Current Genetics

, Volume 65, Issue 5, pp 1229–1242 | Cite as

Cla4 PAK-like kinase is required for pathogenesis, asexual/sexual development and polarized growth in Bipolaris maydis

  • Yuki Kitade
  • Takuya Sumita
  • Kosuke Izumitsu
  • Chihiro TanakaEmail author
Original Article


PAK (p21-activated protein kinases)-like kinases are master regulators of development and morphogenesis, which were conserved among eukaryotes, including fungi. In budding yeast, two types of PAK-like kinases, Ste20 and Cla4 have distinct but shared roles in the regulation of pseudohyphal development, budding and mating. In this study, to examine the broad functions of PAK-like kinases in growth, pathogenicity and asexual/sexual reproduction in filamentous fungi, we identified and characterized two PAK-like kinases, Ste20 and Cla4 in Bipolaris maydis. A single mutant of both Ste20 and Cla4 gene was viable, while the double mutant was not available, possibly because of lethality. In growth, conidiation, and pathogenicity, Δste20 strains showed phenotypes similar to those of the wild-type, while Δcla4 strains showed severely defected phenotypes. In this study, we also clarified that Ste20 is partially involved in pseudothecium development but is dispensable for maternity, while Cla4 is essential for maternal pseudothecium development and also involve in ascospore development in paternal pseudothecium. Fluorescent microscopy visualized the disorder in cell polarity at the hyphal tip in Δcla4. These results suggested that not Ste20 but Cla4 is a master regulator of growth, pathogenicity and asexual/sexual development in B. maydis. In addition, we successfully visualized alternation of branching pattern and distribution of Spitzenkörper at the hyphal tip in Δcla4 strains.


Appressorium Cochliobolus heterostrophus Paternity Spitzenkörper Ste20 Tip splitting 



Part of this work was financially supported by Grants-In-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 15J00381 for YK and no. 15K07311 for CT).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan, where the research was performed.

Supplementary material

294_2019_977_MOESM1_ESM.pptx (102 kb)
Fig. S1 PAK-like kinase gene disruption/reconstitution and confirmation. A: Schematic illustration of PAK-like kinase gene disruption and the location of primers. B: The result of PCR confirmation of Ste20 gene disruption. Each lane shows as follows. λ: λ/StyI digest, W: WT, 1-4: Disruptants, L: 100 bp ladder, R: Reconstituted-strain. C: The result of PCR confirmation of Cla4 gene disruption. Each lane showed as follows. λ: λ/StyI digest, W: WT, 1-4: Disruptants, L: 100 bp ladder, R: Reconstituted strain (PPTX 101 kb)


  1. Boyce KJ, Andrianopoulos A (2007) A p21-activated kinase is required for conidial germination in Penicillium marneffei. PLoS Pathog 3:e162. CrossRefGoogle Scholar
  2. Boyce KJ, Andrianopoulos A (2011) Ste20-related kinases: effectors of signaling and morphogenesis in fungi. Trends Microbiol 19:400–410. CrossRefGoogle Scholar
  3. Boyce KJ, Schreider L, Andrianopoulos A (2009) In vivo yeast cell morphogenesis is regulated by a p21-activated kinase in the human pathogen Penicillium marneffei. PLoS Pathog 5:e1000678. CrossRefGoogle Scholar
  4. Carroll AM, Sweigard JA, Valent B (1994) Improved vectors for selecting resistance to hygromycin. Fungal Genet Newsl 41:22Google Scholar
  5. Cvrcková F, De Virgilio C, Manser E, Pringle JR, Nasmyth K (1995) Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev 9:1817–1830. CrossRefGoogle Scholar
  6. De Souza CP, Hashmi SB, Osmani AH, Andrews P, Ringelberg CS, Dunlap JC, Osmani SA (2013) Functional analysis of the Aspergillus nidulans kinome. PLoS One 8:e58008. CrossRefGoogle Scholar
  7. Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 4:D279–D285. CrossRefGoogle Scholar
  8. Gafur A, Tanaka C, Ouchi S, Tsuda M (1997) A PCR-based method for mating type determination in Cochliobolus heterostrophus. Mycoscience 38:455–458. CrossRefGoogle Scholar
  9. Gatherar IM, Pollerman S, Dunn-Coleman N, Turner G (2004) Identification of a novel gene hbrB required for polarised growth in Aspergillus nidulans. Fungal Genet Biol 41:463–471. CrossRefGoogle Scholar
  10. Harris SD, Morrell JL, Hamer JE (1994) Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. Genetics 136:517–532Google Scholar
  11. Harris SD, Read ND, Roberson RW, Shaw B, Seiler S, Plamann M, Momany M (2005) Polarisome meets spitzenkörper: microscopy, genetics, and genomics converge. Eukaryot Cell 4:225–229. CrossRefGoogle Scholar
  12. Herold I, Yarden O (2017) Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1. Curr Genet 63:145–159. CrossRefGoogle Scholar
  13. Hickey PC, Swift SM, Roca MG, Read ND (2005) Live-cell imaging of filamentous fungi using vital fluorescent dyes. Methods Microbiol 34:63–87. CrossRefGoogle Scholar
  14. Holly SP, Blumer KJ (1999) PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae. J Cell Biol 147:845–856. CrossRefGoogle Scholar
  15. Igbaria A, Lev S, Rose MS, Lee BN, Hadar R, Degani O, Horwitz BA (2008) Distinct and combined roles of the MAP kinases of Cochliobolus heterostrophus in virulence and stress responses. Mol Plant Microbe Interact 21:769–780. CrossRefGoogle Scholar
  16. Izumitsu K, Yoshimi A, Kubo D, Morita A, Saitoh Y, Tanaka C (2009) The MAPKK kinase ChSte11 regulates sexual/asexual development, melanization, pathogenicity, and adaptation to oxidative stress in Cochliobolus heterostrophus. Curr Genet 55:439–448. CrossRefGoogle Scholar
  17. Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A, Gafur A, Ohta A, Kawai M, Yamanaka T, Neda H, Ota Y, Tanaka C (2012) Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 53:396–401. CrossRefGoogle Scholar
  18. Kang H, Lew DJ (2017) How do cells know what shape they are? Curr Genet 63:75–77. CrossRefGoogle Scholar
  19. Kitade Y, Sumita T, Izumitsu K, Tanaka C (2015) MAPKK-encoding gene, Ste7 in Bipolaris maydis is required for development and morphogenesis. Mycoscience 56:150–158. CrossRefGoogle Scholar
  20. Kluge J, Kück U (2018) AcAxl2 and AcMst1 regulate arthrospore development and stress resistance in the cephalosporin C producer Acremonium chrysogenum. Curr Genet 64:713–727. CrossRefGoogle Scholar
  21. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. CrossRefGoogle Scholar
  22. Leberer E, Dignard D, Harcus D, Thomas DY, Whiteway M (1992) The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components. EMBO J 11:4815–4824CrossRefGoogle Scholar
  23. Leberer E, Harcus D, Broadbent ID, Clark KL, Dignard D, Ziegelbauer K, Schmidt A, Gow NA, Brown AJ, Thomas DY (1996) Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc Natl Acad Sci USA 93:13217–13222CrossRefGoogle Scholar
  24. Leberer E, Ziegelbauer K, Schmidt A, Harcus D, Dignard D, Ash J, Johnson L, Thomas DY (1997) Virulence and hyphal formation of Candida albicans require the Ste20p-like protein kinase CaCla4p. Curr Biol 7:539–546. CrossRefGoogle Scholar
  25. Leveleki L, Mahlert M, Sandrock B, Bölker M (2004) The PAK family kinase Cla4 is required for budding and morphogenesis in Ustilago maydis. Mol Microbiol 54:396–406. CrossRefGoogle Scholar
  26. Li L, Xue C, Bruno K, Nishimura M, Xu JR (2004) Two PAK kinase genes, CHM1 and MST20, have distinct functions in Magnaporthe grisea. Mol Plant Microbe Interact 17:547–556. CrossRefGoogle Scholar
  27. Lichius A, Goryachev AB, Fricker MD, Obara B, Castro-Longoria E, Read ND (2014) CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa. J Cell Sci 127:1953–1965. CrossRefGoogle Scholar
  28. Lin X, Momany M (2004) Identification and complementation of abnormal hyphal branch mutants ahbA1 and ahbB1 in Aspergillus nidulans. Fungal Genet Biol 41:998–1006. CrossRefGoogle Scholar
  29. Marcus S, Polverino A, Chang E, Robbins D, Cobb MH, Wigler MH (1995) Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe. Proc Natl acad Sci USA 92:6180–6184CrossRefGoogle Scholar
  30. Martín H, Mendoza A, Rodríguez-Pachón JM, Molina M, Nombela C (1997) Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase. Mol Microbiol 23:431–444. CrossRefGoogle Scholar
  31. Minz-Dub A, Sharon A (2017) The Botrytis cinerea PAK kinase BcCla4 mediates morphogenesis, growth and cell cycle regulating processes downstream of BcRac. Mol Microbiol 104:487–498. CrossRefGoogle Scholar
  32. Nichols CB, Fraser JA, Heitman J (2004) PAK kinases Ste20 and Pak1 govern cell polarity at different stages of mating in Cryptococcus neoformans. Mol Biol Cell 15:4476–4489. CrossRefGoogle Scholar
  33. Ribeiro OK (1978) A source book of the genus Phytophthora. Cramer, VaduzGoogle Scholar
  34. Rolke Y, Tudzynski P (2008) The small GTPase Rac and the p21-activated kinase Cla4 in Claviceps purpurea: interaction and impact on polarity, development and pathogenicity. Mol Microbiol 68:405–423. CrossRefGoogle Scholar
  35. Rossman AY, Manamgoda DS, Hyde KD (2013) Proposal to conserve the name Helminthosporium maydis Y. Nisik. & C. Miyake (Bipolaris maydis) against H. maydis Brond. and Ophiobolus heterostrophus (Ascomycota: Pleosporales: Pleosporaceae). Taxon 62:1332–1333. CrossRefGoogle Scholar
  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  37. Sato H, Sawano T, Shigemori I, Mejima H, Miki M (2008) Breeding of a silage maize hybrid cultivar “Takanestar” Bull Nagano Chushin Agr Exp Sta 18:1–24 (in Japnaese with English summary;
  38. Sells MA, Knaus UG, Bagrodia S, Ambrose DM, Bokoch GM, Chernoff J (1997) Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr Biol 7:202–210. CrossRefGoogle Scholar
  39. Sells MA, Barratt JT, Caviston J, Ottilie S, Leberer E, Chernoff J (1998) Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast. J Biol Chem 273:18490–18498. CrossRefGoogle Scholar
  40. Sells MA, Boyd JT, Chernoff J (1999) p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts. J Cell Biol 145:837–849. CrossRefGoogle Scholar
  41. Smith DG, Garcia-Pedrajas MD, Hong W, Yu Z, Gold SE, Perlin MH (2004) An ste20 homologue in Ustilago maydis plays a role in mating and pathogenicity. Eukaryot Cell 3:180–189. CrossRefGoogle Scholar
  42. Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, Osmani SA, Oakley BR (2006) Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 1:3111–3120. CrossRefGoogle Scholar
  43. Taga M, Nagakubo H, Tsuda M, Ueyama A (1978) Ascospore analysis of kasugamycin resistance in the perfect stage of Pyricularia oryzae. Phytopathology 68:815–817CrossRefGoogle Scholar
  44. Tanaka C, Kubo Y, Tsuda M (1991) Genetic analysis and characterization of Cochliobolus heterostrophus colour mutants. Mycol Res 95:49–56. CrossRefGoogle Scholar
  45. Tatum LA (1971) The southern corn leaf blight epidemic. Science 171:1113–1116. CrossRefGoogle Scholar
  46. Tian H, Zhou L, Guo W, Wang X (2015) Small GTPase Rac1 and its interaction partner Cla4 regulate polarized growth and pathogenicity in Verticillium dahliae. Fungal Genet Biol 74:21–31. CrossRefGoogle Scholar
  47. Versele M, Thorner J (2004) Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. J Cell Biol 164:701–715. CrossRefGoogle Scholar
  48. Wild AC, Yu JW, Lemmon MA, Blumer KJ (2004) The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate. J Biol Chem 279:17101–17110. CrossRefGoogle Scholar
  49. Wu C, Lee SF, Furmaniak-Kazmierczak E, Côté GP, Thomas DY, Leberer E (1996) Activation of myosin-I by members of the Ste20p protein kinase family. J Biol Chem 271:31787–31790. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Yuki Kitade
    • 1
  • Takuya Sumita
    • 1
  • Kosuke Izumitsu
    • 1
    • 2
  • Chihiro Tanaka
    • 1
    Email author
  1. 1.Laboratory of Environmental Mycoscience, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Graduate School of Environmental ScienceThe University of Shiga PrefectureHikone CityJapan

Personalised recommendations