Applied Microbiology and Biotechnology

, Volume 101, Issue 23–24, pp 8571–8584 | Cite as

The Ste12-like transcription factor MaSte12 is involved in pathogenicity by regulating the appressorium formation in the entomopathogenic fungus, Metarhizium acridum

  • Qinglv Wei
  • Yanru Du
  • Kai Jin
  • Yuxian Xia
Applied microbial and cell physiology


Homeodomain transcription factor Ste12 is a key target activated by the pathogenic mitogen-activated-protein kinase pathway, and the activated Ste12p protein regulates downstream gene expression levels to modulate phenotypes. However, the functions of Ste12-like genes in entomopathogenic fungi remain poorly understood and little is known about the downstream genes regulated by Ste12. In this study, we characterized the functions of a Ste12 orthologue in Metarhizium acridum, MaSte12, and identified its downstream target genes. The deletion mutant (ΔMaSte12) is defective in conidial germination but not in hyphal growth, conidiation, or stress tolerance. Bioassays showed that ΔMaSte12 had a dramatically decreased virulence in topical inoculations, but no significant difference was found in intrahemolymph injections when the penetration process was bypassed. The mature appressorium formation rate of ΔMaSte12 was less than 10% on locust wings, with the majority hyphae forming appressorium-like, curved but no swollen structures. Digital gene expression profiling revealed that some genes involved in cell wall synthesis and remodeling, appressorium development, and insect cuticle penetration were downregulated in ΔMaSte12. Thus, MaSte12 has critical roles in the pathogenicity of the entomopathogenic fungus M. acridum, and our study provides some explanations for the impairment of fungal virulence in ΔMaSte12. In addition, virulence is very important for fungal biocontrol agents to control insect pests effectively. This study demonstrated that MaSte12 is involved in fungal virulence but not conidial yield or fungal stress tolerance in M. acridum. Thus, MaSte12 and its downstream genes may be candidates for enhancing fungal virulence to improve mycoinsecticides.


Metarhizium acridum MaSte12 Transcription factor Pathogenicity Appressorium formation 



This work was supported by the Natural Science Foundation of China (nos. 31471820 and 31270040), the Natural Science Foundation Project of Chongqing (cstc 2015jcyjA80037), and the Fundamental Research Funds for the Central Universities (0304005202014).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2017_8569_MOESM1_ESM.pdf (761 kb)
ESM 1 (PDF 761 kb)


