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Functional analysis of a fungal endophyte stress-activated MAP kinase

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Abstract

The ability of fungi to sense and respond rapidly to environmental stress is crucial for their survival in the wild. One of the most important pathways involved in this response is the stress-activated MAP (mitogen-activated protein) kinase pathway. We report here on the isolation of the stress-activated MAP kinase, sakA, from the fungal endophyte Epichloë festucae. Complementation of the stress sensitivity and cell cycle defects of an Schizosaccharomyces pombe sty1Δ mutant with sakA confirmed it encodes a functional MAP kinase. Analysis of an E. festucae ΔsakA mutant revealed sakA is essential for growth under conditions of temperature and osmotic stress in culture, and for sensitivity to the fungicide fludioxonil. However, the ΔsakA mutant shows no increased sensitivity to hydrogen peroxide. Given sakA can rescue the sty1Δ mutant from sensitivity to oxidative stress, SakA has the potential to sense and transduce oxidative stress signals. The ΔsakA mutant is also defective in conidia formation, suggesting a role for SakA in asexual development of E. festucae. The detection of elevated hydrogen peroxide production in the ΔsakA mutant suggests there may be a link between MAP kinase and ROS (reactive oxygen species) signalling pathways in E. festucae.

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References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  PubMed  CAS  Google Scholar 

  • Bahn YS, Kojima K, Cox GM, Heitman J (2005) Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans. Mol Biol Cell 16:2285–2300

    Article  PubMed  CAS  Google Scholar 

  • Banuett F (1998) Signalling in the yeasts: an informational cascade with links to the filamentous fungi. Microbiol Mol Biol Rev 62:249–274

    PubMed  CAS  Google Scholar 

  • Basi G, Schmid E, Maundrell K (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123:131–136

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brewster JL, de Valoir T, Dwyer ND, Winter E, Gustin MC (1993) An osmosensing signal transduction pathway in yeast. Science 259:1760–1763

    Article  PubMed  CAS  Google Scholar 

  • Byrd AD, Schardl CL, Songlin PJ, Mogen KL, Siegel MR (1990) The beta-tubulin gene of Epichloe typhina from perennial ryegrass (Lolium perenne). Curr Genet 18:347–354

    Article  PubMed  CAS  Google Scholar 

  • Daga RR, Bolanos P, Moreno S (2003) Regulated mRNA stability of the Cdk inhibitor Rum1 links nutrient status to cell cycle progression. Curr Biol 13:2015–2024

    Article  PubMed  CAS  Google Scholar 

  • Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H, Read ND, Lee YH, Carbone I, Brown D, Oh YY, Donofrio N, Jeong JS, Soanes DM, Djonovic S, Kolomiets E, Rehmeyer C, Li W, Harding M, Kim S, Lebrun MH, Bohnert H, Coughlan S, Butler J, Calvo S, Ma LJ, Nicol R, Purcell S, Nusbaum C, Galagan JE, Birren BW (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980–986

    Article  PubMed  CAS  Google Scholar 

  • Degols G, Shiozaki K, Russell P (1996) Activation and regulation of the Spc1 stress-activated protein kinase in Schizosaccharomyces pombe. Mol Cell Biol 16:2870–2877

    PubMed  CAS  Google Scholar 

  • Delgado-Jarana J, Sousa S, Gonzalez F, Rey M, Llobell A (2006) ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum. Microbiology 152:1687–1700

    Article  PubMed  CAS  Google Scholar 

  • Dixon KP, Xu JR, Smirnoff N, Talbot NJ (1999) Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea. Plant Cell 11:2045–2058

    Article  PubMed  CAS  Google Scholar 

  • Gaits F, Degols G, Shiozaki K, Russell P (1998) Phosphorylation and association with the transcription factor Atf1 regulate localization of Spc1/Sty1 stress-activated kinase in fission yeast. Genes Dev 12:1464–1473

    PubMed  CAS  Google Scholar 

  • Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, Jedd G, Mewes W, Staben C, Marcotte E, Greenberg D, Roy A, Foley K, Naylor J, Stange-Thomann N, Barrett R, Gnerre S, Kamal M, Kamvysselis M, Mauceli E, Bielke C, Rudd S, Frishman D, Krystofova S, Rasmussen C, Metzenberg RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868

    Article  PubMed  CAS  Google Scholar 

  • Galagan JE, Calvo SE, Cuomo C, Ma LJ, Wortman JR, Batzoglou S, Lee SI, Basturkmen M, Spevak CC, Clutterbuck J, Kapitonov V, Jurka J, Scazzocchio C, Farman M, Butler J, Purcell S, Harris S, Braus GH, Draht O, Busch S, D’Enfert C, Bouchier C, Goldman GH, Bell-Pedersen D, Griffiths-Jones S, Doonan JH, Yu J, Vienken K, Pain A, Freitag M, Selker EU, Archer DB, Penalva MA, Oakley BR, Momany M, Tanaka T, Kumagai T, Asai K, Machida M, Nierman WC, Denning DW, Caddick M, Hynes M, Paoletti M, Fischer R, Miller B, Dyer P, Sachs MS, Osmani SA, Birren BW (2005) Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438:1105–1115

