 construct) was applied as a positive control. Schizosaccharomyces pombe atf1 mutant cells were highly sensitive to the osmotic stress provoked by high concentrations of K+ or Na+ ions. Complementation of this stress sensitivity by AtfA was tested Wrst on agar plates. Moderate expression of AtfA complemented the osmotic stress sensitivity of the atf1 mutant, approximately to the same level as the expression of the native transcription factor Atf1 (Fig. 1). Following that the osmotic stress sensitivity of the strains was also tested by monitoring their propagation in liquid cultures. As shown in Fig. 2a, HM123 control cultures were more sensitive to the selected 1.0 mol l¡1 concentration of K+ ions than to 0.5 mol l¡1

123

294


Table 3 Putative stress-responsive genes tested in RT-PCR experiments Locus ID

Gene’s name and/or putative ortholog’s name (microorganism)2

Gene’s or putative ortholog’s function2

Forward primer

Reverse primer

AN2911.3

atfA atf1 (S. pombe)

bZIP-type transcription factor; Atf1 regulates CESR genes in S. pombe (Chen et al. 2003; Sansó et al. 2008)

5⬘-actactcccccctgccttatc-3⬘

5⬘-aagccccttccgatactgtc-3⬘

AN4896.3

pyp2 (S. pombe)

Tyrosine phosphatase, a negative regulator of Sty1/Spc1 MAPK in S. pombe (Wilkinson et al. 1996)

5⬘-tctacggcaggacaacag-3⬘

5⬘-atcggaacccaatgtgag-3⬘

AN9339.3

catB ctt1 (S. pombe)

CatB is an oxidative stress-responsive catalase (Kawasaki et al. 1997); S. pombe Ctt1 is under Atf1 control in a wide spectrum of stress (Nakagawa et al. 2000)

5⬘-cgtcggtaacaacattcc-3⬘

5⬘-gagcagggtgtgaagagtc-3⬘

AN2846.3

gpx1 (S. pombe)

Glutathione peroxidase, Gpx1 is a scavenger of H2O2 in S.pombe (Yamada et al. 1999)

5⬘-gctaaagggcaaagttatcc-3⬘

5⬘-agggttcgcattatctcc-3⬘

AN0351.3

gfdA gpd1 (S. pombe)

Glycerol-3-phosphate dehydrogenase; regulation is independent of HogA (Han and Prade 2002)

5⬘-atcctgcctcttctctttg-3⬘

5⬘-actccttcctcattcacatc-3⬘

AN6792.3

gfdB gpd2 (S. pombe)

Glycerol-3-phosphate dehydrogenase; regulation is dependent on HogA (Han and Prade 2002; Furukawa et al. 2007)

5⬘-ccagtatcctcgtcttcaac-3⬘

5⬘-tcgtctcgcatagtctttc-3⬘

AN1628.3

cta3 (S. pombe) ena-1 (N. crassa)

Cation-transporting P-type ATPase; adaptation to salt stress in S.pombe and N. crassa (Nishikawa et al. 1999; Benito et al. 2000)

5⬘-cgtctatggctaacctttc-3⬘

5⬘-gtctggagtctctttgctg-3⬘

AN6642.3

cta3 (S. pombe) ena-1 (N. crassa)


5⬘-tatccctgctaccgaaatc-3⬘

5⬘-gtccacgagtaactgttgaag-3

AN10982.3

cta3 (S. pombe) ph-7 (N. crassa)


5⬘-ataggggtctccaacttcac-3⬘

5⬘-gccagcaaatcttaggttag-3⬘

The selected stress-responsive genes are putative AtfA targets as predicted by Miskei et al. (2009) based on yeast stress models a Further putative orthologs and a more detailed functional characterization of the genes listed here are available in the paper of Miskei et al. (2009) and/or in the Aspergillus Stress Database (http://193.6.155.82/AspergillusStress/)

Na+ ion concentration, while the atf1 cells showed similar sensitivity to both agents. Expression of Atf1 in the atf1 strain restored its growth to wild-type level (Fig. 2a–c). Expression of the pREP41-atfA construct has resulted in a growth rate, which was intermediate between the propagation of the Atf1-complemented and atf1 mutant strains, indicating a partial complementation of the osmotic stress sensitivity of the Wssion yeast mutant by the orthologous A. nidulans transcription factor. Complementation of the oxidative-stress-sensitive phenotype of the atf1 mutant was examined after treating the cells with 2.0 mmol l¡1 H2O2 for 1 h (Fig. 2d). The

123

mutant cells (atf1 and pREP41-transformed atf1 strains) were highly sensitive to the agent and were almost unable to divide after the treatment. HM123 cells and those that expressed the pREP41-atf1 or pREP41-atfA constructs resumed propagation after a recovery phase. Expression of the native protein (pREP41-atf1) resulted in signiWcant, though not wild-type level complementation of the oxidative stress sensitivity. Cells expressing the A. nidulans AtfA (pREP41-atfA) showed intermediate sensitivity between the Atf1-expressing and atf1 mutant cells, again, indicating a partial complementation of the phenotype.


295

1.1 mol l-1 KCl

level as recorded with pREP41-atf1, and a partial complementation of the osmotic and oxidative-stress-sensitive phenotypes was also achieved in submerged cultures, we concluded that AtfA was a true functional ortholog of Wssion yeast’s Atf1. Following that, we constructed and phenotypically characterized a large group of A. nidulans atfA mutants to gain a deeper insight into the regulatory functions of this transcription factor in the aspergilli.

0.4 mol l-1 NaCl

control

∆atf1 ∆atf1 + pREP41 ∆atf1 + pREP41 -atf1 ∆atf1 + pREP41 -atfA

Fig. 1 Osmotic stress sensitivities of S. pombe strains on agar plates. A series of tenfold dilutions of the HM123 control, NT146 atf1 mutant, and the atf1 mutant strains expressing diVerent pREP41 constructs were prepared, and the cells were pipetted onto EMM2 plates supplemented with 1.1 mol l¡1 KCl or 0.4 mol l¡1 NaCl. The leftmost drops on both sets of plates contained 105 cells

Because the complementation of S. pombe atf1 mutation with pREP41-atfA construct restored the osmotic stress tolerance of Wssion yeast in surface cultures to the same

The whole coding region of A. nidulans atfA was replaced with the riboB marker gene in atfA mutants using the double-joint PCR method of Yu et al. (2004). Homologous integration of a single copy of the substitution cassette into the atfA locus occurred in several clones as veriWed with atfA gene speciWc PCR and Southern blot experiments. Three single-copy transformants (pabaA1 riboB2 atfA::riboB) 0.5

A

B

0.4

0.4

0.3

0.3

OD630

OD630

0.5

Construction and characterization of A. nidulans atfA deletion strains

0.2

0.2 0.1

0.1

0

0 0

2

4

6

8

10

12

0

2

Cultivation time (h)

6

8

10

12

Cultivation time (h) 1.0

0.5

D

C 0.4

0.8

0.3

0.6

OD630

OD630

4

0.2 0.1

0.4 0.2

0

0 0

2

4

6

8

10

12

Cultivation time (h) Fig. 2 Osmotic and oxidative stress sensitivities of S. pombe strains in liquid cultures. Propagation of the HM123 control (Wlled diamond, hatched diamond, open diamond), the NT146 atf1 mutant (Wlled circle, hatched circle, open circle) and the atf1 mutant strains expressing diVerent constructs including pREP41 (left pointed Wlled inverted triangle, left pointed hatched inverted triangle, left pointed open inverted triangle), pREP41-atf1 (Wlled square, hatched square, open square) and pREP41-atfA (Wlled triangle, hatched triangle, open triangle) in EMM2 medium containing 0.5 mol l¡1 NaCl (Wlled diamond,

0

13

15

17

19

21

Cultivation time (h) Wlled circle, left pointed Wlled inverted triangle, Wlled square, Wlled triangle; a, b), 1.0 mol l¡1 KCl (hatched diamond, hatched circle, left pointed hatched inverted triangle, hatched square, hatched triangle; a, c) or 2 mmol l¡1 H2O2 (open diamond, open circle, left pointed open inverted triangle, open square, open triangle; d). Symbols in a–d represent mean values calculated from three independent experiments. Standard deviations were always less than 10% of the means, and are not shown here for clarity

123

296

were crossed to the CS 2902 strain, and proper deletion of atfA was conWrmed again in several progenies with or without auxotrophy markers. All mutants grew well on MNM agar plates under unstressed conditions with comparable growth rates to those of the control (G1071, HZS 145, FGSC 26, HZS 120, and CS 2902) strains (data not shown). Similar to the Wndings of Hagiwara et al. (2008), there was no signiWcant diVerence in the KCl (1.5 mol l¡1) sensitivities of the tested mutants [7 prototroph (HZS 190, LE 1– 6) and 7 auxotroph (HZS 187–189 and 191–194) atfA progenies were tested, and 55–69% growth inhibitions were recorded] and the control strains (strains G 1071, HZS 145, FGSC 26, HZS 120 and CS 2902 with 48–65% growth reductions). Furthermore, NaCl (1.5 mol l¡1) and sorbitol (2 mol l¡1) also inhibited the growths of selected mutants (HZS 188, HZS 190; 62–67% reductions in growth with NaCl and 35–38% with sorbitol) and the control strains (56–65% growth inhibitions with NaCl and 26–41% with sorbitol) at similar levels. Importantly, the diVerences in the growth rates of the HZS 145 and HZS 190 strains observed at 30°C incubation temperature in the presence of KCl, NaCl or sorbitol were also comparable with those recorded at 37°C. Again, in good accordance with the observations of Hagiwara et al. (2008), HZS 190 mutant conidia possessed a stress-sensitive phenotype. For example, they were highly sensitive to 100–200 mmol l¡1 H2O2 treatments, producing virtually no survivals at the concentration of 200 mmol l¡1 H2O2 (data not shown). Furthermore, mutant conidia lost viability much more signiWcantly than spores from the HZS 145 control strain after 1 week storage at 4°C (84 and 10% losses in the numbers of surviving conidia, respectively). Interestingly, heat stress (50°C) aVected mutant conidia only after prolonged, 60 min, incubation but not at 30 min incubation time (data not shown). It is worth noting that both the HZS 190 atfA mutant and the HZS 145 control strains produced conidiospores in similar numbers within the range of 47–58 £ 107 spores cm¡2 colony area. The deletion of atfA did not inXuence either the PAFtriggered (Leiter et al. 2005; Marx et al. 2008; Binder et al. 2010) or the carbon starvation-triggered (Pócsi et al. 2009) apoptosis of the A. nidulans HZS 190 mutant and HZS 145 control strains (data not shown). As far as the autolysis markers (Emri et al. 2004, 2005; Pócsi et al. 2009) are concerned, there was no signiWcant diVerence between the autolytic loss of biomass and cell vitality and the kinetics of the age-dependent chitinase and proteinase productions of the HZS 190 and the HZS 145 strains up to 168 h of incubation at 37°C (data not shown). Up to this point, the regulatory function of A. nidulans AtfA seemed to be limited to the coordination of the stress defense of conidiospores (Hagiwara et al. 2008). However,

123


this view changed considerably after performing a largescale oxidative stress sensitivity study on the available atfA mutant and control strains. All single-copy transformants (three strains with genotype pabaA1 riboB2 atfA::riboB) and their progenies with diVerent auxotrophy markers or prototrophs [25 prototroph (HZS 190, LE 1–24) and 7 auxotroph (HZS 187–189 and 191–194) strains] were more sensitive to 0.8 mmol l¡1 tBOOH (65–100% growth inhibitions) than the control G1071, HZS 145, HZS 120, CS 2902 and FGSC 26 strains (15–30% growth reductions) (Fig. 3). The appearance of ‘no-growth’ phenotype in tBOOH-exposed mutants was not strain-speciWc because both high growth inhibition and ‘no-growth’ phenotypes were often observed in separate sets of experiments with the same atfA strains (data not shown). The oxidative stress sensitivities of the HZS 188 biotin auxotroph and the HZS 190 prototroph strains were also higher than those of the control strains in the presence of 6.0 mmol l¡1 H2O2 (30–40 and 10–24% growth reductions, respectively) and 0.12 mmol l¡1 MSB (37–58 and 66–78% growth inhibitions, respectively) (Fig. 3). Unexpectedly, the diVerence in the diamide sensitivities of the control and mutant strains recorded at 2.0 mmol l¡1 concentration was surprisingly low with 57–62 and 65–68% growth reductions, respectively. In submerged cultures, the atfA mutant and control strains reached quite similar, 4 ! 8–9 g l¡1 increases in DCM after 10–25 h incubation under unstressed conditions (Fig. 4). The HZS 188 and HZS 190 atfA mutants were more sensitive to H2O2 (150 mmol l¡1), tBOOH (0.75 mmol l¡1) and MSB (0.8 mmol l¡1) initiated oxidative stress at 10 h incubation time than the HZS 145 and FGSC 26 control strains (Fig. 4). Nevertheless, the H2O2treated HZS 188 and HZS 190 and the tBOOH-exposed HZS 190 cultures reached approximately the same DCM production as their unstressed counterparts at 25 h cultivation clearly indicating AtfA-independent cell recovery after peroxide stress (Fig. 4). Importantly, no such recovery was observed in MSB-exposed mutant and control cultures, which can be explained by the redox-cycling character of this chemical, which causes accumulating oxidative stress in sensitive fungi (Emri et al. 1999; Pócsi et al. 2005). Although none of the control strains grew well in the presence of 0.8 mmol l¡1 MSB the increases in DCMs observed with the HZS 145 and FGSC 26 strains at 25 h cultivation time (4 ! 6.1–6.5 g l¡1) exceeded signiWcantly those recorded with the mutants (4 ! 5.1–5.6 g l¡1; Fig. 4). Diamide, when employed in a concentration of 3.0 mmol l¡1, did not inXuence the growth of either the control or the mutant strains (Fig. 4). It is remarkable that the atfA mutant generated by Hagiwara et al. (2008) did not show any oxidative-stresssensitive phenotypes. To try to solve this contradiction, we


297

10

10

8

8

-1

DCM (g l )

-1

DCM (g l )

Fig. 3 Comparison of the oxidative stress sensitivities of the HZS 145 and FGSC 26 control and the HZS 188 and HZS 190 atfA mutant strains in surface cultures. All plates were incubated at 37°C for 5 days. The oxidative stress sensitivities of the G 1071 control, the HZS 120 parental and the CS 2902 crossing strains were very similar to those of the HZS 145 and FGSC 26 control strains and, hence, they are not shown here. Note that ‘no-growth’ phenotype also appeared in tBOOH-exposed HZS 190 cultures in other separate experiments (data not shown)

6 4

6 4

HZS 190

HZS 145 2

2 0

10

20

0

30

10

20

30


10

10

8

8

-1

DCM (g l )

-1

DCM (g l )


6 4

6 4

FGSC 26

HZS 188

2

2 0

10

20

30

Cultivation time (h) Fig. 4 Comparison of the oxidative stress sensitivities of the HZS 145 and FGSC 26 control and the HZS 188 and HZS 190 atfA mutant strains in submerged cultures. Mycelia from 20 h MNM + 0.5% yeast extract pre-cultures were washed and transferred into fresh MNM (Wlled diamond) or MNM supplemented with 150 mmol l¡1 H2O2 (open circle), 0.75 mmol l¡1 tBOOH (open square) or 0.8 mmol l¡1 MSB (open triangle), and were cultivated further for 30 h (Pócsi et al.

0

10

20

30

Cultivation time (h) 2005). Symbols represent means calculated from four independent experiments. SD values were always less than 12% of the means, and are not shown here for clarity. No growth was detected in media containing 2.0 mmol l¡1 tBOOH or 300 mmol l¡1 H2O2, while cultures exposed to 75 mmol l¡1 H2O2, 0.38 mmol l¡1 tBOOH, 0.4 mmol l¡1 MSB or 3.0 mmol l¡1 diamide showed the same growth kinetics independently of the deletion of the atfA gene (data not shown)

123

298


Table 4 Mitigation of the tBOOH-sensitivity of the HZS 190 mutant with auxotrophy supplements Supplementsa

Growthb colony diameter (cm)

Growth inhibition by tBOOHc (%)

Controld

7.03 § 0.05

0

–

4.6 § 0.3

34

Biotin

4.9 § 0.3

31

PABA

5.4 § 0.1***

23

Pyridoxine

5.1 § 0.3**

27

RiboXavin

5.6 § 0.3***

20

Uracil/uridine

4.8 § 0.4

32

Mixturee

6.0 § 0.3***

15

The concentrations of the supplements were 50 g ml¡1 uracil/ 5 mmol l¡1 uridine, 5 mol l¡1 PABA, 0.02 g ml¡1 biotin, 0.05 g ml¡1 pyridoxine and/or 0.1 g ml¡1 riboXavin in accordance with the Aspergillus Media Supplements Recommendations of the Fungal Genetics Stock Center (http://www.gla.ac.uk/ibls/molgen/aspergillus/ supplement.html) b Mean § SD values calculated from nine independent experiments are shown. *P · 5%, **P · 1%, ***P · 0.1%, where P values were calculated using the Student’s t-test c Growth inhibitions with 0.6 mmol l¡1 tBOOH are presented d HZS 190 grown on MNM agar without any tBOOH supplementation was regarded as control e The mixture of all supplements listed earlier a

