Skip to main content
SpringerLink
Log in

Functional Analysis of Arbuscular Mycorrhizal Fungal Genes in Yeast

  • Protocol
  • First Online:
Arbuscular Mycorrhizal Fungi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2146))

  • 1308 Accesses

Abstract

The obligate symbiotic nature of arbuscular mycorrhizal (AM) fungi makes extremely difficult their genetic manipulation or transformation. For this reason, a heterologous system has been traditionally used for functional analysis of AM fungal genes, being the budding yeast Saccharomyces cerevisiae an organism suitable for this purpose. Here we present the yeast methods required for the functional analysis of AM fungal genes, including protocols for yeast transformation, heterologous gene expression, functional complementation assays, preparation of yeast extracts, and subcellular localization of the encoded protein.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 149.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Sanders IR (1999) Evolutionary genetics. No sex please, we're fungi. Nature 399:737–739

    Article  CAS  Google Scholar 

  2. Hinnebusch AG, Johnston M (2011) YeastBook: an encyclopedia of the reference eukaryotic cell. Genetics 189:683–684

    Article  Google Scholar 

  3. Christie KR, Weng S, Balakrishnan R et al (2004) Saccharomyces genome database (SGD) provides tools to identify and analyze sequences from Saccharomyces cerevisiae and related sequences from other organisms. Nucleic Acids Res 32:D311–D314

    Article  CAS  Google Scholar 

  4. Giaever G, Nislow C (2014) The yeast deletion collection: a decade of functional genomics. Genetics 197:451–465

    Article  CAS  Google Scholar 

  5. Maldonado-Mendoza IE, Dewbre GR, Harrison MJ (2001) A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment. Mol Plant-Microbe Interact 14:1140–1148

    Article  CAS  Google Scholar 

  6. Benabdellah K, Merlos MA, Azcón-Aguilar C, Ferrol N (2009) GintGRX1, the first characterized glomeromycotan glutaredoxin, is a multifunctional enzyme that responds to oxidative stress. Fungal Genet Biol 46:94–103

    Article  CAS  Google Scholar 

  7. Helber N, Wippel K, Sauer N et al (2011) A versatile monosaccharide transporter that operates in the arbuscular mycorrhizal fungus Glomus sp is crucial for the symbiotic relationship with plants. Plant Cell 23:3812–3823

    Article  CAS  Google Scholar 

  8. Pérez-Tienda J, Testillano PS, Balestrini R et al (2011) GintAMT2, a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal Genet Biol 48:1044–1055

    Article  Google Scholar 

  9. Ellerbeck M, Schüßler A, Brucker D et al (2013) Characterization of three ammonium transporters of the glomeromycotan fungus Geosiphon pyriformis. Eukaryot Cell 12:1554–1562. https://doi.org/10.1128/EC.00139-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Calabrese S, Pérez-Tienda J, Ellerbeck M et al (2016) GintAMT3—a low-affinity ammonium transporter of the arbuscular mycorrhizal Rhizophagus irregularis. Front Plant Sci 7:679. https://doi.org/10.3389/fpls.2016.00679

    Article  PubMed  PubMed Central  Google Scholar 

  11. Tamayo E, Benabdellah K, Ferrol N (2016) Characterization of three new glutaredoxin genes in the arbuscular mycorrhizal fungus Rhizophagus irregularis: putative role of RiGRX4 and RiGRX5 in iron homeostasis. PLoS One 11(2):e0149606. https://doi.org/10.1371/journal.pone.0149606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tamayo E, Knight SAB, Valderas A et al (2018) The arbuscular mycorrhizal fungus Rhizophagus irregularis uses a reductive iron assimilation pathway for high-affinity iron uptake. Environ Microbiol 20:1857–1872

    Article  CAS  Google Scholar 

  13. Kikuchi Y, Hijikata N, Ohtomo R et al (2016) Aquaporin-mediated long-distance polyphosphate translocation directed towards the host in arbuscular mycorrhizal symbiosis: application of virus-induced gene silencing. New Phytol 211:1202–1208

