Introduction of Genetic Material in Ralstonia solanacearum Through Natural Transformation and Conjugation

  • Anthony Perrier
  • Patrick Barberis
  • Stéphane GeninEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1734)


Ralstonia solanacearum is a soil-borne plant pathogen, responsible of the bacterial wilt disease. Its unusual wide host range (more than 250 plant species), aggressiveness, and broad geographic distribution have made of this bacterium the main plant pathogenic model in the beta-Proteobacteria class. Many R. solanacearum strains have the ability to internalize exogenous DNA through natural transformation. This property is widely used in reverse genetics studies to create mutants or reporter gene constructs, in the aim to study the molecular bases of pathogenesis of this bacterium. In this chapter, we describe three in vitro methods (natural transformation, electrotransformation, and conjugation) commonly used to produce recombinant R. solanacearum cells after introduction of exogenous DNA.

Key words

Natural competence Bacterial conjugation Electroporation Plant pathogen Reverse genetics 


  1. 1.
    Prior P, Ailloud F, Dalsing BL et al (2016) Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species. BMC Genomics 17:90CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Genin S, Denny TP (2012) Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol 50:67–89CrossRefPubMedGoogle Scholar
  3. 3.
    Coupat B, Chaumeille-Dole F, Fall S et al (2008) Natural transformation in the Ralstonia solanacearum species complex: number and size of DNA that can be transferred. FEMS Microbiol Ecol 66:14–24CrossRefPubMedGoogle Scholar
  4. 4.
    Le T, D Leccas D, Boucher C (1978) Transformation of Pseudomonas solanacearum strain K60. In: proceedings of the 4th international conference on plant pathogenic bacteria. Angers (INRA ed). pp 819–822 Google Scholar
  5. 5.
    Liu H, Zhang S, M a S, Denny TP (2005) Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence. Mol Plant-Microbe Interact 18:1296–1305CrossRefPubMedGoogle Scholar
  6. 6.
    Cunnac S, Occhialini A, Barberis P et al (2004) Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum: identification of novel effector proteins translocated to plant host cells through the type III secretion system. Mol Microbiol 53:115–128CrossRefPubMedGoogle Scholar
  7. 7.
    Monteiro F, Solé M, van Dijk I, Valls M (2012) A chromosomal insertion toolbox for promoter probing, mutant complementation, and pathogenicity studies in Ralstonia solanacearum. Mol Plant-Microbe Interact 25:557–568CrossRefPubMedGoogle Scholar
  8. 8.
    Kang Y, Liu H, Genin S et al (2002) Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence. Mol Microbiol 46:427–437CrossRefPubMedGoogle Scholar
  9. 9.
    Barman A, Buragohain C, Ray SK (2017) Disruption of comA homolog in Ralstonia solanacearum does not impair its twitching motility. J Basic Microbiol 57:218–227CrossRefPubMedGoogle Scholar
  10. 10.
    Bertolla F, Van Gijsegem F, Nesme X, Simonet P (1997) Conditions for natural transformation of Ralstonia solanacearum. Appl Environ Microbiol 63:4965–4968PubMedPubMedCentralGoogle Scholar
  11. 11.
    Guidot A, Coupat B, Fall S et al (2009) Horizontal gene transfer between Ralstonia solanacearum strains detected by comparative genomic hybridization on microarrays. ISME J 3:549–562CrossRefPubMedGoogle Scholar
  12. 12.
    González A, Plener L, Restrepo S et al (2011) Detection and functional characterization of a large genomic deletion resulting in decreased pathogenicity in Ralstonia solanacearum race 3 biovar 2 strains. Environ Microbiol 13:3172–3185CrossRefPubMedGoogle Scholar
  13. 13.
    Schäfer A, Tauch A, Jäger W et al (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73CrossRefPubMedGoogle Scholar
  14. 14.
    Boucher CA, Barberis PA, Trigalet AP, Demery DA (1985) Transposon mutagenesis of pseudomonas solanacearum : isolation of Tn5- induced avirulent mutants. J Gen Microbiol 131:2449–2457Google Scholar
  15. 15.
    Friedman AM, Long SR, Brown SE et al (1982) Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296CrossRefPubMedGoogle Scholar
  16. 16.
    Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845PubMedPubMedCentralGoogle Scholar
  17. 17.
    Marchetti M, Capela D, Glew M et al (2010) Experimental evolution of a plant pathogen into a legume symbiont. PLoS Biol 8:e1000280CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Anthony Perrier
    • 1
  • Patrick Barberis
    • 1
  • Stéphane Genin
    • 1
    Email author
  1. 1.LIPMUniversité de Toulouse, INRA, CNRSCastanet-TolosanFrance

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