riboXavin, PABA and pyridoxine moderated signiWcantly the tBOOH sensitive phenotype. On the other hand, supplementation of MNM agar with the combination of these compounds did not aVect the ‘no-growth’ phenotype of HZS 190 when this phenotype appeared in the presence of 0.8 mmol l¡1 tBOOH (data not shown). Importantly, A. nidulans HZS 190 mycelia pre-grown on MNM agar under unstressed conditions grew more slowly than their HZS 145 counterparts or even did not grow at all when they were exposed to high concentrations (2.0– 3.0 mmol l¡1) of tBOOH in MNM surface cultures (Fig. 5). This means that, in addition to mutant conidia, vegetative tissues of atfA mutants also had an oxidative-stress-sensitive phenotype. AtfA-regulated stress-responsive genes

tested the possibilities that auxotrophy supplements may interfere with oxidative stress plate assays and/or that oxidative-stress-sensitive phenotypes may originate in the increased stress sensitivity of atfA mutant conidia. As presented in Table 4, a mixture of commonly used auxotrophy supplements in A. nidulans genetics mitigated the oxidative stress sensitivity of the HZS 190 mutant exposed to 0.6 mmol l¡1 tBOOH. When added separately,

As shown in Fig. 6a–c, AtfA regulated the transcription of several stress-responsive genes under versatile stress conditions including 75 mmol l¡1 H2O2 [catB catalase, AN2846.3 putative gpx1 (S. pombe) glutathione peroxidase ortholog, AN6642.3 putative cta3 (S. pombe) and ena-1 (N. crassa) cation-transporting P-type ATPase ortholog], 1.0 mmol l¡1 tBOOH (catB, AN 2846.3 putative gpx1 ortholog, AN1628.3 and AN6642.3 putative cta3 and ena-1 cation-transporter orthologs) and 0.6 mol l¡1 NaCl [gfdB glycerol-3-phosphate dehydrogenase, AN10982.3 putative cta3 and ph-7 (N. crassa) cation-transporter ortholog] treatments. The AN6642.3 putative cta3 and ena-1 cation-transporting P-type ATPase ortholog was equally induced in control and atfA mutant strains, which indicated a salt-stress-responsive but AtfA-independent regulation for this gene. Among the tested genes, the transcriptions of AN4896.3 putative pyp2 (S. pombe) tyrosine phosphatase ortholog, gfdA glycerol-3-phosphate dehydrogenase and

Fig. 5 DiVerences in the oxidative stress sensitivities of A. nidulans HZS 145 and HZS 190 mycelia exposed to increasing concentrations of tBOOH. Conidia (105) were incubated on cellophane sheets placed on the surfaces of MNM agar plates at 37°C for 24 h. Following that

colonies were lifted and laid on MNM stress plates supplemented with tBOOH and were incubated for 5 days. The tBOOH sensitivity of the G 1071 control strain was identical to that of the HZS 145 strain (data not shown)

123


299 -1

*

6 2

Locus ID

-1

HZS 145

14

HZS 190

**

**

10 *

6

*

AN 10982.3

AN 6642.3

AN 1628.3

AN 6792.3 (gfdB)

AN 0351.3 (gfdA)

AN 2846.3

-6

AN 9339.3 (catB)

-2

AN 4896.3

2

Locus ID

Relative transcript levels

D

18

AN 2911.3 ( atfA))


Oxidative stress

NaCl stress (0.6 mol l )

C

AN 10982.3

-6

AN 6642.3

-2 AN 1628.3

Locus ID

AN 10982.3

AN 6642.3

AN 1628.3

AN 6792.3 (gfdB)

AN 0351.3 (gfdA)

AN 2846.3

-6

AN 4896.3

-2

AN 9339.3 (catB))

2

*

AN 6792.3 (gfdB)

*

**

AN 0351.3 (gfdA)

*

6

**

10

AN 2846.3

*

HZS 190

AN 9339.3 (catB)

10

HZS 145

14

AN 4896.3

HZS 190

18

AN 2911.3 atfA)


HZS 145 14

t BOOH stress (1.0 mmol l )

B

18

AN 2911.3 (atfA)


-1

H2 O2 stress (75 mmol l )

A

18 **

HZS 145

14 * 10

* *

AN 9339.3 (catB) AN 2846.3

HZS 190

6 2 -2 -6

H2O2 tBOOH MSB

H 2O2 tBOOH MSB

Stress generating agents

Fig. 6 Relative transcript levels of selected stress-responsive genes determined in oxidative and high osmotic stress exposed HZS 145 control and HZS 190 atfA mutant mycelia. a–c Transcript levels recorded in the presence of 75 mmol l¡1 H2O2, 1.0 mmol l¡1 tBOOH and 0.6 mol l¡1 NaCl are presented. d The experimental conditions of Asano et al. (2007) were employed including 5.0 mmol l¡1 H2O2, 0.8 mmol l¡1 tBOOH, and 60 mol l¡1 MSB treatments. Relative transcript levels were characterized with ¡CP = CPtarget gene (control ¡ sample) ¡ CP18S rRNA (control ¡ sample) RT-PCR cycle-number diVerences (PfaZ 2001), and positive and negative

¡CP values indicate gene inductions and repressions, respectively. Symbols represent means calculated from three independent experiments and bars show SD values. *P · 5%, **P · 1%, ***P · 0.1%. P values were calculated using Student’s t-test, and the probability levels of P · 5% were regarded as indicative of statistical signiWcance. Similar gene expression patterns were observed for catB (AN 9339.3) and the AN 10982.3 putative cta3, and ph-7 (N. crassa) cation-transporter ortholog when FGSC 26 and HZS 188 mycelia were exposed to 75 mmol l¡1 H2O2 or 0.6 mol l¡1 NaCl (data not shown)

atfA itself were not inXuenced signiWcantly by any stress generating agents (Fig. 6a–c). In a separate experiment, the oxidants H2O2, tBOOH, and MSB were employed in concentrations used by Asano et al. (2007) to screen for the target genes of NapA bZIP-type transcription factor. Under these conditions, catB was up-regulated by 5.0 mmol l¡1 H2O2, 0.8 mmol l¡1 tBOOH, and 60 mol l¡1 MSB in an AtfA-dependent manner, while the AtfA-regulated induction of the putative gpx1 ortholog was only demonstrated when mycelia were exposed to 0.8 mmol l¡1 tBOOH (Fig. 6d).

kinases (Vargas-Pérez et al. 2007; Suzuki et al. 2008) and MAPKs (HogA/SakA; MpkC; Furukawa et al. 2005) and the abundance of stress response transcriptional regulators including the bZIP-type AtfA (Aguirre et al. 2005; Hagiwara et al. 2008), AtfB (Sakamoto et al. 2008) and NapA (Asano et al. 2007), the C2H2 zinc-Wnger type MsnA (Han and Prade 2002; Miskei et al. 2009) transcription factors and the SrrA response regulator (Hagiwara et al. 2007; Vargas-Pérez et al. 2007). The remarkable complexity of stress sensing, signaling and stress response in A. nidulans with considerable redundancies and functional overlaps (Miskei et al. 2009) itself may hinder researchers’ attempts to Wnd clear-cut phenotypes of gene deletion mutants in the stress signaling and regulatory pathways, e.g., under certain types of high-osmolarity or oxidative stress (Fig. 3; Han and Prade 2002; Furukawa et al. 2002; Kawasaki et al. 2002;

Discussion The stress–response systems of the aspergilli can be characterized with the copiousness of stress-sensing histidine

123

300

Vargas-Pérez et al. 2007). When faced with the complexity of stress signaling and regulation of stress response in the aspergilli (Miskei et al. 2009), complementation studies in yeast mutants, where orthologs of the corresponding Aspergillus genes have been deleted, are of especially great value in the functional analyses of the gene products (Kawasaki et al. 2002; Furukawa et al. 2002, 2005). In this study, the successful complementation of the osmotic and oxidativestress-sensitive phenotypes of the S. pombe atf1 strain with atfA (Figs. 1, 2) demonstrated that AtfA is a true functional ortholog of Wssion yeast’s Atf1. This observation suggests that AtfA functions downstream of the SskB– PbsB–HogA/SakA stress signaling pathway and transmits diVerent types of stress signals to an array of stress-responsive genes (Pócsi et al. 2005; Miskei et al. 2009). The pivotal role of AtfA in the regulation of the osmotic and oxidative stress responses of A. nidulans was demonstrated by the observation that the A. nidulans orthologs of well-known target genes of Wssion yeast Atf1 did not respond to stress in atfA mutants grown in liquid MNM cultures (Fig. 6). Among the putative S. pombe orthologs of the tested A. nidulans genes, atf1, pyp2, ctt1, gpx1, and gpd1 were induced in Wssion yeast by diVerent types of stress as part of the CESR (Chen et al. 2003, 2008). In A. nidulans, the induction of the majority of the selected genes was stress-type-speciWc rather than stress-responsive in general, and the transcription of the putative pyp2 ortholog, and atfA itself did not respond signiWcantly to stress (Fig. 6). These Wndings do not support the idea that a Wssion yeast-like CESR may exist in A. nidulans, but whole-genome expression proWling studies have not been performed yet in this fungus to answer the important question of whether a group of genes signiWcantly induced or repressed by all or most types of stress also operates in the genome of A. nidulans (Miskei et al. 2009). Interestingly, the expression of the putative gpx1 ortholog (locus ID AN 2846.3) was induced by tBOOH and high (75 mmol l¡1) concentration H2O2 treatments in A. nidulans but was not aVected signiWcantly by MSB and 5.0 mmol l¡1 H2O2 (Fig. 6). These Wndings indicate that, similar to S. pombe (Chen et al. 2008), diverse gene expression programs may exist in A. nidulans to protect the cells from oxidative damage in a stress-type and dose-dependent manner. In Wssion yeast, mutants defective in the Sty1/Spc1-Atf1 regulatory pathway are hypersensitive to diVerent types of environmental stress (Nguyen et al. 2000; Quinn et al. 2002; Chen et al. 2008). In good accordance with this but contrary to the previous Wndings of Hagiwara et al. (2008), the deletion of atfA resulted in oxidative-stress-sensitive phenotypes in A. nidulans (Figs. 3, 4, 5). On the other hand, the atfA mutations did not aVect signiWcantly the osmotic and diamide stress tolerance of the fungus, which is in agreement with the observations of Hagiwara et al. (2008).

123


Contradictory phenotypes have also been presented for the A. nidulans sskA mutants by Hagiwara et al. (2007) and Vargas-Pérez et al. (2007), and these discrepancies remain yet to be explained. In addition to likely genotypic variations and the lack of interlaboratory standardization of stress-sensitivity assays, commonly used auxotrophy supplements and their combinations (Table 4) may mitigate or even mask stresssensitive phenotypes, which subsequently may lead to ambiguous phenotype descriptions. Among the additives tested, riboXavin, which may protect cells from oxidative injuries (Sugiyama 1991; Perumal et al. 2005), considerably decreased the tBOOH-sensitivity of the HZS 190 mutant (Table 4). Considering the antioxidant eVect of pyridoxine, pyridoxine derivatives have been demonstrated to prevent the death of MSB-exposed Wssion yeast cells (Chumnantana et al. 2005). The oxidative stress protective eVect of PABA may originate in its precursor function in folic acid biosynthesis. Folic acid plays a pivotal role in the prevention of mitochondria-associated oxidative injuries (Huang et al. 2004; Chang et al. 2007), which are intensiWed in tBOOH-exposed fungal cultures (Fekete et al. 2007). Relying on the outcomes of these studies, we argue here for the use of prototrophic strains to promote interlaboratory standardization of future stress assays. In good accordance with the Wndings of Hagiwara et al. (2008), conidia of HZS 190 were highly sensitive to H2O2treatments, heat stress, and storage in glucose-free MNM at 4°C for 1 week. Importantly, the oxidative-stress-sensitive phenotypes observed with atfA strains were not a mere consequence of the increased stress sensitivity of mutant conidia as demonstrated by transferring pre-grown A. nidulans mycelial mats onto tBOOH-supplemented stress plates (Fig. 5). Nevertheless, colonies growing out from the mats (either control or mutant) tolerated tBOOH-treatments at approximately twice higher concentrations than their conidia-inoculated counterparts (Figs. 3, 5). This phenomenon may be explained by the bioWlm-like texture of the mycelial mats pre-grown on cellophane sheets. More recently, baker’s yeast cells growing as bioWlms were characterized with an increased oxidative stress tolerance (Gales et al. 2008). Because the generation of stress signal transduction pathway mutants frequently leads to the formation of stress-sensitive conidia (Hagiwara et al. 2007, 2008; Vargas-Pérez et al. 2007) and the contribution of the increased stress-sensitivity of conidiospores to the apparent stress-sensitive phenotypes of the mutants cannot be quantiWed properly (especially not on stress agar plates when ‘no-growth’ phenotypes may appear unpredictably; Fig. 3), we also recommend the use of mycelium-inoculated liquid cultures to compare the stresssensitivities of mutant and control strains (Fig. 4). There are numerous papers reporting on the apoptosiseliciting eVect of the phosphorylated form of ATF-2