    Article  PubMed  CAS  Google Scholar 

  • Gehmann K, Nyfeler R, Leadbeater AJ, Nevill D, Sozzi D (1990) CGA 173506: a new phenylpyrrole fungicide for broad-spectrum disease control. Brighton Crop Prot Conf Pests Dis 2:399–406

    Google Scholar 

  • Gustin MC, Albertyn J, Alexander M, Davenport K (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1264–1300

    PubMed  CAS  Google Scholar 

  • Hamer L, Pan H, Adachi K, Orbach MJ, Page A, Ramamurthy L, Woessner JP (2001) Regions of microsynteny in Magnaporthe grisea and Neurospora crassa. Fungal Genet Biol 33:137–143

    Article  PubMed  CAS  Google Scholar 

  • Hanks SK, Hunter T (1995) Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9:576–596

    PubMed  CAS  Google Scholar 

  • Itoh Y, Johnson R, Scott B (1994) Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli. Curr Genet 25:508–513

    Article  PubMed  CAS  Google Scholar 

  • Kawasaki L, Sanchez O, Shiozaki K, Aguirre J (2002) SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans. Mol Microbiol 45:1153–1163

    Article  PubMed  CAS  Google Scholar 

  • Kobayashi T, Abe K, Asai K, Gomi K, Juvvadi PR, Kato M, Kitamoto K, Takeuchi M, Machida M (2007) Genomics of Aspergillus oryzae. Biosci Biotechnol Biochem 71:646–670

    Article  PubMed  CAS  Google Scholar 

  • Kojima K, Bahn YS, Heitman J (2006) Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans. Microbiology 152:591–604

    Article  PubMed  CAS  Google Scholar 

  • Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4:181–189

    Article  PubMed  CAS  Google Scholar 

  • Lara-Ortíz T, Riveros-Rosas H, Aguirre J (2003) Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans. Mol Microbiol 50:1241–1255

    Article  PubMed  CAS  Google Scholar 

  • Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, Kusumoto K, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161

    Article  PubMed  Google Scholar 

  • Maundrell K (1990) nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem 265:10857–10864

    PubMed  CAS  Google Scholar 

  • Mehrabi R, Zwiers LH, de Waard MA, Kema GH (2006) MgHog1 regulates dimorphism and pathogenicity in the fungal wheat pathogen Mycosphaerella graminicola. Mol Plant Microbe Interact 19:1262–1269

    Article  PubMed  CAS  Google Scholar 

  • Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Millar JB, Buck V, Wilkinson MG (1995) Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev 9:2117–2130

    Article  PubMed  CAS  Google Scholar 

  • Moreno S, Klar A, Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Meth Enzymol 194:795–823

    PubMed  CAS  Google Scholar 

  • Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J, Berriman M, Abe K, Archer DB, Bermejo C, Bennett J, Bowyer P, Chen D, Collins M, Coulsen R, Davies R, Dyer PS, Farman M, Fedorova N, Fedorova N, Feldblyum TV, Fischer R, Fosker N, Fraser A, Garcia JL, Garcia MJ, Goble A, Goldman GH, Gomi K, Griffith-Jones S, Gwilliam R, Haas B, Haas H, Harris D, Horiuchi H, Huang J, Humphray S, Jimenez J, Keller N, Khouri H, Kitamoto K, Kobayashi T, Konzack S, Kulkarni R, Kumagai T, Lafon A, Latge JP, Li W, Lord A, Lu C, Majoros WH, May GS, Miller BL, Mohamoud Y, Molina M, Monod M, Mouyna I, Mulligan S, Murphy L, O’Neil S, Paulsen I, Penalva MA, Pertea M, Price C, Pritchard BL, Quail MA, Rabbinowitsch E, Rawlins N, Rajandream MA, Reichard U, Renauld H, Robson GD, Rodriguez de Cordoba S, Rodriguez-Pena JM, Ronning CM, Rutter S, Salzberg SL, Sanchez M, Sanchez-Ferrero JC, Saunders D, Seeger K, Squares R, Squares S, Takeuchi M, Tekaia F, Turner G, Vazquez de Aldana CR, Weidman J, White O, Woodward J, Yu JH, Fraser C, Galagan JE, Asai K, Machida M, Hall N, Barrell B, Denning DW (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151–1156

    Article  PubMed  CAS  Google Scholar 

  • Norbury C, Moreno S (1997) Cloning cell cycle regulatory genes by transcomplementation in yeast. Meth Enzymol 283:44–59

