Skip to main content
Log in

The role of three calcineurin subunits and a related transcription factor (Crz1) in conidiation, multistress tolerance and virulence in Beauveria bassiana

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The eukaryotic calcineurin (CN) pathway comprising catalytic A (CnA) and regulatory B subunits (CnB) is crucial for many biological processes but functionally unexplored in entomopathogenic fungi. Here, we characterise three CN subunits (CnA1, CnA2 and CnB) and a downstream CN-responsive zinc finger transcription factor (Crz1) in Beauveria bassiana. CN-mediated phosphatase activity decreased by 16–38 % in all deletion mutants compared with wild type. Growth and conidiation were most defective in ΔcnB, which showed a large proportion of abnormally branched germlings but were less defective in ΔcnA1 and ΔcnA2. Conidiation defects also occurred in Δcrz1, uniquely accompanied with slower germination. Compared with wild type, the four deletion mutants became, to varying degrees, more sensitive to Ca2+, Mn2+, Zn2+, Mg2+, two oxidants, three cell wall stressors, carbendazim, heat shock and ultraviolet (UV)-B irradiation. They were also less virulent to Spodoptera litura larvae. Only ΔcnB and Δcrz1 were less tolerant to high osmolarity. The altered phenotypes of the deletion mutants were associated with lower intracellular mannitol and trehalose levels, reduced overall activity of superoxide dismutases and catalases, altered cell wall composition and down-regulation of numerous phenotype-influencing genes. Additionally, the transcription of six cascaded genes in two stress-responsive mitogen-activated protein kinase (MAPK) pathways and the phosphorylation of hallmarking Hog1 and Slt2 were largely down-regulated in all the deletion mutants under osmotic and cell wall stresses, respectively. All the changes were restored by gene complementation. Taken together, three calcineurin subunits and Crz1 play vital, but variable, roles in B. bassiana responses to environmental stresses during development and host signals during infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Aramburu J, Rao A, Klee CB (2000) Calcineurin: from structure to function. Curr Top Cell Regul 36:237–295

    Article  CAS  PubMed  Google Scholar 

  • Bader T, Bodendorfer B, Schröppel K, Morschhäuser J (2003) Calcineurin is essential for virulence in Candida albicans. Infect Immun 71:5344–5354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blankenship JR, Heitman J (2005) Calcineurin is required for Candida albicans to survive calcium stress in serum. Infect Immun 73:5767–5774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blankenship JR, Wormley FL, Boyce MK, Schell WA, Filler SG, Perfect JR, Heitman J (2003) Calcineurin is essential for Candida albicans survival in serum and virulence. Eukaryot Cell 2:422–430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao YQ, Du MJ, Luo S, Xia YX (2014) Calcineurin modulates growth, stress tolerance, and virulence in Metarhizium acridum and its regulatory network. Appl Microbiol Biotechnol 98:8253–8265

    Article  CAS  PubMed  Google Scholar 

  • Cervantes-Chávez JA, Ali S, Bakkeren G (2011) Response to environmental stresses, cell-wall integrity, and virulence are orchestrated through the calcineurin pathway in Ustilago hordei. Mol Plant Microbe Interact 24:219–232

    Article  PubMed  Google Scholar 

  • Chen YL, Brand A, Morrison EL, Silao FG, Bigol UG, Malbas FF Jr, Nett JE, Andes DR, Solis NV, Filler SG, Averette A, Heitman J (2011) Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis. Eukaryot Cell 10:803–819

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi J, Kim Y, Kim S, Park J, Lee YH (2009) MoCRZ1, a gene encoding a calcineurin-responsive transcription factor, regulates fungal growth and pathogenicity of Magnaporthe oryzae. Fungal Genet Biol 46:243–254

    Article  CAS  PubMed  Google Scholar 

  • Cramer RA Jr, Perfect BZ, Pinchai N, Park S, Perlin DS, Asfaw YG, Heitman J, Perfect JR, Steinbach WJ (2008) Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus. Eukaryot Cell 7:1085–1097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cruz MC, Sia RA, Olson M, Cox GM, Heitman J (2000) Comparison of the roles of calcineurin in physiology and virulence inserotype D and serotype A strains of Cryptococcus neoformans. Infect Immun 68:982–985

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cruz MC, Fox DS, Heitman J (2001) Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans. EMBO J 20:1020–1032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cunningham KW, Fink GR (1994) Ca2+ transport in Saccharomyces cerevisiae. J Exp Biol 196:157–166

    CAS  PubMed  Google Scholar 

  • Cyert MS (2003) Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress. Biochem Biophys Res Commun 311:1143–1150

    Article  CAS  PubMed  Google Scholar 

  • da Silva Ferreira ME, Heinekamp T, Härtl A, Brakhage AA, Semighini CP, Harris SD, Savoldi M, de Gouvêa PF, de Souza Goldman MH, Goldman GH (2007) Functional characterization of the Aspergillus fumigatus calcineurin. Fungal Genet Biol 44:219–230

    Article  PubMed  Google Scholar 

  • de Faria M, Wraight SP (2007) Mycoinsecticides and Mycoacaricides: a comprehensive list with worldwide coverage and international classification of formulation types. Biol Control 43:237–256

    Article  Google Scholar 

  • Dürr G, Strayle J, Plemper R, Elbs S, Klee SK, Catty P, Wolf DH, Rudolph HK (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell 9:1149–1162

    Article  PubMed  PubMed Central  Google Scholar 

  • Elbein AD, Pan YT, Pastuszak I, Carroll D (2003) New insights on trehalose: a multifunctional molecule. Glycobiology 13:17R–27R

    Article  CAS  PubMed  Google Scholar 

  • Fang WG, St Leger RJ (2012) Enhanced UV resistance and improved killing of malaria mosquitoes by photolyase transgenic entomopathogenic fungi. PLoS ONE 7:e43069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fang WG, Zhang YJ, Yang XY, Zheng XL, Duan H, Li Y, Pei Y (2004) Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marks. J Invertebr Pathol 85:18–24

    Article  CAS  PubMed  Google Scholar 

  • Fox DS, Heitman J (2002) Good fungi gone bad: the corruption of calcineurin. Bioassays 24:894–903

    Article  CAS  Google Scholar 

  • Free SJ (2013) Fungal cell wall organization and biosynthesis. Adv Genet 81:33–82

    CAS  PubMed  Google Scholar 

  • Harel A, Bercovich S, Yarden O (2006) Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner. Mol Plant Microbe Interact 19:682–693

    Article  CAS  PubMed  Google Scholar 

  • Harren K, Schumacher J, Tudzynski B (2012) The Ca2+/calcineurin-dependent signaling pathway in the gray mold Botrytis cinerea: the role of calcipressin in modulating calcineurin activity. PLoS ONE 7:e41761. doi:10.1371/journal.pone.0041761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hemenway CS, Heitman J (1999) Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae. Mol Gen Genet 261:388–401

  • Hirayama S, Sugiura R, Lu Y, Maeda T, Kawagishi K, Yokoyama M, Tohda H, Giga-Hama Y, Shuntoh H, Kuno T (2003) Zinc finger protein Prz1 regulates Ca2+ but not Cl homeostasis in fission yeast. Identification of distinct branches of calcineurin signaling pathway in fission yeast. J Biol Chem 278:18078–18084

    Article  CAS  PubMed  Google Scholar 

  • Jayashree T, Praveen Rao J, Subramanyam C (2000) Regulation of aflatoxin production by Ca2+/calmodulin-dependent protein phosphorylation and dephosphorylation. FEMS Microbiol Lett 183:215–219

    Article  CAS  PubMed  Google Scholar 

  • Juvvadi PR, Kuroki Y, Arioka M, Nakajima H, Kitamoto K (2003) Functional analysis of the calcineurin-encoding gene cnaA from Aspergillus oryzae: evidence for its putative role in stress adaptation. Arch Microbiol 179:416–422

    Article  CAS  PubMed  Google Scholar 

  • Juvvadi PR, Fortwendel JR, Rogg LE, Burns KA, Randell SH, Steinbach WJ (2011) Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus. Mol Microbiol 82:1235–1259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kashyap RS, Rajan AN, Ramteke SS, Agrawal VS, Kelkar SS, Purohit HJ, Taori GM, Daginawala HF (2007) Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study. BMC Infect Dis 7:74

  • Kim S, Hu J, Oh Y, Park J, Choi J, Lee YH, Dean RA, Mitchell TK (2010) Combining ChIP-chip and expression profiling to model the MoCRZ1 mediated circuit for Ca/calcineurin signaling in the rice blast fungus. PLoS Pathog 6:e1000909

    Article  PubMed  PubMed Central  Google Scholar 

  • Kothe GO, Free SJ (1998) Calcineurin subunit B is required for normal vegetative growth in Neurospora crassa. Fungal Genet Biol 23:248–258

    Article  CAS  PubMed  Google Scholar 

  • Kraus PR, Heitman J (2003) Coping with stress: calmodulin and calcineurin in model and pathogenic fungi. Biochem Biophys Res Commun 311:1151–1157

    Article  CAS  PubMed  Google Scholar 

  • Lee SC, Li A, Calo S, Heitman J (2013) Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic Zygomycete Mucor circinelloides. PLoS Genet 9:e1003625. doi:10.1371/journal.ppat.1003625

    Article  CAS  Google Scholar 

  • Liu Q, Ying SH, Feng MG, Jiang XH (2009) Physiological implication of intracellular trehalose and mannitol changes in response of entomopathogenic fungus Beauveria bassiana to thermal stress. Antonie Van Leeuwenhoek 95:65–75

    Article  CAS  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Matheos DP, Kingsbury TJ, Ahsan US, Cunningham KV (1997) Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression in Saccharomyces cerevisise. Genes Dev 11:345–3458

    Article  Google Scholar 

  • Miyakawa T, Mizunuma M (2007) Physiological roles of calcineurin in Saccharomyces cerevisiae with special emphasis on its roles in G2/M cell-cycle regulation. Biosci Biotechnol Biochem 71:633–645

    Article  CAS  PubMed  Google Scholar 

  • Nanthakumar NN, Dayton JS, Means AR (1996) Role of Ca2+/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation. Prog Cell Cycle Res 2:217–228

    Article  CAS  PubMed  Google Scholar 

  • Odom A, Muir S, Lim E, Toffaletti DL, Perfect J, Heitman J (1997) Calcineurin is required for virulence of Cryptococcus neoformans. EMBO J 16:2576–2589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Prokisch H, Yarden O, Dieminger M, Tropschug M, Barthelmess IB (1997) Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient. Mol Gen Genet 256:104–114

    Article  CAS  PubMed  Google Scholar 

  • Reedy JL, Filler SG, Heitman J (2010) Elucidating the Candida albicans calcineurin signaling cascade controlling stress response and virulence. Fungal Genet Biol 47:107–116

    Article  CAS  PubMed  Google Scholar 

  • 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 signaling components in plant and human pathogenic fungi. Fungal Genet Biol 46:287–298

    Article  CAS  PubMed  Google Scholar 

  • Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiol Rev 80:1483–1521

    CAS  PubMed  Google Scholar 

  • Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J (2003) Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 48:959–976

    Article  CAS  PubMed  Google Scholar 

  • Santos M, de Larrinoa IF (2005) Functional characterization of the Candida albicans CRZ1 gene encoding a calcineurin-regulated transcription factor. Curr Genet 48:88–100

    Article  CAS  PubMed  Google Scholar 

  • Schumacher J, de Larrinoa IF, Tudzynski B (2008) Calcineurin-responsive zinc finger transcription factor CRZ1 of Botrytis cinerea is required for growth, development and full virulence on bean plants. Eukaryot Cell 7:584–601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seo JA, Guan Y, Yu JH (2006) FluG-dependent asexual development in Aspergillus nidulans occurs via derepression. Genetics 172:1535–1544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song TT, Zhao J, Ying SH, Feng MG (2013) Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus. PLoS ONE 8:e62179

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Soriani FM, Malavazi I, da Silva Ferreira ME, Savoldi M, Von Zeska Kress MR, de Souza Glodman MH, Loss O, Bignell E, Goldman GH (2008) Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA. Mol Microbiol 67:1274–1291

    Article  CAS  PubMed  Google Scholar 

  • Soriani FM, Malavazi I, Savoldi M, Espeso E, Dinamarco TM, Bernardes LA, Ferreira ME, Goldman MH, Goldman GH (2010) Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA. BMC Microbiol 15:10–12

    Google Scholar 

  • Steinbach WJ, Cramer RA, Perfect BZ, Asfaw YG, Sauer TC, Najvar LK, Kirkpatrick WR, Patterson TF, Benjamin DK, Heitman J, Perfect JR (2006) Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5:1091–1103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stie J, Fox D (2008) Calcineurin regulation in fungi and beyond. Eukaryot Cell 7:177–186

    Article  CAS  PubMed  Google Scholar 

  • Viaud M, Brunet-Simon A, Brygoo Y, Pradier JM, Levis C (2003) Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporine A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea. Mol Microbiol 50:1451–1465

    Article  CAS  PubMed  Google Scholar 

  • Wanchoo A, Lewis MW, Keyhani NO (2009) Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana. Microbiology SGM 155:3121–3133

    Article  CAS  Google Scholar 

  • Wang CS, Feng MG (2014) Advances in fundamental and applied studies in China of fungal biocontrol agents for use against arthropod pests. Biol Control 68:129–135

    Article  Google Scholar 

  • Wang CS, St Leger RJ (2007) The MAD1 adhesin of use of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants. Eukaryot Cell 6:808–816

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang ZL, Lu JD, Feng MG (2012) Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana. Environ Microbiol 14:2139–2150

    Article  CAS  PubMed  Google Scholar 

  • Wang ZL, Zhang LB, Ying SH, Feng MG (2013a) Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol 15:409–418

    Article  CAS  PubMed  Google Scholar 

  • Wang J, Zhou G, Ying SH, Feng MG (2013b) P-type calcium ATPase functions as a core regulator of Beauveria bassiana growth, conidiation and responses to multiple stressful stimuli through cross-talk with signalling networks. Environ Microbiol 15:967–979

    Article  CAS  PubMed  Google Scholar 

  • West S, Bamborough P, Tully R (1993) Tertiary structure of calcineurin B by homology modeling. J Mol Graph 11:47–52

    Article  CAS  PubMed  Google Scholar 

  • 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 2:483

    PubMed  PubMed Central  Google Scholar 

  • 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:e30298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu JH, Mah JH, Seo JA (2005) Growth and developmental control in the model and pathogenic aspergilli. Eukaryot Cell 5:1577–1584

    Article  Google Scholar 

  • Zhang H, Zhao Q, Liu K, Zhang Z, Wang Y, Zheng X (2009) MgCRZ1, a transcription factor of Magnaporthe grisea, controls growth, development and is involved in full virulence. FEMS Microbiol Lett 293:160–169

    Article  CAS  PubMed  Google Scholar 

  • Zhang SZ, Xia YX, Kim B, Keyhani 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 80:811–826

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was financially supported by the Ministry of Science and Technology of China (grant no. 2011AA10A204) and the Natural Science Foundation of China (grant nos. 31270537 and 31321063). The authors declare no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ming-Guang Feng.

Additional information

Fang Li and Zheng-Liang Wang contributed equally to this paper.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 331 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, F., Wang, ZL., Zhang, LB. et al. The role of three calcineurin subunits and a related transcription factor (Crz1) in conidiation, multistress tolerance and virulence in Beauveria bassiana . Appl Microbiol Biotechnol 99, 827–840 (2015). https://doi.org/10.1007/s00253-014-6124-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-6124-6

Keywords

Navigation