Skip to main content
SpringerLink
Log in

Gene Silencing for Functional Analysis: Assessing RNAi as a Tool for Manipulation of Gene Expression

  • Protocol
  • First Online:
Molecular and Cell Biology Methods for Fungi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 638))

Abstract

The availability of a large number of gene-disrupted mutants (either from natural mutants’ collections or from knockout projects) is a great advantage for functional analysis studies. However, disfunction of many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes in cell morphology, conferring a major difficulty in studying null mutants. Therefore, obtaining variable levels of reduction in gene expression, especially of essential genes or genes whose impaired expression confers a pleiotropic phenotype, is extremely beneficial for studying their function. Here, we describe the use of RNAi as a gene silencing mechanism, in a manner that might facilitate the functional analysis of such essential genes. Two alternative strategies for the construction of an RNAi-induced inverted-repeat construct are demonstrated and a third alternative is suggested. In addition, DNA-mediated transformation of conidia by electroporation, RNA extraction from fungal mycelium and northern blot analysis are described in detail.

The experimental results presented, demonstrate that RNAi can be employed as a gene silencing tool in Neurospora crassa, both for nonessential (al-2) and essential (cot-1) genes, resulting in a range of stable, partially silenced mutants, exhibiting different phenotypes.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Davis RH, Perkins DD (2002) Timeline: Neurospora: a model of model microbes. Nat Rev Genet 3:397–403

    Article  CAS  PubMed  Google Scholar 

  2. Davis RH (2000) Neurospora: contributions of a model organism. Oxford University Press, Oxford, UK

    Google Scholar 

  3. Divon HH, Ziv C, Davydov O, Yarden O, Fluhr R (2006) The global nitrogen regulator, FNR1, regulates fungal nutrition-genes and fitness during Fusarium oxysporum pathogenesis. Mol Plant Pathol 7:485–497

    Article  CAS  PubMed  Google Scholar 

  4. Scheffer J, Ziv C, Yarden O, Tudzynski P (2005) The COT1 homologue CPCOT1 regulates polar growth and branching and is essential for pathogenicity in Claviceps purpurea. Fungal Genet Biol 42:107–118

    Article  CAS  PubMed  Google Scholar 

  5. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D et al (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422:859–868

    Article  CAS  PubMed  Google Scholar 

  6. Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read N 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

    Article  CAS  PubMed  Google Scholar 

  7. Kasuga T, Townsend JP, Tian C, Gilbert LB, Mannhaupt G, Taylor J et al (2005) Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination. Nucleic Acids Res 33:6469–6485

    Article  CAS  PubMed  Google Scholar 

  8. Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L et al (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci U S A 103:10352–10357

    Article  CAS  PubMed  Google Scholar 

  9. Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529

    Article  CAS  PubMed  Google Scholar 

  10. Cottrell TR, Doering TL (2003) Silence of the strands: RNA interference in eukaryotic pathogens. Trends Microbiol 11:37–43

    Article  CAS  PubMed  Google Scholar 

  11. Hutvagner G, Zamore PD (2002) RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12:225–232

    Article  CAS  PubMed  Google Scholar 

  12. Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353

    Article  CAS  PubMed  Google Scholar 

  13. Catalanotto C, Azzalin G, Macino G, Cogoni C (2002) Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora. Genes Dev 16:790–795

    Article  CAS  PubMed  Google Scholar 

  14. Fulci V, Macino G (2007) Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa. Curr Opin Microbiol 10:199–203

    Article  CAS  PubMed  Google Scholar 

  15. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J 21:4671–4679

    Article  CAS  PubMed  Google Scholar 

  16. Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33

    Article  CAS  PubMed  Google Scholar 

  17. Raponi M, Arndt GM (2003) Double-stranded RNA-mediated gene silencing in fission yeast. Nucleic Acids Res 31:4481–4489

    Article  CAS  PubMed  Google Scholar 

  18. Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2003) RNA silencing in the phytopathogenic fungus Magnaporthe oryzae. Mol Plant Microbe Interact 16:769–776

    Article  CAS  PubMed  Google Scholar 

  19. Cogoni C, Macino G (1999) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399:166–169

    Article  CAS  PubMed  Google Scholar 

  20. Catalanotto C, Azzalin G, Macino G, Cogoni C (2000) Gene silencing in worms and fungi. Nature 404:245

    Article  CAS  PubMed  Google Scholar 

  21. Cogoni C, Macino G (1999) Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase. Science 17:2342–2344

    Article  Google Scholar 

  22. Catalanotto C, Pallotta M, ReFalo P, Sachs MS, Vayssie L, Macino G, Cogoni C (2004) Redund-ancy of the two Dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 24:2536–2545

    Article  CAS  PubMed  Google Scholar 

  23. Maiti M, Lee HC, Liu Y (2007) QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Genes Dev 21:590–600

    Article  CAS  PubMed  Google Scholar 

  24. Goldoni M, Azzalin G, Macino G, Cogoni C (2004) Efficient gene silencing by expression of double stranded RNA in Neurospora crassa. Fungal Genet Biol 41:1016–1024

    Article  CAS  PubMed  Google Scholar 

  25. Erental A, Harel A, Yarden O (2007) Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum. Mol Plant Microbe Interact 20:944–954

    Article  CAS  PubMed  Google Scholar 

  26. Khatri M, Rajam MV (2007) Targeting polyamines of Aspergillus nidulans by siRNA specific to fungal ornithine decarboxylase gene. Med Mycol 45:211–220

    Article  CAS  PubMed  Google Scholar 

  27. Liu H, Cottrell TR, Pierini LM, Goldman WE, Doering TL (2002) RNA interference in the pathogenic fungus Cryptococcus neoformans. Genetics 160:463–470

    CAS  PubMed  Google Scholar 

  28. Namekawa SH, Iwabata K, Sugawara H, Hamada FN, Koshiyama A, Chiku H et al (2005) Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing. Microbiology 151:3669–3678

    Article  CAS  PubMed  Google Scholar 

  29. Matityahu A, Hadar Y, Dosoretz CG, Belinky PA (2008) Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol 74:5359–5365

    Article  CAS  PubMed  Google Scholar 

  30. Nakayashiki H, Nguyen QB (2008) RNA interference: roles in fungal biology. Curr Opin Microbiol 11:494–502

    Article  CAS  PubMed  Google Scholar 

  31. Nakayashiki H (2005) RNA silencing in fungi: mechanisms and applications. FEBS Lett 579:5950–5957

    Article  CAS  PubMed  Google Scholar 

  32. Aravind L, Watanabe H, Lipman DJ, Koonin EV (2000) Lineage-specific loss and divergence of functionally linked genes in eukaryotes. Proc Natl Acad Sci U S A 97:11319–11324

    Article  CAS  PubMed  Google Scholar 

  33. Kamper J, Kahmann R, Bolker M, Ma L-J, Brefort T, Saville BJ et al (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444:97–101

    Article  PubMed  Google Scholar 

  34. Feldbrugge M, Zarnack K, Vollmeister E, Baumann S, Koepke J, Konig J et al (2008) The posttranscriptional machinery of Ustilago maydis. Fungal Genet Biol 45:S40-S46

    Article  PubMed  Google Scholar 

  35. Nakayashiki H, Hanada S, Nguyen BQ, Kadotani N, Tosa Y, Mayama S (2005) RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 42:275–283

    Article  CAS  PubMed  Google Scholar 

  36. Stoutjesdijk PA, Singh SP, Liu Q, Hurlstone CJ, Waterhouse PA, Green AG (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723–31

    Article  CAS  PubMed  Google Scholar 

  37. Han Y, Grierson D (2002) The influence of inverted repeats on the production of small antisense RNAs involved in gene silencing. Mol Genet Genomics 267:629–635