  1. Audic S, Claverie JM (1997) The significance of digital gene expression profiles. Genome Res 7(10):986–995CrossRefPubMedGoogle Scholar
  2. Bischoff JF, Rehner SA, Humber RA (2009) A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101(4):512–530CrossRefPubMedGoogle Scholar
  3. Braga GU, Rangel DE, Flint SD, Anderson AJ, Roberts DW (2006) Conidial pigmentation is important to tolerance against solar-simulated radiation in the entomopathogenic fungus Metarhizium anisopliae. Photochem Photobiol 82(2):418–422CrossRefPubMedGoogle Scholar
  4. Calcagno AM, Bignell E, Warn P, Jones MD, Denning DW, Mühlschlegel FA, Rogers TR, Haynes K (2003) Candida glabrata STE12 is required for wild-type levels of virulence and nitrogen starvation induced filamentation. Mol Microbiol 50(4):1309–1318CrossRefPubMedGoogle Scholar
  5. Cao Y, Zhu X, Jiao R, Xia Y (2012) The Magas1 gene is involved in pathogenesis by affecting penetration in Metarhizium acridum. J Microbiol Biotechnol 22(7):889–893CrossRefPubMedGoogle Scholar
  6. Chang YC, Penoyer LA, Kwon-Chung KJ (2001) The second STE12 homologue of Cryptococcus neoformans is MATa-specific and plays an important role in virulence. Proc Natl Acad Sci U S A 98(6):3258–3263CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chang YC, Wickes BL, Miller GF, Penoyer A, Kwon-Chung KJ (2000) Cryptococcus neoformans STE12α regulates virulence but is essential for mating. J Exp Med 191(5):871–881CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chang YC, Wright LC, Tscharke RL, Sorrell TC, Wilson CF, Kwon-Chung KJ (2004) Regulatory roles for the homeodomain and C2H2 zinc finger regions of Cryptococcus neoformans Ste12αp. Mol Microbiol 53(5):1385–1396CrossRefPubMedGoogle Scholar
  9. Chen X, Xu C, Qian Y, Liu R, Zhang Q, Zeng G, Zhang X, Zhao H, Fang W (2016) MAPK cascade-mediated regulation of pathogenicity, conidiation and tolerance to abiotic stresses in the entomopathogenic fungus Metarhizium robersii. Environ Microbiol 18(3):1048-1062Google Scholar
  10. Chen Y, Duan Z, Chen P, Shang Y, Wang C (2015) The Bax inhibitor MrBI-1 regulates heat tolerance, apoptotic-like cell death, and virulence in Metarhizium robertsii. Sci Rep 5:10625–10635CrossRefPubMedPubMedCentralGoogle Scholar
  11. Clarkson JM, Charnley AK (1996) New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol 4(5):197–203CrossRefPubMedGoogle Scholar
  12. Dean RA (1997) Signal pathways and appressorium morphogenesis. Annu Rev Phytopathol 35:211–234CrossRefPubMedGoogle Scholar
  13. Di Pietro A, García-Maceira FI, Méglecz E, Roncero MIG (2001) A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis. Mol Microbiol 39(5):1140–1152CrossRefPubMedGoogle Scholar
  14. dos Reis MC, Pelegrinelli Fungaro MH, Delgado Duarte RT, Furlaneto L, Furlaneto MC (2004) Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana. J Microbiol Methods 58(2):197–202CrossRefPubMedGoogle Scholar
  15. Errede B, Ammerer G (1989) STE12, a protein involved in cell-type-specific transcription and signal transduction in yeast, is part of protein-DNA complexes. Genes Dev 3:1349–1361CrossRefPubMedGoogle Scholar
  16. Fang W, Fernandes EK, Roberts DW, Bidochka MJ, St Leger RJ (2010) A laccase exclusively expressed by Metarhizium anisopliae during isotropic growth is involved in pigmentation, tolerance to abiotic stresses and virulence. Fungal Genet Biol 47(7):602–607CrossRefPubMedGoogle Scholar
  17. Gao Q, Jin K, Ying SH, Zhang Y, Xiao G, Shang Y, Duan Z, Hu X, Xie XQ, Zhou G, Peng G, Luo Z, Huang W, Wang B, Fang W, Wang S, Zhong Y, Ma LJ, St Leger RJ, Zhao GP, Pei Y, Feng MG, Xia Y, Wang C (2011) Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet 7(1):e1001264CrossRefPubMedPubMedCentralGoogle Scholar
  18. García-Sánchez MA, Martín-Rodrigues N, Ramosa B, JJd V-B, Perlin MH, Díaz-Mínguez JM (2010) Fost12, the Fusarium oxysporum homolog of the transcription factor Ste12, is upregulated during plant infection and required for virulence. Fungal Genet Biol 47(3):216–225CrossRefGoogle Scholar
  19. Gavrias V, Andrianopoulos A, Gimeno CJ, Timberlake WE (1996) Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth. Mol Microbiol 15:1255–1263CrossRefGoogle Scholar
  20. Gu Q, Zhang C, Liu X, Ma Z (2015) A transcription factor FgSte12 is required for pathogenicity in Fusarium graminearum. Mol Plant Pathol 16(1):1–13CrossRefPubMedGoogle Scholar
  21. Gu SQ, Li P, Wu M, Hao ZM, Gong XD, Zhang XY, Tian L, Zhang P, Wang Y, Cao ZY, Fan YS, Han JM, Dong JG (2014) StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration. Microbiol Res 169(11):817–823CrossRefPubMedGoogle Scholar
  22. Hall RA, Gow NA (2013) Mannosylation in Candida albicans: role in cell all function and immune recognition. Mol Microbiol 90(6):1147–1161CrossRefPubMedPubMedCentralGoogle Scholar
  23. Hoi JWS, Dumas B (2010) Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity. Eukaryot Cell 9(4):480–485CrossRefGoogle Scholar
  24. Hoi JWS, Herbert G, Bacha N, O’Connell R, Lafitte C, Borderies G, Rossignol M, Rougé P, Dumas B (2007) Regulation and role of a STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum. Mol Microbiol 64(1):68–82CrossRefGoogle Scholar
  25. Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci U S A 88:11281–11284CrossRefPubMedPubMedCentralGoogle Scholar
  26. Jacobson ES (2000) Pathogenic roles for fungal melanins. Clin Microbiol Rev 13(4):708–717CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jarrold SL, Moore D, Potter U, Charnley AK (2007) The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol Res 111(Pt 2):240–249CrossRefPubMedGoogle Scholar
  28. Jin K, Han L, Xia Y (2014) MaMk1, a FUS3/KSS1-type mitogen-activated protein kinase gene, is required for appressorium formation, and insect cuticle penetration of the entomopathogenic fungus Metarhizium acridum. J Invertebr Pathol 115:68–75CrossRefPubMedGoogle Scholar
  29. Jin K, Ming Y, Xia YX (2012) MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum. Microbiology 158(12):2987–2996CrossRefPubMedGoogle Scholar
  30. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9(10):963–967CrossRefPubMedGoogle Scholar
  31. Leng Y, Peng G, Cao Y, Xia Y (2011) Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum. BMC Microbiol 11:32CrossRefPubMedPubMedCentralGoogle Scholar
  32. Li D, Bobrowicz P, Wilkinson HH, Ebbole DJ (2005) A mitogen-activated protein kinase pathway essential for mating and contributing to vegetative growth in Neurospora crassa. Genetics 170(3):1091–1104CrossRefPubMedPubMedCentralGoogle Scholar
  33. Liu H, Köhler J, Fink G (1995) Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266(5191):1723–1726CrossRefGoogle Scholar
  34. Lomer CJ, Bateman RP, Johnson DL, Langewald J, Thomas M (2001) Biological control of locusts and grasshoppers. Annu Rev Entomol 46:667–702CrossRefPubMedGoogle Scholar
  35. Luo S, He M, Cao Y, Xia Y (2013) The tetraspanin gene MaPls1 contributes to virulence by affecting germination, appressorial function and enzymes for cuticle degradation in the entomopathogenic fungus, Metarhizium acridum. Environ Microbiol 15(11):2966–2979PubMedGoogle Scholar
  36. Madhani HD, Fink GR (1997) Combinatorial control required for the specificity of yeast MAPK signaling. Science 275:1315–1317CrossRefGoogle Scholar
  37. Ming Y, Wei Q, Jin K, Xia Y (2014) MaSnf1, a sucrose non-fermenting protein kinase gene, is involved in carbon source utilization, stress tolerance, and virulence in Metarhizium acridum. Appl Microbiol Biotechnol 98(24):10153–10164CrossRefPubMedGoogle Scholar
  38. Morita H, Hatamoto O, Masuda T, Sato T, Takeuchi M (2007) Function analysis of steA homolog in Aspergillus oryzae. Fungal Genet Biol 44(5):330–338CrossRefPubMedGoogle Scholar
  39. Park G, Bruno KS, Staiger CJ, Talbot NJ, Xu JR (2004) Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus. Mol Microbiol 53(6):1695–1707CrossRefPubMedGoogle Scholar
  40. Park G, Xue C, Zheng L, Lam S, JR X (2002) MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. Mol Plant-Microbe Interact 15(3):183–192CrossRefPubMedGoogle Scholar
  41. Peng G, Wang Z, Yin Y, Zeng D, Xia Y (2008) Field trials of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against oriental migratory locusts, Locusta migratoria manilensis (Meyen) in Northern China. Crop Prot 27(9):1244–1250CrossRefGoogle Scholar
  42. Peng GX, Xia YX (2014) Expression of scorpion toxin LqhIT2 increases the virulence of Metarhizium acridum towards Locusta migratoria manilensis. J Ind Microbiol Biotechnol 41(11):1659–1666CrossRefPubMedGoogle Scholar
  43. Rangel DE, Alston DG, Roberts DW (2008) Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus. Mycol Res 112(11):1355–1361CrossRefPubMedGoogle Scholar
  44. Ren P, Springer DJ, Behr MJ, Samsonoff WA, Chaturvedi S, Chaturvedi V (2006) Transcription factor STE12α has distinct roles in morphogenesis, virulence, and ecological fitness of the primary pathogenic yeast Cryptococcus gattii. Eukaryot Cell 5(7):1065–1080CrossRefPubMedPubMedCentralGoogle Scholar
  45. Rispail N, Di Pietro A (2009) Fusarium oxysporum Ste12 controls invasive growth and virulence downstream of the Fmk1 MAPK cascade. Mol Plant-Microbe Interact 22(7):830–839CrossRefPubMedGoogle Scholar
  46. Rispail N, Di Pietro A (2010) The homeodomain transcription factor Ste12 connecting fungal MAPK signalling to plant pathogenicity. Commun Integr Biol 3(4):327–332CrossRefPubMedPubMedCentralGoogle Scholar
  47. Ryder LS, Talbot NJ (2015) Regulation of appressorium development in pathogenic fungi. Curr Opin Plant Biol 26:8–13CrossRefPubMedPubMedCentralGoogle Scholar
  48. 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(1):105–119CrossRefPubMedGoogle Scholar
  49. St Leger RJ, Charnley AK, Cooper RM (1986) Cuticle-degrading enzymes of entomopathogenic fungi: mechanisms of interaction between pathogen enzymes and insect cuticle. J Invertebr Pathol 47:295–302CrossRefGoogle Scholar
  50. Takano Y, Kikuchi T, Kubo Y, Hamer JE, Mise K, Furusawa I (2000) The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis. Mol Plant-Microbe Interact 13(4):374–383CrossRefPubMedGoogle Scholar
  51. Tang QY, Zhang CX (2013) Data processing system (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci 20(2):254–260CrossRefPubMedGoogle Scholar
  52. Thomas MB, Read AF (2007) Can fungal biopesticides control malaria? Nat Rev Microbiol 5(5):377–383CrossRefPubMedGoogle Scholar
  53. Tsuji G, Fujii S, Tsuge S, Shiraishi T, Kubo Y (2003) The Colletotrichum lagenarium Ste12-like gene CST1 is essential for appressorium penetration. Mol Plant-Microbe Interact 16(4):315–325CrossRefPubMedGoogle Scholar
  54. Vallim MA, Miller KY, Miller BL (2000) Aspergillus SteA (Sterile12-like) is a homeodomain-C2/H2-Zn+2 finger transcription factor required for sexual reproduction. Mol Microbiol 36(2):290–301CrossRefPubMedGoogle Scholar
  55. Wang C, St Leger RJ (2007) The Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure, and virulence. J Biol Chem 282(29):21110–21115CrossRefPubMedGoogle Scholar
  56. Werner S, Sugui JA, Steinberg G, Deising HB (2007) A chitin synthase with a myosin-like motor domain is essential for hyphal growth, appressorium differentiation, and pathogenicity of the maize anthracnose fungus Colletotrichum graminicola. Mol Plant-Microbe Interact 20(12):1555–1567CrossRefPubMedGoogle Scholar
  57. Winnenburg R, Urban M, Beacham A, Baldwin TK, Holland S, Lindeberg M, Hansen H, Rawlings C, Hammond-Kosack KE, Kohler J (2008) PHI-base update: additions to the pathogen host interaction database. Nucleic Acids Res 36(Database issue):D572–D576PubMedGoogle Scholar
  58. Xu JR (2000) MAP kinases in fungal pathogens. Fungal Genet Biol 31(3):137–152CrossRefPubMedGoogle Scholar
  59. Yuan YL, Fields S (1991) Properties of the DNA-binding domain of the Saccharomyces cerevisiae STE12 protein. Mol Cell Biol 11(12):5910–5918CrossRefPubMedPubMedCentralGoogle Scholar
  60. Zhang S, Peng G, Xia Y (2010) Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium. Biocontrol Sci Tech 20(8):809–819CrossRefGoogle Scholar
  61. Zhang S, Xia Y, Kim B, Keyhan NO (2011) Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development and pathogenesis in the entomopathogenic fungus, Beauveria bassiana. Mol Microbiol 80:811–826CrossRefPubMedGoogle Scholar
  62. Zhang Y, Zhang J, Jiang X, Wang G, Luo Z, Fan Y, Wu Z, 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(7):2262–2270CrossRefPubMedPubMedCentralGoogle Scholar
  63. Zhao X, Mehrabi R, Xu JR (2007) Mitogen-activated protein kinase pathways and fungal pathogenesis. Eukaryot Cell 6(10):1701–1714CrossRefPubMedPubMedCentralGoogle Scholar
  64. Zheng L, Campbell M, Murphy J, Lam S, Xu JR (2000) The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea. Mol Plant-Microbe Interact 13(7):724–732CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Genetic Engineering Research Center, School of Life SciencesChongqing UniversityChongqingPeople’s Republic of China
  2. 2.Chongqing Engineering Research Center for Fungal InsecticideChongqingPeople’s Republic of China

Personalised recommendations