    Article  PubMed  CAS  Google Scholar 

  • Payne DM, Rossomando AJ, Martino P, Erickson AK, Her JH, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW (1991) Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10:885–892

    PubMed  CAS  Google Scholar 

  • Roux PP, Blenis J (2004) ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev 68:320–344

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Scott B, Takemoto D, Tanaka A, Young CA, Bryant MK, May KJ (2007) Functional analysis of the Epichloë festucae-perennial ryegrass symbiosis. In: Popay AJ, Thom ER (eds) 6th International symposium on fungal endophytes of grasses. New Zealand Grassland Association, pp 443–441

  • Segmüller N, Ellendorf U, Tudzynski B, Tudzynski P (2007) BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea. Eukaryot Cell 6:211–221

    Article  PubMed  CAS  Google Scholar 

  • Shiozaki K, Russell P (1995a) Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature 378:739–743

    Article  PubMed  CAS  Google Scholar 

  • Shiozaki K, Russell P (1995b) Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast. EMBO J 14:492–502

    PubMed  CAS  Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Article  PubMed  CAS  Google Scholar 

  • Tanaka A, Tapper BA, Popay A, Parker EJ, Scott B (2005) A symbiosis expressed non-ribosomal peptide synthetase from a mutualistic fungal endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory. Mol Microbiol 57:1036–1050

    Article  PubMed  CAS  Google Scholar 

  • Tanaka A, Christensen MJ, Takemoto D, Park P, Scott B (2006) Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. Plant Cell 18:1052–1066

    Article  PubMed  CAS  Google Scholar 

  • Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11:1187–1194

    Article  CAS  Google Scholar 

  • Toda T, Shimanuki M, Yanagida M (1991) Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes Dev 5:60–73

    Article  PubMed  CAS  Google Scholar 

  • Toda T, Dhut S, Superti-Furga G, Gotoh Y, Nishida E, Sugiura R, Kuno T (1996) The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway. Mol Cell Biol 16:6752–6764

    PubMed  CAS  Google Scholar 

  • Young CA, Bryant MK, Christensen MJ, Tapper BA, Bryan GT, Scott B (2005) Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Mol Genet Genomics 274:13–29

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Lamm R, Pillonel C, Lam S, Xu JR (2002) Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue. Appl Environ Microbiol 68:532–538

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This research was supported by a grant from the Lincoln Bioprotection CoRE (Centre of Research Excellence) and by a Top Achiever Doctoral Scholarship to CE from the Tertiary Education Commission (TEC). E. festucae DNA sequence was provided by Professor Chris Schardl through grant EF-0523661 from the US National Science Foundation. The S. pombe sty1Δ strain was provided by Professor Jonathan Millar. The authors thank Dr Michelle Bryant (Massey University) for advice on sequence analysis, and Gemma Cartwright for technical assistance.

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  1. Simon J. Foster

    Present address: The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

Authors and Affiliations

  1. Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand

    Carla J. Eaton, Isabelle Jourdain, Simon J. Foster, Jeremy S. Hyams & Barry Scott

  2. National Centre for BioProtection, Massey University, Private Bag 11 222, Palmerston North, New Zealand

    Carla J. Eaton & Barry Scott

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  1. Carla J. Eaton
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Correspondence to Barry Scott.

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Communicated by K. Borkovich.

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294_2007_174_MOESM3_ESM.tif

Amino acid sequence alignment of fungal MAP kinases. The predicted amino acid sequence of E. festucae SakA (EfSakA) is aligned with stress MAP kinases from F. graminearum (FGSG_09612), N. crassa (NcOs-2), M. grisea (MgOSM1), and S. pombe (SpSty1); and two non-stress activated MAP kinases from F. graminearum (FGSG_06385 and FGSG_10313). Dashed black boxing indicates regions to which degenerate primers sak1 and sak2 were designed. Solid boxing indicates the position of the highly conserved TGY lip

294_2007_174_MOESM4_ESM.tif

Agarose gel electrophoresis of sakA RT-PCR products. RT-PCR amplification with primers sak24 and sak39 was performed on cDNA isolated from sty1D expressing sakA gDNA (lane 1), no RT control (lane 2), E. festucae genomic DNA (lane 3) and E. festucae cDNA (lane 4). M, 1 kb plus ladder (Invitrogen)

294_2007_174_MOESM5_ESM.tif

An E. festucae sakA C-terminal GFP fusion protein rescues the S. pombe sty1Δ mutant from its osmosensitivity defect. Wild-type and the sty1Δ mutant carrying pREP81-GFP or sakA-GFP were streaked on minimal media (EMM) containing either no stress agents (A) or 1.5 M sorbitol (B)

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Eaton, C.J., Jourdain, I., Foster, S.J. et al. Functional analysis of a fungal endophyte stress-activated MAP kinase. Curr Genet 53, 163–174 (2008). https://doi.org/10.1007/s00294-007-0174-6

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