(Cho et al. 2001; Makino et al. 2006; Breitwieser et al. 2007), the human ortholog of S. pombe Atf1 (Degols and Russell 1997; Lawrence et al. 2007). On the other hand, the overexpression of another bZIP-type oxidative stress response transcription factor, Yap1p, strongly delayed chronological aging-induced apoptosis in S. cerevisiae (Herker et al. 2004). We addressed therefore the question of whether the deletion of atfA would have any impact on the apoptotic cell death processes in A. nidulans. Contrary to our expectations, the deletion of atfA did not aVect either PAF-elicited or carbon-starvation-triggered apoptosis in A. nidulans. The unaVected apoptosis rates indicate that cell-death signals may be transmitted by signal transduction pathways other than the SskA–SskB–PbsB–HogA/SakA–AtfA system, e.g. via FadA heterotrimeric G-protein signaling (Leiter et al. 2005; Marx et al. 2008). It is important to note that neither SskA (Hagiwara et al. 2007) nor AtfA (Figs. 3, 5) are necessary to respond to GSH/GSSG redox imbalance in A. nidulans, which is a well-known elicitor of programmed cell death in fungi (Madeo et al. 1999; Pócsi et al. 2004). The deletion of atfA did not aVect the autolysis of the fungus in submerged cultures either and, therefore, its participation in the regulation of autolysis also seems to be unlikely. This is understandable because the autolytic phase of growth is preceded by a severe GSH/GSSG redox imbalance in carbon-depleted submerged cultures of A. nidulans (i.e. by a physiological change that the atfA mutant is not largely sensitive to), but not by the accumulation of reactive oxygen species (i.e. not by changes that the atfA mutant is largely sensitive to; Fig. 3; Emri et al. 2004). In A. nidulans, autolysis is initiated by the FluG-BrlA conidiation initiation pathway (Emri et al. 2005; Pócsi et al. 2009), while the age-dependent degradation of GSH and, hence, the pre-autolytic changes in the GSH/GSSG redox balance is under the control of FadA/FlbA and GanB/RgsA heterotrimer G-protein signaling pathways (Molnár et al. 2004, 2006; Emri et al. 2008). In conclusion, the AtfA bZIP-type transcription factor, which is a true ortholog of the Wssion yeast’s ‘general stress’ transcription factor Atf1, plays an important role in the regulation of the elements of the oxidative and osmotic stress responses in the vegetative tissues of A. nidulans. AtfA is also involved in stress-tolerant conidia formation (Hagiwara et al. 2008) but does not seem to play a major role in the regulation of the GSH/GSSG redox balance of the cells or any role in the initiation and regulation of programmed cell death and autolysis. The atfA mutants and control strains, which were physiologically characterized in this study, will be used in genome-wide gene expression experiments with oligomer-based DNA microarrays to map the elements of general and stress-type-speciWc stress responses in A. nidulans.

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During the revision of this paper, Hagiwara et al. (2009) published DNA microarray data on Xudioxonil (this antimycotic exerts its action through Hog MAPK signaling pathway) and sorbitol-responsive A. nidulans genes using a series of mutants impaired in SskA—HogA/SakA—AtfA signaling. The authors found considerable overlaps between sskA-, hogA/sakA-, and atfA-dependent genes, which were up-regulated by Xudioxonil and sorbitol treatments. Moreover, the Xudioxonil-triggered induction of atfA itself was both sskA- and hogA/sakA-dependent. Based on these observations, Hagiwara et al. (2009) reached the conclusions that AtfA functions downstream of HogA/ SakA, and SskA—HogA/SakA—AtfA signaling is implicated in the transcriptional response to Xudioxonil and osmotic stress. In another recent study, Sakamoto et al. (2009) demonstrated that some oxidative stress response genes encoding catalases, thioredoxin, -glutamylcysteine synthase, 6-phosphogluconate dehydrogenase, etc. were downregulated in the colony surface cells (aerial hyphae and conidia) of an Aspergillus oryzae atfA mutant. The A. oryzae atfA mutant conidia were also sensitive to oxidative stress and possessed a low germination ratio even without any stress (Sakamoto et al. 2009). Acknowledgments The Authors are indebted to Mrs. Lászlóné Gábor Tóth for her valuable technical assistance and Mr. Matthew Britschgi for editing the English of the paper. One of us (ZH) was awarded a János Bolyai Research Scholarship by the Hungarian Academy of Sciences. This work was supported Wnancially by the Hungarian National OYce for Research and Technology (grant reference number OMFB 01501/2006) and by the GENOMNANOTECH-DEBRET (RET-06/2004).

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