    Article  CAS  Google Scholar 

  14. Tsuzuki S, Handa Y, Takeda N, Kawaguchi M (2016) Strigolactone-induced putative secreted protein 1 is required for the establishment of symbiosis by the arbuscular mycorrhizal fungus Rhizophagus irregularis. Mol Plant-Microbe Interact 29:277–286

    Article  CAS  Google Scholar 

  15. Xie X, Lin H, Peng X et al (2016) Arbuscular mycorrhizal symbiosis requires a phosphate transceptor in the Gigaspora margarita fungal symbiont. Mol Plant 9:1583–1608

    Article  CAS  Google Scholar 

  16. Sun Z, Song J, Xin X et al (2018) Arbuscular mycorrhizal fungal 14-3-3 proteins are involved in arbuscule formation and responses to abiotic stresses during AM symbiosis. Front Microbiol. https://doi.org/10.3389/fmicb.2018.00091

  17. Voß S, Betz R, Heidt S et al (2018) RiCRN1, a crinkler effector from the arbuscular mycorrhizal fungus Rhizophagus irregularis, functions in arbuscule development. Front Microbiol 9:2068. https://doi.org/10.3389/fmicb.2018.02068

    Article  PubMed  PubMed Central  Google Scholar 

  18. Minet M, Dufour M, Lacroute F (1992) Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J 2:417–422

    CAS  PubMed  Google Scholar 

  19. Rentsch D, Laloi M, Rouhara I et al (1995) NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis. FEBS Lett 370:264–268

    Article  CAS  Google Scholar 

  20. Sauer N, Stolz J (1994) SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker’s yeast and identification of the histidine-tagged protein. Plant J 6:67–77

    Article  CAS  Google Scholar 

  21. Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167

    Article  CAS  Google Scholar 

  22. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34

    Article  CAS  Google Scholar 

  23. Hamilton NA (2012) Open source tools for fluorescent imaging. In: Conn PM (ed) Methods in enzymology, vol 504. Elsevier Inc., Amsterdam, pp 393–417

    Google Scholar 

  24. Knight SAB, Vilaire G, Lesuisse E, Dancis A (2005) Iron acquisition from transferrin by Candida albicans depends on the reductive pathway. Infect Immun 73:5482–5492

    Article  CAS  Google Scholar 

  25. Çağlayan M, Wilson SH (2014) Enzymatic activity assays in yeast cell extracts. Bio Protoc 4:23. https://doi.org/10.21769/BioProtoc.1312

    Article  Google Scholar 

Download references

Acknowledgments

Tamara Gómez-Gallego was supported by a PhD contract (FPI) from the MINECO and Elisabeth Tamayo was supported by a postdoctoral fellowship from the Alfonso Martin Escudero Foundation. We are grateful to Andrew Dancis for the laboratory protocol of iron transport assays. Research was funded by project RTI2018-098756-B-I00 (MCIU/AEI/FEDER, UE). Elisabeth Tamayo and Tamara Gómez-Gallego contributed equally to this work.

Author information

Author notes

  1. Elisabeth Tamayo and Tamara Gómez-Gallego contributed equally to this work.

Authors and Affiliations

  1. Molecular Phytopathology Department, Botanical Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

    Elisabeth Tamayo

  2. Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Granada, Spain

    Tamara Gómez-Gallego & Nuria Ferrol

Authors
  1. Elisabeth Tamayo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tamara Gómez-Gallego
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nuria Ferrol
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elisabeth Tamayo .

Editor information

Editors and Affiliations

  1. Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Granada, Spain

    Nuria Ferrol

  2. Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, Torino, Italy

    Luisa Lanfranco

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tamayo, E., Gómez-Gallego, T., Ferrol, N. (2020). Functional Analysis of Arbuscular Mycorrhizal Fungal Genes in Yeast. In: Ferrol, N., Lanfranco, L. (eds) Arbuscular Mycorrhizal Fungi. Methods in Molecular Biology, vol 2146. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0603-2_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0603-2_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0602-5

  • Online ISBN: 978-1-0716-0603-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation