Applied Microbiology and Biotechnology

, Volume 102, Issue 16, pp 6973–6986 | Cite as

C-terminal Ser/Thr residues are vital for the regulatory role of Ste7 in the asexual cycle and virulence of Beauveria bassiana

  • Zhi-Kang Wang
  • Qing Cai
  • Sen-Miao Tong
  • Sheng-Hua Ying
  • Ming-Guang FengEmail author
Biotechnologically relevant enzymes and proteins


The mitogen-activated protein kinase (MAPK) kinase Ste7 has a conserved Ser/Thr loop (S/T-X4(6)-S/T) that can activate the MAPK Fus3 or Kss1 for the regulation of pheromone response and filamentous growth in model yeast. Here, we show that not only the loop but also four C-terminal Ser/Thr residues are essential for Ste7 to function in the Fus3 cascade of Beauveria bassiana, a filamentous fungal insect pathogen. Mutagenesis of either looped S216/T220 or C-terminal S362 resulted in the same severe defects in conidial germination, hyphal growth, aerial conidiation, and submerged blastospore production as the ste7 deletion, followed by a complete loss of virulence and similarly increased cell sensitivities to osmotic salts, oxidants, heat shock and UV-B irradiation. Mutagenesis of three other Ser/Thr residues (S391, S440, and T485) also caused severe defects in most of the mentioned phenotypes. These defects correlated well with dramatically reduced transcript levels of some phenotype-related genes. These genes encode a transcription factor (CreA) essential for carbon/nitrogen assimilation, developmental activators (BrlA, AbaA, and WetA) and upstream transcription factor (FluG) required for conidiation, P-type N+/K+ ATPases (Ena1–5) required for intracellular N+/K+ homeostasis, and antioxidant enzymes involved in multiple stress responses. Our study unveils that the loop and four C-terminal Ser/Thr residues are all vital for the regulatory role of Ste7 in the growth, conidiation, virulence, and/or stress tolerance of B. bassiana and perhaps other filamentous fungi.


Entomopathogenic fungi MAPK cascade Fus3-cascaded component Phosphorylation sites Gene expression and regulation Asexual cycle Stress response Virulence 



This work was supported by the National Natural Science Foundation of China (Grant No. 31572054), the Ministry of Science and Technology of the People’s Republic of China (Grant No. 2017YFD0201202), and the Fundamental Research Funds for the Central Universities (Grant No. 2018FZA6003).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9148_MOESM1_ESM.pdf (4.2 mb)
ESM 1 (PDF 4295 kb)


