Advertisement

Gene Silencing in Medicago truncatula Roots Using RNAi

  • Daniela S. Floss
  • Alexa M. Schmitz
  • Colby G. Starker
  • J. Stephen Gantt
  • Maria J. Harrison
Part of the Methods in Molecular Biology book series (MIMB, volume 1069)

Abstract

Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.

Key words

Legume Gene knockdown Functional genomics 

Notes

Acknowledgments

The authors thank Armando Bravo and Sergey Ivanov for their reviews and useful comments on the manuscript. Financial support for research was provided by the US National Science Foundation, grants DBI-0421676 and IOS-1127155.

References

  1. 1.
    Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM (2002) High-throughput vectors for efficient gene silencing in plants. Funct Plant Biol 29:1217–1225CrossRefGoogle Scholar
  2. 2.
    Pasquinelli AE, Ruvkun G (2002) Control of developmental timing by microRNAs and their targets. Annu Rev Cell Dev Biol 18:495–513PubMedCrossRefGoogle Scholar
  3. 3.
    Hannon GJ (2002) RNA interference. Nature 418:244–251PubMedCrossRefGoogle Scholar
  4. 4.
    Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690PubMedCrossRefGoogle Scholar
  5. 5.
    Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590PubMedCrossRefGoogle Scholar
  6. 6.
    Gubler F, Hughes T, Waterhouse P, Jacobsen J (2008) Regulation of dormancy in barley by blue light and after-ripening: effects on abscisic acid and gibberellin metabolism. Plant Physiol 147:886–896PubMedCrossRefGoogle Scholar
  7. 7.
    Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R (2003) LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 302:630–633PubMedCrossRefGoogle Scholar
  8. 8.
    Javot H, Penmetsa RV, Terzaghi N, Cook DR, Harrison MJ (2007) A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis. Proc Natl Acad Sci U S A 104:1720–1725PubMedCrossRefGoogle Scholar
  9. 9.
    Subramanian S, Graham MY, Yu O, Graham TL (2005) RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and to enhanced susceptibility to Phytophthora sojae. Plant Physiol 137:1345–1353PubMedCrossRefGoogle Scholar
  10. 10.
    Dunoyer P, Himber C, Ruiz-Ferrer V, Alioua A, Voinnet O (2007) Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat Genet 39:848–856PubMedCrossRefGoogle Scholar
  11. 11.
    Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30:289–295PubMedCrossRefGoogle Scholar
  12. 12.
    Helliwell CA, Waterhouse PM (2005) Constructs and methods for hairpin RNA-mediated gene silencing in plants. In: Engelke D, Rossi J (eds) Methods in enzymology. Academic, San Diego, pp 24–35Google Scholar
  13. 13.
    Hartley J, Temple G, Brasch M (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795PubMedCrossRefGoogle Scholar
  14. 14.
    Boisson-Dernier A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker DG (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant Microbe Interact 14:695–700PubMedCrossRefGoogle Scholar
  15. 15.
    Collier R, Fuchs B, Walter N, Kevin Lutke W, Taylor CG (2005) Ex vitro composite plants: an inexpensive, rapid method for root biology. Plant J 43:449–457PubMedCrossRefGoogle Scholar
  16. 16.
    Floss DS, Hause B, Lange PR, Kuster H, Strack D, Walter MH (2008) Knock-down of the MEP pathway isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes. Plant J 56:86–100PubMedCrossRefGoogle Scholar
  17. 17.
    Pumplin N, Mondo SJ, Topp S, Starker CG, Gantt JS, Harrison MJ (2010) Medicago truncatula vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis. Plant J 61:482–494PubMedCrossRefGoogle Scholar
  18. 18.
    Vieweg MF, Fruhling M, Quandt HJ, Heim U, Baumlein H, Puhler A, Kuster H, Perlick AM (2004) The promoter of the Vicia faba L. leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and nonlegume plants. Mol Plant Microbe Interact 17:62–69PubMedCrossRefGoogle Scholar
  19. 19.
    Ivashuta S, Liu J, Liu J, Lohar DP, Haridas S, Bucciarelli B, VandenBosch KA, Vance CP, Harrison MJ, Gantt JS (2005) RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. Plant Cell 17:2911–2921PubMedCrossRefGoogle Scholar
  20. 20.
    Liu J, Blaylock L, Endre G, Cho J, Town CD, VandenBosch K, Harrison MJ (2003) Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis. Plant Cell 15: 2106–2123PubMedCrossRefGoogle Scholar
  21. 21.
    Quandt HJ, Puhler A, Broer I (1993) Transgenic root nodules of Vicia hirsuta: a fast and efficient system for the study of gene expression in indeterminate-type nodules. MPMI 6:699–706CrossRefGoogle Scholar
  22. 22.
    Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H, Bisseling T, Geurts R (2004) RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J Exp Bot 55:983–992PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Daniela S. Floss
    • 1
  • Alexa M. Schmitz
    • 1
  • Colby G. Starker
    • 2
    • 3
    • 4
  • J. Stephen Gantt
    • 2
  • Maria J. Harrison
    • 1
  1. 1.Boyce Thompson Institute for Plant ResearchIthacaUSA
  2. 2.Department of Plant BiologyUniversity of MinnesotaSt. PaulUSA
  3. 3.Department of GeneticsCell Biology, and Development University of MinnesotaMinneapolisUSA
  4. 4.Center for Genome EngineeringUniversity of MinnesotaMinneapolisUSA

Personalised recommendations