    Article  CAS  PubMed  Google Scholar 

  38. Tavernarakis N, Wang SL, Dorovkov M, Ryazanov A, Driscoll M (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat Genet 24:180–183

    Article  CAS  PubMed  Google Scholar 

  39. Ngo H, Tschudi C, Gull K, Ullu E (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci U S A 95:14687–14692

    Article  CAS  PubMed  Google Scholar 

  40. Yarden O, Plamann M, Ebbole DJ, Yanofsky C (1992) cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. EMBO J 11:2159–2166

    CAS  PubMed  Google Scholar 

  41. Gorovits R, Sjollema KA, Sietsma JH, Yarden O (2000) Cellular distribution of COT1 kinase in Neurospora crassa. Fungal Genet Biol 30:63–70

    Article  CAS  PubMed  Google Scholar 

  42. Steele GC, Trinci AP (1977) Effect of temperature and temperature shifts on growth and branching of a wild type and a temperature sensitive colonial mutant (Cot 1) of Neurospora crassa. Arch Microbiol 113:43–48

    Article  CAS  PubMed  Google Scholar 

  43. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. CSH Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  44. Perkins DD, Radford A, Sachs MS (2001) The Neurospora compendium. Academic, San Diego, California

    Google Scholar 

  45. Li C, Schmidhauser TJ (1995) Developmental and photoregulation of al-1 and al-2, structural genes for two enzymes essential for carotenoid biosynthesis in Neurospora. Dev Biol 169:90–95

    Article  CAS  PubMed  Google Scholar 

  46. Schmidhauser TJ, Lauter FR, Schumacher M, Zhou W, Russo VE, Yanofsky C (1994) Characterization of al-2, the phytoene synthase gene of Neurospora crassa. Cloning, sequence analysis, and photoregulation. J Biol Chem 269:12060–12066

    CAS  PubMed  Google Scholar 

  47. Carroll AM, Sweigard JA, Valent B (1994) Improved vectors for selecting resistance to hygromycin. Fungal Genet Newsl 41:22

    Google Scholar 

  48. Margolin BS, Freitag M, Selker EU (1997) Improved plasmids for gene targeting at the his-3 locus of Neurospora crassa by electroporation. Fungal Genet Newsl 44:34–36

    Google Scholar 

  49. Linden H, Rodriguez Franco M, Macino G (1997) Mutants of Neurospora crassa defective in regulation of blue light perception. Mol Gen Genet 254:111–118

    Article  CAS  PubMed  Google Scholar 

  50. Ebbole DJ, Sachs MS (1990) A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia. Fungal Genet Newsl 37:17–18

    Google Scholar 

  51. Gorovits R, Yarden O (2003) Environmental suppression of Neurospora crassa cot-1 hyperbranching: a link between COT1 kinase and stress-sensing. Eukaryot Cell 2:699–707

    Article  CAS  PubMed  Google Scholar 

  52. Henry C, Mouyna I, Latge JP (2007) Testing the efficacy of RNA interference constructs in Aspergillus fumigatus. Curr Genet 51:277–284

    Article  CAS  PubMed  Google Scholar 

  53. Agrawal N, Dasaradhi PVN, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67:657–685

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

    Carmit Ziv & Oded Yarden

Authors
  1. Carmit Ziv
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oded Yarden
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Dept. Plant Sciences, Tel Aviv University, Tel Aviv, 69978, Israel

    Amir Sharon

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Ziv, C., Yarden, O. (2010). Gene Silencing for Functional Analysis: Assessing RNAi as a Tool for Manipulation of Gene Expression. In: Sharon, A. (eds) Molecular and Cell Biology Methods for Fungi. Methods in Molecular Biology, vol 638. Humana Press. https://doi.org/10.1007/978-1-60761-611-5_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-611-5_6

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-610-8

  • Online ISBN: 978-1-60761-611-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation