Novel Reporter Gene Expression Systems for Monitoring Activation of ...

Download PDF
1 downloads 0 Views 279KB Size Report
Comment
The Aspergillus nidulans high-osmolarity glycerol re- sponse (AnHOG) pathway is involved in osmoadapta- tion. We found that fludioxonil, a fungicide, causes ...
Biosci. Biotechnol. Biochem., 71 (7), 1724–1730, 2007

Novel Reporter Gene Expression Systems for Monitoring Activation of the Aspergillus nidulans HOG Pathway Kentaro F URUKAWA,1; y Akira Y OSHIMI,2 Takako F URUKAWA,1 Yukiko H OSHI,1 Daisuke H AGIWARA,3 Natsuko S ATO,1 Tomonori F UJIOKA,1 Osamu M IZUTANI,1 Takeshi M IZUNO,3 Tetsuo K OBAYASHI,4 and Keietsu A BE1;2; y 1

Laboratory of Enzymology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan 2 New Industry Creation Hatchery Center, Tohoku University, 6-6-10 Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan 3 Laboratory of Molecular Microbiology, Graduate School of Agriculture, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Japan 4 Laboratory of Gene Regulation, Graduate School of Agriculture, Nagoya University, Furocho, Chikusa-ku, Nagoya 464-8601, Japan Received March 7, 2007; Accepted April 12, 2007; Online Publication, July 7, 2007 [doi:10.1271/bbb.70131]

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogA. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogA mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds. Key words:

Aspergillus nidulans; high-osmolarity glycerol (HOG) pathway; reporter system; osmotic stress; fludioxonil

In the yeast Saccharomyces cerevisiae, the highosmolarity glycerol (HOG) response MAPK pathway is activated by increased external osmolarity (Fig. 1A).1) The HOG pathway consists of two upstream osmoy

sensing branches (Sln1p and Sho1p) and downstream elements, including Ssk2p/Ssk22p MAPKKKs, Ste11p MAPKKK, Pbs2p MAPKK, and Hog1p MAPK.2,3) The two-component signaling system (Sln1p-Ypd1p-Ssk1p) regulates the downstream Hog1p MAPK cascade.4) Under osmotic stress conditions, Ssk1p activates Ssk2p and Ssk22p, which in turn activate Pbs2p.2) Pbs2p is activated also by the Sho1p branch via Ste11p in a quite complex manner.3,5) Activation of the HOG pathway results in the induction of genes required for osmotic adaptation, for instance glycerol biosynthesis genes such as GPD1 (glycerol-3-phosphate dehydrogenase) and GPP2 (glycerol-3-phosphatase).6) S. cerevisiae has another response regulator, Skn7p, that is also phosphorylated via Sln1p-Ypd1p. Skn7p is involved in multiple physiological processes, such as maintenance of cell wall integrity and the oxidative stress response.1) Recently, osmotic stress signaling pathways corresponding to the yeast HOG pathway have been described in filamentous fungi, especially Aspergillus nidulans (Fig. 1B)7–11) and Neurospora crassa,12–15) and several differences between yeast and filamentous fungi were found: (i) Activation of the filamentous fungal Hog1ptype MAPK in response to osmotic stress depends fully on the two-component signaling pathway, while in S. cerevisiae the Sho1p branch can also support Hog1p activation.10,14) (ii) Filamentous fungi have multiple putative histidine kinases, 15 and 11 in A. nidulans and

To whom correspondence should be addressed. Keietsu ABE, Tel: +81-22-717-8777; Fax: +81-22-717-8778; E-mail: kabe@biochem. tohoku.ac.jp; Kentaro FURUKAWA, Present address: Department of Cell and Molecular Biology/Microbiology, Go¨teborg University, Box 462, 405 30 Go¨teborg, Sweden; Tel: +46-31-786-3911; Fax +46-31-786-2599; E-mail: [email protected] Abbreviations: HOG, high-osmolarity glycerol; MAPK, mitogen-activated protein kinase

Reporter Systems for AnHOG Pathway Activation

1725

Fig. 1. S. cerevisiae and A. nidulans HOG Pathways. For the sake of clarity, simplified models of the pathways are shown. A, The yeast HOG pathway consists of two upstream osmosensing branches and a downstream MAPK cascade. Under the osmotic stress condition, the Sln1p branch activates Ssk2p and Ssk22p, which in turn activate Pbs2p. Pbs2p is activated also by the Sho1p branch via Ste11p in a complex manner. Skn7p is another response regulator. Importantly, fludioxonil dose not activate the HOG pathway.16) B, A. nidulans has genes orthologous to all those of the yeast HOG pathway, but several differences have been reported.10) Although A. nidulans ShoA (unlike yeast Sho1p) might not be involved in osmoresponsive activation of HogA, it appears that the two-component signaling system is more complex than that of yeast.

N. crassa respectively.11,14) (iii) S. cerevisiae sln1 is lethal, while filamentous fungal Group VI (TcsB type) histidine kinase is not essential for growth or osmotic response.9,15) However, Group III (Nik-1/Os-1 type) histidine kinase, which does not exist in S. cerevisiae, functions as a positive regulator of Hog1p-type MAPK.11,16,17) (iv) Some filamentous fungal Skn7p orthologs are involved not only in the oxidative stress response but also in osmoadaptation and fungicide sensitivity.11,18,19) Thus filamentous fungi appear to have more complex two-component signaling systems than S. cerevisiae. Magnaporthe grisea and Aspergillus flavus are major plant pathogens, and Aspergillus fumigatus is a human pathogen. Genome sequencing of these filamentous fungi has revealed that these fungi possess similar HOG signaling pathways. Hyperactivation of the yeast HOG pathway by deletion of SLN1 or YPD1 causes lethality.4) Mutants of the human pathogen Candida albicans lacking histidine kinase or response regulator display diminished virulence.20) We found recently that downregulation of gene expression of A. nidulans ypdA (YPD1 ortholog) causes severe growth inhibition (Sato et al., unpublished data), and Banno et al., have reported that hpt-1 (YPD1 ortholog) is essential for growth.15) Therefore, the filamentous fungal HOG pathways, especially two-component signaling systems, which do not exist in mammals, are potential drug targets. Fludioxonil is a phenylpyrrole fungicide derived from the antibiotic pyrrolnitrin and it is now used to control a variety of harmful plant-pathogenic fungi,21) but appearance of fludioxonil resistant mutants, mostly caused by mutations in the Group III hisitidine kinase, is a serious

problem.22) Therefore, development of new antifungal chemicals against other proteins, such as filamentous fungal Ypd1p orthologs, and improvement of currently used chemicals are needed. For these purposes, a reliable monitoring (measuring) method for the filamentous fungal HOG pathway activation is required. Although immunoblot detection of phosphorylated MAPKs is established, this approach takes a long time and requires skilled techniques. Recently, Tatebayashi et al. developed a reporter gene for yeast HOG pathway activation, termed 8xCRE-lacZ, which contains eight tandem repeats of the ENA1-derived cAMP responsive element (CRE) sequence,5) but since filamentous fungi have original components which do not exist in S. cerevisiae, fungal specific reporter systems should be constructed. In the present study, we found that fludioxonil causes improper activation of the HogA MAPK in A. nidulans, too. Since A. nidulans is a model filamentous fungus, and it is safe and easy to study fungicide effects on A. nidulans, we employed A. nidulans as a model system to develop novel reporter gene expression systems to monitor activation of the filamentous fungal HOG pathway. We fused the promoter of the A. nidulans glycerol 3-phosphate dehydrogenase gene (gfdB) to Escherichia coli uidA or EGFP-LacI-NLS, encoding a fusion protein of EGFP and E. coli LacI with a nuclear localization signal. We show for the first time that our reporter systems successfully detected the filamentous fungal HOG pathway activation in response to osmotic stress and fludioxonil treatment. Our reporter systems should be useful in screening new antifungal chemicals that inhibit or activate the filamentous fungal HOG pathway.

1726

K. FURUKAWA et al.

Materials and Methods Fungal strains and media. The Aspergillus nidulans strains used in this study were RB89 (biA1 argB2 argB+),9) hogA (biA1 argB2 hogA::argB),10) RB89GU (RB89, [pNAgfdB::uidA]), RB89-GU3 (RB89, [pNAgfdB3::uidA]), hogA-GU (hogA, [pNAgfdB:: uidA]), RB89-VE (RB89, [pNA(N)EGFP]), and RB89GE3 (RB89, [pNAgfdB3::EGFP]). A. nidulans was grown on potato dextrose (PD) medium (Nissui, Tokyo) or on Czapek-Dox (CD) minimal medium23) supplemented with 0.02 mg/ml biotin (CD/biotin) for preparation of the conidial suspension. To compare the growth of RB89 with that of hogA, 104 conidia were grown on YPD (1% yeast extract, 2% polypeptone, 2% glucose) agar medium containing 0.8 M NaCl or 2 mg/ml fludioxonil (kindly provided by T. Yoshimura) at 30  C for 5 d. RB89 and hogA were transformed as described previously,24) with indicated reporter plasmids digested with BssHII or NdeI for recombination at the aurA locus. PD medium containing 2 mg/ml Aureobasidin A (Takara, Tokyo) was used as a selection medium for A. nidulans transformation. Plasmids. (i) pNAgfdB::uidA: A fragment containing E. coli uidA (from pNGAG1, kindly provided by Professor K. Gomi), which has an NotI site in the 50 -upstream region, was ligated into the PstI and XbaI site of pUC142,10) resulting in pUC(N)GUS. A fragment containing uidA and the agdA gene terminator was obtained from pUC(N)GUS by digestion with BamHI, and was ligated into the BglII site of pNA316 (kindly provided by Professor Gomi), which has the A. nidulans Aureobasidin A resistance gene (aurAR ), resulting in pNA(N)GUS. The gfdB (AN6792.3) promoter fragment (1;000 to 1 relative to +1 of the ATG) was amplified by PCR using A. nidulans genomic DNA as a template and primers 50 -GGATGCCGTGGAATTCGCGAAGAAATGTACG-30 and 50 -CTTTGTTGGGAATTCAGTGTGCGGGATGTC-30 . This fragment was digested with EcoRI and ligated into the same site of pGEM-T Easy (Promega, Tokyo), resulting in pGEM-PgfdB. The gfdB promoter fragment was obtained from pGEM-PgfdB by digestion with NotI and inserted into the same site of pNA(N)GUS, resulting in pNAgfdB::uidA. To construct a gfdB promoter deletion mutant (gfdB3), a second Aor51HI site was introduced into pNAgfdB::uidA by QuikChange site-directed mutagenesis with primers 50 GATGGGCAGCCGGGGAGCGCTGTTTCTTATCAGTTATC-30 and 50 -GATAACTGATAAGAAACAGCGCTCCCCGGCTGCCCATC-30 , and then the resulting plasmid was digested with Aor51HI and self-ligated to excise the 1000 to 401 region, resulting in pNAgfdB3::uidA. (ii) pNAgfdB3::EGFP: A fragment of the EGFP-LacI-NLS fusion gene was amplified by PCR using p30 SSdimer-C1-EGFP25) (kindly provided by Professor A. S. Belmont) as a template and primers 50 GATCGTCTAGAGTCGACCTGCAG-30 and 50 -GGT-

GCCTCTAGAGTGAGCTAACTTAC-30 . The fragment was digested with SalI and XbaI and ligated into the same sites of pUC(N)GUS, resulting in pUC(N)EGFP. Here, an internal BamHI site in the EGFP gene was deleted by QuikChange site-directed mutagenesis with primers 50 -GCAGCCCGGGGGCTCCATGGTGAAAC30 and 50 -GTTTCACCATGGAGCCCCCGGGCTGC-30 . A fragment containing EGFP-LacI-NLS and the agdA terminator was obtained from the mutated pUC(N)EGFP by digestion with BamHI, and it was ligated into the BglII site of pNA316, resulting in pNA(N)EGFP. The gfdB promoter fragment was inserted into the NotI site of pNA(N)EGFP, resulting in pNAgfdB::EGFP. Then pNAgfdB3::EGFP, which has a truncated gfdB promoter, was constructed as described above. Immunoblot detection of HogA phosphorylation. Conidia of RB89 (108 ) were inoculated into 200 ml of CD/biotin liquid medium and grown at 30  C with shaking (160 rpm). After 24 h, a 50-ml culture aliquot was frozen in liquid nitrogen. NaCl or fludioxonil solution was added to 150 ml of the remaining culture (final conc, 0.5 M or 1 M NaCl; 2 or 20 mg/ml fludioxonil), and the mixture was incubated for 10 min. Preparation of samples, determination of protein concentration, and immunoblot detection were performed as described previously.10) -Glucronidase reporter assay. RB89-GU and hogA-GU cells were treated with osmotic stress or fludioxonil, as described above. The frozen mycelia were ground to a fine powder in a mortar and immediately resuspended in protein extraction buffer (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, and 10 mM 2-mercaptoethanol), and then cell debris was removed by centrifugation for 10 min at 15;000  g. The GUS activity of the cell-free extract was assayed in a buffer (50 mM sodium phosphate, pH 7.0, 0.1% Triton X-100, and 1 mM p-nitrophenyl-D-glucuronide, Nacalai Tesque, Kyoto). Reactions occurred in 200 ml volumes at 37  C and were terminated by the addition of 80 ml 1 N NaOH. p-nitrophenol absorbance was measured at 415 nm, and one unit was defined as the amount of enzyme that produced 1 nmol/ min of p-nitrophenol at 37  C. The protein concentration of the supernatant was determined using a BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). Blue-white assay with X-Gluc. Conidia of RB89-GU and RB89-GU3 (105 ) were inoculated into 300 ml of CD/biotin liquid medium containing 5-Bromo-4-chloro3-indoxyl--D-glucuronide (X-Gluc, final conc, 50 mg/ ml) in a 96-well microplate. Cells were cultivated at 30  C for 40 h. Fludioxonil was added to a final concentration of 0.1 to 20 mg/ml, and the cells were further incubated at 30  C for 24 h.

Reporter Systems for AnHOG Pathway Activation

1727

Detection of EGFP-LacI-NLS using fluorescence microscopy. Conidia of RB89-VE and RB89-GE3 (105 ) were inoculated into 150 ml of CD/biotin liquid medium in an 8-well microplate, and the cells were cultured at 30  C for 24 h. Then fludioxonil was added to a final concentration of 2 mg/ml. The cells were observed using an FV1000 confocal laser scanning microscope (Olympus, Tokyo).

Results and Discussion Fludioxonil caused severe growth inhibition through improper HogA activation in A. nidulans It has been reported that mutants lacking a HOG1type MAPK gene in Neurospora crassa (os-2), Colletotrichum lagenarium (OSC1), and Cochliobolus heterostrophus (BmHOG1) are sensitive to osmotic stress but resistant to fludioxonil.12,17,26) In this study, we investigated the effects of fludioxonil on A. nidulans HogA MAPK. The A. nidulans wild-type strain (RB89) showed severe growth inhibition in the presence of fludioxonil (2 mg/ml) (Fig. 2A). In addition to slight sensitivity to high osmolarity, hogA did not exhibit resistance to fludioxonil as strongly as it does in other fungi (Fig. 2A).12,26) This difference can be explained by the fact that the A. nidulans Skn7p ortholog, SrrA, is also involved in fungicide sensitivity.11) Next we examined whether HogA was phosphorylated (i.e., activated) following fludioxonil treatment using anti-phospho-p38 antibody. HogA in RB89 was activated not only by exposure to high osmolarity (0.5 or 1 M NaCl) but also by fludioxonil treatment (2 or 20 mg/ml) (Fig. 2B). These results indicate that HogA activation by fludioxonil treatment causes severe growth inhibition in A. nidulans, too. Construction of a reporter system to monitor activation of the AnHOG pathway In order to develop a method to investigate activation of the AnHOG pathway as a replacement for immunoblot detection of phosphorylated (i.e., activated) HogA, we constructed a novel reporter gene expression system by fusing the promoter of a HogA-dependent gene promoter to a reporter gene. A. nidulans has two glycerol-3phosphate dehydrogenase genes (gfdA and gfdB) homologous to the yeast GPD1/GPD2 genes.7,27) We analyzed transcriptional changes of gfdB in RB89 and hogA in response to osmotic stress by quantitative real-time PCR. The mRNA level of gfdB in RB89 was upregulated in response to osmotic stress, while that in hogA was not upregulated (data not shown). Hence we decided to employ the gfdB promoter for construction of the reporter system. The promoter region (1;000 to 1 relative to +1 of the ATG) of gfdB was fused to an E. coli uidA gene (GUS) to construct a gfdB::uidA reporter plasmid. Then we introduced it into the aurA locus of RB89 and hogA, resulting in RB89-GU and hogA-GU respec-

Fig. 2. Phosphorylation of HogA MAPK after Osmotic Shock or Fludioxonil Treatment in A. nidulans Wild Type and Their Effects on the Growth of Wild-Type and hogA Strains. A, A. nidulans wild-type (RB89) and hogA strains (104 conidia) were cultured on YPD medium, YPD containing 0.8 M NaCl, or YPD containing 2 mg/ml fludioxonil at 30  C for 5 d. B, RB89 was grown in CD/biotin liquid medium at 30  C for 24 h and then treated with either NaCl (0.5 and 1 M) or fludioxonil (2 and 20 mg/ml). Aliquots of cells were harvested after 10 min, immediately frozen in liquid nitrogen, and used to prepare total protein extracts, followed by immunoblotting with anti-phospho-p38 MAPK or anti-Hog1p antibodies. C indicates untreated control. hogA was used as a negative control in phospho-HogA detection.

tively. First we investigated the time course of gfdB:: uidA reporter induction by hyper-osmotic shock (final conc, 1 M NaCl) or fludioxonil treatment (final conc, 20 mg/ml) in RB89-GU. RB89-GU showed quite low basal GUS activity, and increased GUS activity was detected only when it was treated by hyper-osmotic shock or fludioxonil treatment (Fig. 3A and B). Expectedly, GUS activity in hogA-GU was scarcely stimulated (Fig. 3B). These results indicate that the reporter activity is controlled via HogA activation. Thus we succeeded for the first time in the construction of a reporter system for AnHOG pathway activation. Visualization of A. nidulans cells in response to fludioxonil treatment As mentioned in the introduction, the filamentous fungal HOG pathways are potential drug targets, and the gfdB::uidA reporter appears to be available for the screening of new antifungal chemicals that inhibit or activate the fungal HOG pathway. During deletion analysis of the gfdB promoter, we found that the uidA

1728

K. FURUKAWA et al.

Fig. 3. GUS Assay Using the gfdB::uidA Reporter. A, Time course of gfdB::uidA reporter induction by osmotic stress ( , 1 M NaCl) and fludioxonil treatment ( , 20 mg/ml fludioxonil). RB89GU (104 conidia) was grown in CD/biotin liquid medium at 30  C for 24 h and then treated with either NaCl or fludioxonil. Aliquots of cells were harvested at the indicated times, immediately frozen in liquid nitrogen, and used to prepare total protein extracts. GUS assay was performed as described in ‘‘Materials and Methods.’’ B, RB89-GU and hogA-GU were treated with 1 M NaCl or 20 mg/ml fludioxonil for 150 min and analyzed as described in (A). White and gray bars indicate GUS activity in RB89-GU and hogA-GU respectively. Error bars indicate standard deviation (n > 3).

reporter, driven by a truncated gfdB promoter region (gfdB3; 400 to 1 relative to +1 of the ATG), showed a high level of GUS activity after fludioxonil treatment, although its basal level was also higher than that of the complete promoter (unpublished data). We were able to visualize the blue color of A. nidulans wild-type cells transformed with improved gfdB3::uidA reporter (RB89GU3 strain) using the GUS substrate (X-Gluc) only when the cells were treated with fludioxonil (Fig. 4A). Despite the usefulness of the gfdB3::uidA reporter for fungicide detection, it appears to have limitations: visualization of fungal cells requires incubation of cells with X-Gluc, and real-time observation is difficult due to the low sensitivity of the X-Gluc assay in vivo. An EGFP-LacI-NLS reporter driven by the gfdB promoter To overcome the above limitations, we developed another reporter system, termed gfdB3::EGFP-LacINLS. We used an enhanced green fluorescent protein (EGFP)-LacI-NLS fusion gene as a reporter; it has been reported to localize to the nucleus.25) The gfdB3 promoter was fused to the EGFP-LacI-NLS fusion gene, resulting in the gfdB3::EGFP-LacI-NLS reporter plasmid. Then we introduced the empty vector and the reporter plasmid into the aurA locus of A. nidulans wild type, resulting in RB89-VE and RB89-GE3 respectively. As shown in Fig. 4B, RB89-VE did not show any detectable signals, while RB89-GE3 had no background signals under the normal condition and showed EGFP accumulation in the nucleus following fludioxonil treatment. These results indicate that the reporter system might be useful in screening new antifungal chemicals that alter the activity of the fungal HOG pathway.

Kojima et al. have reported that the C. lagenarium Osc1(Hog1p-type MAPK)-GFP fusion protein was translocated to the nucleus after the addition of fludioxonil (final conc, 100 mg/ml),26) but this method suffers from a high background of the fusion protein in the cytoplasm of unstimulated cells. Hence we constructed the gfdB3::EGFP-LacI-NLS reporter system, which resulted in EGFP accumulation in the nucleus following fludioxonil treatment. When fluorescence data of expressed EGFP was collected by imaging analysis by fluorescence microscopy, EGFP accumulated in the nucleus allowed more accurate quantification as compared to EGFP dispersed in the cytoplasm. Our approach may be more useful for high-throughput screening than employing the GUS reporter. In summary, we constructed novel reporter systems to monitor activation of the filamentous fungal HOG pathway. We expect that our reporter systems will be useful in analysis of the disregulated fungal HOG pathway and in screening new antifungal chemicals. Improvement of the reporter systems for practical use and drug discovery using these systems are goals for future research in our laboratory.

Acknowledgments We thank Stefan Hohmann, Katsuya Gomi, Andrews S. Belmont, and Masaki Yamamoto for critical reading of the manuscript, discussion, and providing plasmids. We also thank Takumi Yoshimura for providing fludioxonil, and Masayuki Sato for technical assistance with fluorescence microscopy. This study was supported in part by a grant (to K.A.) from the Research and Development Program for New-Bio-industry Initiatives of

Reporter Systems for AnHOG Pathway Activation

1729

Fig. 4. Visualization of A. nidulans Cells and EGFP Accumulation in the Nucleus in Response to Fludioxonil Treatment. A, Conidia of RB89-GU and RB89-GU3 (105 ) were inoculated into 300 ml of CD/biotin liquid medium containing X-Gluc (final conc, 50 mg/ ml) in a 96-well microplate and cultured at 30  C for 40 h. Then fludioxonil was added (final conc, 0.1 to 20 mg/ml), and the plate was incubated at 30  C for 24 h. B, Conidia of RB89-VE and RB89-GE3 (105 ) were inoculated into 150 ml of CD/biotin liquid medium in an 8-well microplate and cultured at 30  C for 24 h, and then fludioxonil was added (final conc, 2 mg/ml). At the indicated times, the cells were observed using an FV1000 confocal laser scanning microscope.

Japan. K.F. was supported by a research fellowship from the Japan Society for the Promotion of Science.

7)

References

8)

1)

2)

3)

4)

5)

6)

Hohmann, S., Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev., 66, 300–372 (2002). Maeda, T., Takekawa, M., and Saito, H., Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. Science, 269, 554–558 (1995). Posas, F., and Saito, H., Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science, 276, 1702–1705 (1997). Posas, F., Wurgler-Murphy, S. M., Maeda, T., Witten, E. A., Thai, T. C., and Saito, H., Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 ‘two-component’ osmosensor. Cell, 86, 865–875 (1996). Tatebayashi, K., Yamamoto, K., Tanaka, K., Tomida, T., Maruoka, T., Kasukawa, E., and Saito, H., Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway. EMBO J., 25, 3033–3044 (2006). Akhtar, N., Blomberg, A., and Adler, L., Osmoregulation and protein expression in a pbs2delta mutant of Saccharomyces cerevisiae during adaptation to hypersa-

9)

10)

11)

12)

line stress. FEBS Lett., 403, 173–180 (1997). Han, K. H., and Prade, R. A., Osmotic stress-coupled maintenance of polar growth in Aspergillus nidulans. Mol. Microbiol., 43, 1065–1078 (2002). Kawasaki, L., Sa´nchez, O., Shiozaki, K., and Aguirre, J., SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans. Mol. Microbiol., 45, 1153–1163 (2002). Furukawa, K., Katsuno, Y., Urao, T., Yabe, T., YamadaOkabe, T., Yamada-Okabe, H., Yamagata, Y., Abe, K., and Nakajima, T., Isolation and functional analysis of a gene, tcsB, encoding a transmembrane hybrid-type histidine kinase from Aspergillus nidulans. Appl. Environ. Microbiol., 68, 5304–5310 (2002). Furukawa, K., Hoshi, Y., Maeda, T., Nakajima, T., and Abe, K., Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress. Mol. Microbiol., 56, 1246–1261 (2005). Hagiwara, D., Matsubayashi, Y., Marui, J., Furukawa, K., Yamashino, T., Kanamaru, K., Kato, M., Abe, K., Kobayashi, T., and Mizuno, T., Characterization of the NikA histidine kinase implicated in the phosphorelay signal transduction of Aspergillus nidulans, with special reference to fungicide responses. Biosci. Biotechnol. Biochem., 71, 844–847 (2007). Zhang, Y., Lamm, R., Pillonel, C., Lam, S., and Xu, J. R., Osmoregulation and fungicide resistance: the Neuro-

1730

13)

14)

15)

16)

17)

18)

19)

K. FURUKAWA et al.

spora crassa os-2 gene encodes a HOG1 mitogenactivated protein kinase homologue. Appl. Environ. Microbiol., 68, 532–538 (2002). Fujimura, M., Ochiai, N., Oshima, M., Motoyama, T., Ichiishi, A., Usami, R., Horikoshi, K., and Yamaguchi, I., Putative homologs of SSK22 MAPKK kinase and PBS2 MAPK kinase of Saccharomyces cerevisiae encoded by os-4 and os-5 genes for osmotic sensitivity and fungicide resistance in Neurospora crassa. Biosci. Biotechnol. Biochem., 67, 186–191 (2003). Noguchi, R., Banno, S., Ichikawa, R., Fukumori, F., Ichiishi, A., Kimura, M., Yamaguchi, I., and Fujimura, M., Identification of OS-2 MAP kinase-dependent genes induced in response to osmotic stress, antifungal agent fludioxonil, and heat shock in Neurospora crassa. Fungal Genet. Biol., 44, 208–218 (2007). Banno, S., Noguchi, R., Yamashita, K., Fukumori, F., Kimura, M., Yamaguchi, I., and Fujimura, M., Roles of putative His-to-Asp signaling modules HPT-1 and RRG2, on viability and sensitivity to osmotic and oxidative stresses in Neurospora crassa. Curr. Genet., 51, 197– 208 (2007). Motoyama, T., Ohira, T., Kadokura, K., Ichiishi, A., Fujimura, M., Yamaguchi, I., and Kudo, T., An Os-1 family histidine kinase from a filamentous fungus confers fungicide-sensitivity to yeast. Curr. Genet., 47, 298–306 (2005). Yoshimi, A., Kojima, K., Takano, Y., and Tanaka, C., Group III histidine kinase is a positive regulator of Hog1-type mitogen-activated protein kinase in filamentous fungi. Eukaryot. Cell, 4, 1820–1828 (2005). Izumitsu, K., Yoshimi, A., and Tanaka, C., Two-component response regulators Ssk1p and Skn7p additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus. Eukaryot. Cell, 6, 171–181 (2007). Hagiwara, D., Asano, Y., Marui, J., Furukawa, K., Kanamaru, K., Kato, M., Abe, K., Kobayashi, T., Yamashino, T., and Mizuno, T., The SskA and SrrA response regulators are implicated in oxidative stress responses of hyphae and asexual spores in the phospho-

20)

21)

22)

23)

24)

25)

26)

27)

relay signaling network of Aspergillus nidulans. Biosci. Biotechnol. Biochem., 71, 1003–1014 (2007). Calera, J. A., Zhao, X. J., and Calderone, R., Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans. Infect. Immun., 68, 518–525 (2000). Gehmann, K., Nyfeler, R., Leadbeater, A. J., Nevill, D., and Sozzi, D., CGA 173506: a new phenylpyrrole fungicide for broad-spectrum disease control. Brighton Crop Prot. Conf. Pests Dis., 2, 399–406 (1990). Ochiai, N., Fujimura, M., Motoyama, T., Ichiishi, A., Usami, R., Horikoshi, K., and Yamaguchi, I., Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa. Pest Manag. Sci., 57, 437–442 (2001). Nakajima, K., Kunihiro, S., Sano, M., Zhang, Y., Eto, S., Chang, Y. C., Suzuki, T., Jigami, Y., and Machida, M., Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus, Aspergillus oryzae. Curr. Genet., 37, 322–327 (2000). Gomi, K., Iimura, Y., and Hara, S., Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem., 51, 2549–2555 (1987). Robinett, C. C., Straight, A., Li, G., Willhelm, C., Sudlow, G., Murray, A., and Belmont, A. S., In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/ repressor recognition. J. Cell Biol., 135, 1685–1700 (1996). Kojima, K., Takano, Y., Yoshimi, A., Tanaka, C., Kikuchi, T., and Okuno, T., Fungicide activity through activation of a fungal signalling pathway. Mol. Microbiol., 53, 1785–1796 (2004). Fillinger, S., Ruijter, G., Tamas, M. J., Visser, J., Thevelein, J. M., and d’Enfert, C., Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans. Mol. Microbiol., 39, 145– 157 (2001).