Skip to main content
SpringerLink
Log in

Calmodulin, Calcium/Calmodulin-Dependent Kinases-1 and 2 Regulate Expression of the Heat Shock Proteins for Heat Shock Tolerance and Pheromone Signaling Genes for Sexual Development in Neurospora crassa

  • ORIGINAL RESEARCH ARTICLE
  • Published:
Indian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Calmodulin (CaM) is a primary Ca2+ sensor that binds and activates numerous target proteins and regulates several cellular processes in eukaryotes. CaM is essential in Neurospora crassa; therefore, we generated a CaM mutant using repeat-induced point (RIP) mutation and investigated the cmdRIP mutant phenotypes. We also studied knockout mutants of four Ca2+/CaM kinases (camk-1, 2, 3, and 4) for their role during stress conditions and sexual development. The cmdRIP, ∆camk-1, and ∆camk-2 mutants showed reduced survival and growth rates under heat stress, oxidative stress, pH, and ER stress conditions. In addition, under the heat stress conditions, expression of the heat shock protein genes hsp70 and hsp80 was reduced in the cmdRIP, ∆camk-1, and ∆camk-2 mutants. The cmdRIP mutant was also defective in cell fusion, its vegetative hyphae could not support the fertilized wild type perithecia graft, and female sterile. Furthermore, the expression of pheromone signaling genes pre-1, pre-2, ccg-4, mfa-1, and fmf-1 was reduced in the cmdRIP, ∆camk-1, and ∆camk-2 mutants. Therefore, CaM, Ca2+/CaMK-1 and 2 are involved in the tolerance to heat stress conditions and sexual development by regulating the heat shock and pheromone response pathways, respectively, in N. crassa.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Freitas FZ, Virgilio S, Cupertino FB, Kowbel DJ et al (2016) The SEB-1 transcription factor binds to the STRE motif in Neurospora crassa and regulates a variety of cellular processes including the stress response and reserve carbohydrate metabolism. G3 (Bethesda) 6:1327–1343. https://doi.org/10.1534/g3.116.028506

    Article  CAS  PubMed  Google Scholar 

  2. Borkovich KA, Alex LA, Yarden O et al (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68:1–108. https://doi.org/10.1128/MMBR.68.1.1-108.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Roy A, Kumar A, Baruah D, Tamuli R (2021) Calcium signaling is involved in diverse cellular processes in fungi. Mycology 12:10–24. https://doi.org/10.1080/21501203.2020.1785962

    Article  CAS  Google Scholar 

  4. Bootman MD, Collin TJ, Peppiatt CM et al (2001) Calcium signalling–an overview. Semin Cell Dev Biol 12:3–10. https://doi.org/10.1006/scdb.2000.0211

    Article  CAS  PubMed  Google Scholar 

  5. Tamuli R, Kumar R, Srivastava DA, Deka R (2013) Calcium signaling. In: Kasbekar DP, McCluskey K (eds) Neurospora: genomics and molecular biology. Caister Academic Press, Norfolk, pp 35–57

    Google Scholar 

  6. Chin D, Means AR (2000) Calmodulin: a prototypical calcium sensor. Trends Cell Biol 10:322–328. https://doi.org/10.1016/s0962-8924(00)01800-6

    Article  CAS  PubMed  Google Scholar 

  7. Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J 405:199–221. https://doi.org/10.1042/BJ20070255

    Article  CAS  PubMed  Google Scholar 

  8. Halling DB, Liebeskind BJ, Hall AW, Aldrich RW (2016) Conserved properties of individual Ca2+-binding sites in calmodulin. Proc Natl Acad Sci USA 113:E1216–E1225. https://doi.org/10.1073/pnas.1600385113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Laxmi V, Tamuli R (2017) The calmodulin gene in Neurospora crassa is required for normal vegetative growth, ultraviolet survival, and sexual development. Arch Microbiol 199:531–542. https://doi.org/10.1007/s00203-016-1319-0

    Article  CAS  PubMed  Google Scholar 

  10. Melnick MB, Melnick C, Lee M, Woodward DO (1993) Structure and sequence of the calmodulin gene from Neurospora crassa. Biochem Biophys Acta 1171:334–336. https://doi.org/10.1016/0167-4781(93)90079-s

    Article  CAS  PubMed  Google Scholar 

  11. Laxmi V, Tamuli R (2015) The Neurospora crassa cmd, trm-9, and nca-2 play a role in growth, development, and survival in stress conditions. Genom Appl Biol 6:1–12

    CAS  Google Scholar 

  12. Swulius MT, Waxham MN (2008) Ca2+/calmodulin-dependent protein kinases. Cell Mol Life Sci 65:2637–2657. https://doi.org/10.1007/s00018-008-8086-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tamuli R, Kumar R, Deka R (2011) Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi. J Basic Microbiol 51:120–128. https://doi.org/10.1002/jobm.201000184

    Article  CAS  PubMed  Google Scholar 

  14. Kumar R, Tamuli R (2014) Calcium/calmodulin-dependent kinases are involved in growth, thermotolerance, oxidative stress survival, and fertility in Neurospora crassa. Arch Microbiol 196:295–305. https://doi.org/10.1007/s00203-014-0966-2

    Article  CAS  PubMed  Google Scholar 

  15. McCluskey K, Wiest A, Plamann M (2010) The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research. J Biosci 35:119–126. https://doi.org/10.1007/s12038-010-0014-6

    Article  CAS  PubMed  Google Scholar 

  16. Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implications. Am Nat 98:435–446

    Article  CAS  Google Scholar 

  17. Westergaard M, Mitchell HK (1947) Neurospora V. A Synthetic medium favoring sexual reproduction. Am J Bot 34:573–577

    Article  Google Scholar 

  18. Yang Q, Borkovich KA (1999) Mutational activation of a Gαi causes uncontrolled proliferation of aerial hyphae and increased sensitivity to heat and oxidative stress in Neurospora crassa. Genetics 151:107–117. https://doi.org/10.1093/genetics/151.1.107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kapoor M, Lewis J (1987) Alteration of the protein synthesis pattern in Neurospora crassa cells by hyperthermal and oxidative stress. Can J Microbiol 33:162–168

    Article  CAS  Google Scholar 

  20. Barman A, Tamuli R (2017) The pleiotropic vegetative and sexual development phenotypes of Neurospora crassa arise from double mutants of the calcium signaling genes plc-1, splA2, and cpe-1. Curr Genet 63:861–875. https://doi.org/10.1007/s00294-017-0682-y

    Article  CAS  PubMed  Google Scholar 

  21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262

    Article  CAS  PubMed  Google Scholar 

  22. Cusick KD, Fitzgerald LA, Pirlo RK, Cockrell AL, Petersen ER, Biffinger JC (2014) Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture. PloS One 9:e112706. https://doi.org/10.1371/journal.pone.0112706

  23. Kim H, Wright SJ, Park G, Ouyang S, Krystofova S, Borkovich KA (2012) Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa. Genetics 190:1389–1404. https://doi.org/10.1534/genetics.111.136358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Roychowdhury HS, MacAlister TJ, Costerton JW, Kapoor M (1992) Induction and intracellular localization of the 80-kilodalton heat-shock protein of Neurospora crassa. Biochem Cell Biol 70:1347–1355. https://doi.org/10.1139/o92-183

    Article  CAS  PubMed  Google Scholar 

  25. Kapoor M, Curle CA, Runham C (1995) The hsp70 gene family of Neurospora crassa: cloning, sequence analysis, expression, and genetic mapping of the major stress-inducible member. J Bacteriol 177:212–221. https://doi.org/10.1128/jb.177.1.212-221.1995

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Freitag DG, Ouimet PM, Girvitz TL, Kapoor M (1997) Heat shock protein 80 of Neurospora crassa, a cytosolic molecular chaperone of the eukaryotic stress 90 family, interacts directly with heat shock protein 70. Biochemistry 36:10221–10229. https://doi.org/10.1021/bi963030g

    Article  CAS  PubMed  Google Scholar 

  27. Sharma SK, Christen P, Goloubinoff P (2009) Disaggregating chaperones: an unfolding story. Curr Prot Pept Sci 10:432–446. https://doi.org/10.2174/138920309789351930

    Article  CAS  Google Scholar 

  28. Kumar A, Roy A, Deshmukh MV, Tamuli R (2020) Dominant mutants of the calcineurin catalytic subunit (CNA-1) showed developmental defects, increased sensitivity to stress conditions, and CNA-1 interacts with CaM and CRZ-1 in Neurospora crassa. Arch Microbiol 202:921–934. https://doi.org/10.1007/s00203-019-01768-z

    Article  CAS  PubMed  Google Scholar 

  29. Kraus PR, Heitman J (2003) Coping with stress: calmodulin and calcineurin in model and pathogenic fungi. Biochem Biophys Res Commun 311:1151–1157. https://doi.org/10.1016/s0006-291x(03)01528-6

    Article  CAS  PubMed  Google Scholar 

  30. Li B, Liu HT, Sun DY, Zhou RG (2004) Ca2+ and calmodulin modulate DNA-binding activity of maize heat shock transcription factor in vitro. Plant Cell Physiol 45:627–634. https://doi.org/10.1093/pcp/pch074

    Article  CAS  PubMed  Google Scholar 

  31. Virdi AS, Pareek A, Singh P (2011) Evidence for the possible involvement of calmodulin in regulation of steady state levels of Hsp90 family members (Hsp87 and Hsp85) in response to heat shock in sorghum. Plant Signal Behav 6:393–399. https://doi.org/10.4161/psb.6.3.13867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Peng W, Zhang Y, Zheng M et al (2010) Cardioprotection by CaMKII-deltaB is mediated by phosphorylation of heat shock factor 1 and subsequent expression of inducible heat shock protein 70. Circ Res 106:102–110. https://doi.org/10.1161/CIRCRESAHA.109.210914

    Article  CAS  PubMed  Google Scholar 

  33. Rodriguez-Caban J, Gonzalez-Velazquez W, Perez-Sanchez L et al (2011) Calcium/calmodulin kinase1 and its relation to thermotolerance and HSP90 in Sporothrix schenckii: An RNAi and yeast two-hybrid study. BMC Microbiol 11:162. https://doi.org/10.1186/1471-2180-11-162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Perkins DD, Barry EG (1977) The cytogenetics of Neurospora. Adv Genet 19:133–285. https://doi.org/10.1016/s0065-2660(08)60246-1

    Article  CAS  PubMed  Google Scholar 

  35. Nelson MA, Metzenberg RL (1992) Sexual development genes of Neurospora crassa. Genetics 132:149–162. https://doi.org/10.1093/genetics/132.1.149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pöggeler S, Kück U (2001) Identification of transcriptionally expressed pheromone receptor genes in filamentous ascomycetes. Gene 280:9–17. https://doi.org/10.1016/s0378-1119(01)00786-7

    Article  PubMed  Google Scholar 

  37. Iyer SV, Ramakrishnan M, Kasbekar DP (2009) Neurospora crassa fmf-1 encodes the homologue of the Schizosaccharomyces pombe Ste11p regulator of sexual development. J Genet 88:33–39. https://doi.org/10.1007/s12041-009-0005-2

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Kevin McCluskey and Eric at the Fungal Genetics Stock Center (FGSC) for generously waiving charges for some of the strains. CM was supported by a Research Fellowship from the Ministry of Human Resource Development (MHRD), Government of India. We thank the MHRD and IIT Guwahati for partial financial support. We also thank the Department of Biotechnology, Govt. of India, for partial financial support through the DBT-NER twinning grant BT/PR24473/NER/95/737/2017.

Author information

Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781 039, India

    Christy Noche K. Marak & Ranjan Tamuli

Authors
  1. Christy Noche K. Marak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ranjan Tamuli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ranjan Tamuli.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 64849 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Marak, C.N.K., Tamuli, R. Calmodulin, Calcium/Calmodulin-Dependent Kinases-1 and 2 Regulate Expression of the Heat Shock Proteins for Heat Shock Tolerance and Pheromone Signaling Genes for Sexual Development in Neurospora crassa. Indian J Microbiol 63, 317–323 (2023). https://doi.org/10.1007/s12088-023-01091-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12088-023-01091-8

Keywords

Search

Navigation