  1. Andrews DL, Egan JD, Mayorga ME, Gold SE (2000) The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth. Mol Plant-Microbe Interact 13:781–786CrossRefPubMedGoogle Scholar
  2. Banuett F, Herskowitz I (1994) Identification of fuz7, a Ustilago maydis MEK/MAPKK homolog required for a-locus-dependent and -independent steps in the fungal life cycle. Genes Dev 8:1367–1378CrossRefPubMedGoogle Scholar
  3. Bardwell AJ, Flatauer LJ, Matsukuma K, Thorner J, Bardwell L (2001) A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission. J Biol Chem 276:10374–10386CrossRefPubMedGoogle Scholar
  4. Bayram O, Bayram OS, Ahmed YL, Maruyama J, Valerius O, Rizzoli SO, Ficner R, Irniger S, Braus GH (2012) The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism. PLoS Genet 8:e1002816CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bhattacharyya RP, Remenyi A, Good MC, Bashor CJ, Falick AM, Lim WA (2006) The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway. Science 311:822–826CrossRefPubMedGoogle Scholar
  6. Bryksin AV, Matsumura I (2010) Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. Biotechniques 48:463–465CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cai Q, Wang JJ, Fu B, Ying SH, Feng MG (2018) Gcn5-dependent histone H3 acetylation and gene activity is required for the asexual development and virulence of Beauveria bassiana. Environ Microbiol 20:1484–1497CrossRefPubMedGoogle Scholar
  8. Chen RE, Thorner J (2007) Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisia. Biochim Biophys Acta-Mol. Cell Res 1773:1311–1340Google Scholar
  9. Chen JY, Chen J, Lane S, Liu HP (2002) A conserved mitogen-activated protein kinase pathway is required for mating in Candida albicans. Mol Microbiol 46:1335–1344CrossRefPubMedGoogle Scholar
  10. Davanture M, Dumur J, Bataillé-Simoneau N, Campion C, Valot B, Zivy M, Simoneau P, Fillinger S (2014) Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures. Proteomics 14:1639–1645CrossRefPubMedGoogle Scholar
  11. Davidson RC, Nicholls CB, Cox GM, Perfect JR, Heitman J (2003) A MAP kinase cascade composed of cell type specific and non-specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans. Mol Microbiol 49:469–485CrossRefPubMedGoogle Scholar
  12. Errede B, Ge QY (1996) Feedback regulation of map kinase signal pathways. Philos Trans R Soc Lond Ser B Biol Sci 351:143–148CrossRefGoogle Scholar
  13. Etxebeste O, Garzia A, Espeso EA, Ugalde U (2010) Aspergillus nidulans asexual development: making the most of cellular modules. Trends Microbiol 18:569–576CrossRefPubMedGoogle Scholar
  14. Franck WL, Gokce E, Randall SM, Oh Y, Eyre A, Muddiman DC, Dean RA (2015) Phosphoproteome analysis links protein phosphorylation to cellular remodeling and metabolic adaptation during Magnaporthe oryzae appressorium development. J Proteome Res 14:2408–2424CrossRefPubMedPubMedCentralGoogle Scholar
  15. Good M, Tang G, Singleton J, Remenyi A, Lim WA (2009) The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation. Cell 136:1085–1097CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gu Q, Chen Y, Liu Y, Zhang CQ, Ma ZH (2015) The transmembrane protein FgSho1 regulates fungal development and pathogenicity via the MAPK module Ste50-Ste11-Ste7 in Fusarium graminearum. New Phytol 206:315–328CrossRefPubMedGoogle Scholar
  17. Gustin MC, Albertyn J, Alexander M, Davenport K (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1264–1300PubMedPubMedCentralGoogle Scholar
  18. Hansen KC, Schmitt-Ulms G, Chalkley RJ, Hirsch J, Baldwin MA, Burlingame AL (2003) Mass spectrometric analysis of protein mixtures at low levels using cleavable C-13-isotope-coded affinity tag and multidimensional chromatography. Mol Cell Proteomics 2:299–314CrossRefPubMedGoogle Scholar
  19. Holder DJ, Keyhani NO (2005) Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Appl Environ Microbiol 71:5260–5266CrossRefPubMedPubMedCentralGoogle Scholar
  20. Huang BF, Feng MG (2009) Comparative tolerances of various Beauveria bassiana isolates to UV-B irradiation with a description of a modeling method to assess lethal dose. Mycopathologia 168:145–152CrossRefPubMedGoogle Scholar
  21. Kim YK, Kawano T, Li DX, Kolattukudy PE (2000) A mitogen-activated protein kinase kinase required for induction of cytokinesis and appressorium formation by host signals in the conidia of Colletotrichum gloeosporioides. Plant Cell 12:1331–1343CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kitade Y, Sumita T, Izumitsu K, Tanaka C (2015) MAPKK-encoding gene Ste7 in Bipolaris maydis is required for development and morphogenesis. Mycoscience 56:150–158CrossRefGoogle Scholar
  23. Li YF, Ye ZJ, Nie YF, Zhang J, Wang GL, Wang ZZ (2015a) Comparative phosphoproteome analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars. J Proteome 115:66–80CrossRefGoogle Scholar
  24. Li F, Shi HQ, Ying SH, Feng MG (2015b) WetA and VosA are distinct regulators of conidiation capacity, conidial quality, and biological control potential of a fungal insect pathogen. Appl Microbiol Biotechnol 99:10069–10081CrossRefPubMedGoogle Scholar
  25. Li F, Shi HQ, Ying SH, Feng MG (2015c) Distinct contributions of one Fe- and two Cu/Zn-cofactored superoxide dismutases to antioxidation, UV tolerance and virulence of Beauveria bassiana. Fungal Genet Biol 81:160–171CrossRefPubMedGoogle Scholar
  26. Liñeiro E, Chiva C, Cantoral JM, Sabido E, Fernández-Acero FJ (2016) Phosphoproteome analysis of B. cinerea in response to different plant-based elicitors. J Proteome 139:84–94CrossRefGoogle Scholar
  27. Liu J, Wang ZK, Sun HH, Ying SH, Feng MG (2017) Characterization of the Hog1 MAPK pathway in the entomopathogenic fungus Beauveria bassiana. Environ Microbiol 19:1808–1821CrossRefPubMedGoogle Scholar
  28. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  29. Luo ZB, Qin YQ, Pei Y, Keyhani NO (2014) Ablation of the creA regulator results in amino acid toxicity, temperature sensitivity, pleiotropic effects on cellular development and loss of virulence in the filamentous fungus Beauveria bassiana. Environ Microbiol 16:1122–1136CrossRefPubMedGoogle Scholar
  30. Ma QS, Jin K, Peng GX, Xia YX (2015) An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na+-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance. Fungal Genet Biol 83:68–77CrossRefPubMedGoogle Scholar
  31. Maleri S, Ge Q, Hackett EA, Wang Y, Dohlman HG, Errede B (2004) Persistent activation by constitutive Ste7 promotes Kss1-mediated invasive growth but fails to support Fus3-dependent mating in yeast. Mol Cell Biol 24:9221–9238CrossRefPubMedPubMedCentralGoogle Scholar
  32. Martin H, Flandez M, Nombela C, Molina M (2005) Protein phosphatases in MAPK signalling: we keep learning from yeast. Mol Microbiol 58:6–16CrossRefPubMedGoogle Scholar
  33. Müller P, Weinzierl G, Brachmann A, Feldbrügge M, Kahmann R (2003) Mating and pathogenic development of the Smut fungus Ustilago maydis are regulated by one mitogen-activated protein kinase cascade. Eukaryot Cell 2:1187–1199CrossRefPubMedPubMedCentralGoogle Scholar
  34. Park HS, Yu JH (2012) Genetic control of asexual sporulation in filamentous fungi. Curr Opin Microbiol 15:669–677CrossRefPubMedGoogle Scholar
  35. Rampitsch C, Subramaniam R, Djuric-Ciganovic S, Bykova NV (2010) The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvation. Proteomics 10:124–140CrossRefPubMedGoogle Scholar
  36. Rampitsch C, Tinker NA, Subramaniam R, Barkow-Oesterreicher S, Laczko E (2012) Phosphoproteome profile of Fusarium graminearum grown in vitro under nonlimiting conditions. Proteomics 12:1002–1005CrossRefPubMedGoogle Scholar
  37. Ramsubramaniam N, Harris SD, Marten MR (2014) The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion. Proteomics 14:2454–2459CrossRefPubMedGoogle Scholar
  38. Rispail N, Soanes DM, Ant C, Czajkowski R, Grünler A, Huguet R, Perez-Nadales E, Poli A, Sartorel E, Valiante V, Yang M, Beffa R, Brakhage AA, Gow NAR, Kahmann R, Lebrun MH, Lenasi H, Perez-Martin J, Talbot NJ, Wendland J, Di Pietro A (2009) Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi. Fungal Genet Biol 46:287–298CrossRefPubMedGoogle Scholar
  39. Rodriguez-Navarro A, Benito B (2010) Sodium or potassium efflux ATPase: a fungal, bryophyte, and protozoal ATPase BBA. Biomembranes 1798:1841–1853CrossRefGoogle Scholar
  40. Sakaguchi A, Tsuji G, Kubo Y (2010) A yeast STE11 homologue CoMEKK1 is essential for pathogenesis-related morphogenesis in Colletotrichum orbiculare. Mol Plant-Microbe Interact 23:1563–1572CrossRefPubMedGoogle Scholar
  41. Schamber A, Leroch M, Diwo J, Mendgen K, Hahn M (2010) The role of mitogen-activated protein (MAP) kinase signalling components and the Ste12 transcription factor in germination and pathogenicity of Botrytis cinerea. Mol Plant Pathol 11:105–119CrossRefPubMedGoogle Scholar
  42. Tong SM, Zhang AX, Guo CT, Ying SH, Feng MG (2018) Daylight length-dependent translocation of VIVID photoreceptor in cells and its essential role in conidiation and virulence of Beauveria bassiana. Environ Microbiol 20:169–185CrossRefPubMedGoogle Scholar
  43. van Drogen F, Stucke VM, Jorritsma G, Peter M (2001) MAP kinase dynamics in response to pheromones in budding yeast. Nat Cell Biol 3:1051–1059CrossRefPubMedGoogle Scholar
  44. Wang J, Liu J, Hu Y, Ying SH, Feng MG (2013a) Cytokinesis-required Cdc14 is a signaling hub of asexual development and multi-stress tolerance in Beauveria bassiana. Sci Rep 3:3086CrossRefPubMedPubMedCentralGoogle Scholar
  45. Wang ZL, Zhang LB, Ying SH, Feng MG (2013b) Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol 15:409–418CrossRefPubMedGoogle Scholar
  46. Wang ZK, Wang J, Liu J, Ying SH, Peng XJ, Feng MG (2016a) Proteomic and phosphoproteomic insights into a signaling hub role for Cdc14 in asexual development and multiple stress responses in Beauveria bassiana. PLoS One 11:e0153007CrossRefPubMedPubMedCentralGoogle Scholar
  47. Wang J, Ying SH, Hu Y, Feng MG (2016b) Mas5, a homologue of bacterial DnaJ, is indispensable for the host infection and environmental adaptation of a filamentous fungal insect pathogen. Environ Microbiol 18:1037–1047CrossRefPubMedGoogle Scholar
  48. Xiao GH, Ying SH, Zheng P, Wang ZL, Zhang SW, Xie XQ, Shang YF, St Leger RJ, Zhao GP, Wang CS, Feng MG (2012) Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep 2:483CrossRefPubMedPubMedCentralGoogle Scholar
  49. Xie XQ, Li F, Ying SH, Feng MG (2012) Additive contributions of two manganese-cored superoxide dismutases (MnSODs) to antioxidation, UV tolerance and virulence of Beauveria bassiana. PLoS One 7:e30298CrossRefPubMedPubMedCentralGoogle Scholar
  50. Xiong Y, Coradetti ST, Li X, Gritsenko MA, Clauss T, Petyuk V, Camp D, Smith R, Cate JHD, Yang F, Glass NL (2014) The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation. Fungal Genet Biol 72:21–33CrossRefPubMedPubMedCentralGoogle Scholar
  51. Ying SH, Feng MG (2006) Novel blastospore-based transformation system for integration of phosphinothricin resistance and green fluorescence protein genes into Beauveria bassiana. Appl Microbiol Biotechnol 72:206–210CrossRefPubMedGoogle Scholar
  52. Ying SH, Feng MG, Keyhani NO (2013) Use of uridine auxotrophy (ura3) for markerless transformation of the mycoinsecticide Beauveria bassiana. Appl Microbiol Biotechnol 97:3017–3025CrossRefPubMedGoogle Scholar
  53. Zhang LB, Feng MG (2018) Antioxidant enzymes and their contributions to biological control potential of fungal insect pathogens. Appl Microbiol Biotechnol 102:4995–5004CrossRefPubMedGoogle Scholar
  54. Zhang YJ, Zhang JQ, Jiang XD, Wang GJ, Luo ZB, Fan YH, Wu ZQ, Pei Y (2010) Requirement of a mitogen-activated protein kinase for appressorium formation and penetration of insect cuticle by the entomopathogenic fungus Beauveria bassiana. Appl Environ Microbiol 76:2262–2270CrossRefPubMedPubMedCentralGoogle Scholar
  55. Zhao X, Kim Y, Park G, Xu JR (2005) A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea. Plant Cell 17:1317–1329CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zheng CF, Guan KL (1994) Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J 13:1123–1131PubMedPubMedCentralCrossRefGoogle Scholar
  57. Zhu J, Ying SH, Feng MG (2016) The Na+/H+ antiporter Nhx1 controls vacuolar fusion indispensible for the life cycle in vitro and in vivo of a fungal insect pathogen. Environ Microbiol 18:3884–3895CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Coastal EcologyLudong UniversityYantaiChina
  2. 2.Institute of Microbiology, College of Life SciencesZhejiang UniversityZhejiangChina
  3. 3.School of Agricultural and Food ScienceZhejiang A&F UniversityZhejiangChina

Personalised recommendations