Genetic Manipulation of Agrobacterium

Elise R. Morton1, Clay Fuqua1

1 Department of Biology, Indiana University, Bloomington, Indiana
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3D.2
DOI:  10.1002/9780471729259.mc03d02s25
Online Posting Date:  May, 2012
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Agrobacterium species are plant‐associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens, this is called the Ti (tumor‐inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host‐microbe associations, horizontal gene transfer, cell‐cell communication, and biofilm formation. This unit describes standard protocols for genetic manipulation of A. tumefaciens. Curr. Protoc. Microbiol. 25:3D.2.1‐3D.2.15. © 2012 by John Wiley & Sons, Inc.

Keywords: Agrobacterium; growth media; genetic analyses; taxonomy; opines; plant association; virulence; plasmids; attachment; transposon

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Table of Contents

  • Introduction
  • Basic Protocol 1: Transformation of Agrobacterium by Electroporation
  • Basic Protocol 2: Plasmid Introduction by Conjugation
  • Basic Protocol 3: Transposon Mutagenesis of Agrobacterium
  • Basic Protocol 4: Touchdown PCR
  • Basic Protocol 5: Creation of Markerless Deletions by Allelic Replacement
  • Support Protocol 1: Generating an Allelic Replacement Construct
  • Support Protocol 2: Introducing a Gene into the tetRA Locus
  • Basic Protocol 6: Plasmid Curing
  • Basic Protocol 7: Eckhardt Gel for Determining Plasmid Composition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Transformation of Agrobacterium by Electroporation

  • Electrocompetent cells
  • LB broth ( appendix 4A) supplemented with appropriate antibiotics
  • Sterile water, ice cold
  • 30% glycerol, ice cold
  • DNA
  • ATGN plates in 10‐cm petri dishes supplemented with appropriate antibiotics (see unit 3.1 for recipe) or LB plates (optional)
  • 2‐ and 5‐ml culture tubes
  • Spectrophotometer
  • Microcentrifuge
  • 1.6‐ml microcentrifuge tubes, sterile
  • Electroporation cuvettes (e.g., Harvard Apparatus, cat. no. 45‐0124)
  • Electroporator (e.g., Bio‐Rad E. coli Pulser)
  • 28°C incubator with shaker

Basic Protocol 2: Plasmid Introduction by Conjugation

  • Donor strain of E. coli carrying plasmid of interest (plasmid must be self‐conjugal or donor strain must encode appropriate conjugal machinery, tra+)
  • Recipient strain of A. tumefaciens
  • LB medium ( appendix 4A)
  • LB plates (10‐cm, no antibiotics; appendix 4A)
  • ATGN medium
  • ATGN plates supplemented with appropriate antibiotics
  • Sterile hydrophilic polyethersulfone or cellulose acetate filters (25‐mm, 0.2‐µm)
  • 28°C and 37°C incubators
  • Sterile forceps
  • 1.5‐ml tubes

Basic Protocol 3: Transposon Mutagenesis of Agrobacterium

  • Donor strain: E. coli carrying pFD1
  • Recipient strain (to be mutated) must be sensitive to kanamycin
  • 30% sterile glycerol
  • ATGN plates supplemented with kanamycin (10‐cm plates)
  • Additional reagents and equipment (see protocol 2)

Basic Protocol 4: Touchdown PCR

  • Genomic DNA from transposon mutant(s) (30 to 120 ng; Promega Wizard Genomic DNA purification)
  • TD‐PCR primers:
    • Set 1 (amplifies from within the left side of the transposon):
    • MarLseq (specific primer) 5′GGG AAT CAT TTG AAG GTT GGT 3′
    • MarTDL2 (arbitrary primer) 5′GACACGGGCCTCGANGNNNCNTNGG 3′
    • Set 2 (amplifies from within the right side of the transposon):
    • MarRseq (specific primer) 5′ CGG GTA TCG CTC TTG AAG GGA 3′
    • MarTDR1 (arbitrary primer) 5′ CAACCGTGGCGGGGNTNCNNGNCNCG 3′
  • 10 mM dNTP mix
  • Taq polymerase and 10× tricine buffer
  • 20 mM MgCl 2
  • 1.0% agarose gel
  • PCR purification kit (e.g., QIAquick PCR Cleanup, cat. no. 28104)
  • 0.2‐ml PCR tubes
  • Thermal cycler
  • Gel electrophoresis apparatus and supplies

Basic Protocol 5: Creation of Markerless Deletions by Allelic Replacement

  • Plasmid to generate primary integrants (see Support Protocols protocol 61 and protocol 72)
  • ATGN plates supplemented with appropriate antibiotics (10‐cm plates)
  • ATSN (5% sucrose) plates with and without antibiotics (10‐cm plates)
  • ATGN liquid medium
  • 28°C incubator
  • 1.6‐ml microcentrifuge tubes
  • 5‐ml glass culture tubes

Support Protocol 1: Generating an Allelic Replacement Construct

  • Primers to amplify repABC (incompatibility) region of plasmid to be cured
  • Vector (lacking replication genes suitable for multiplication in Agrobacterium, contains a selectable marker such as antibiotic‐resistance gene, and a counter‐selectable marker such as sacB, e.g., pNPTS138)
  • A. tumefaciens strain
  • ATGN liquid and plates (with and without antibiotics)
  • ATSN (5% sucrose) 10‐cm plates (with and without antibiotics)
  • Primers to amplify non‐repABC sequences of the plasmid to be cured (for diagnostics)
  • 30% sterile glycerol
  • 28°C incubator with shaking
  • Grid for patching cells onto two types of media
  • Additional reagents and equipment for electroporation (see protocol 1) and conjugation (see protocol 2)

Support Protocol 2: Introducing a Gene into the tetRA Locus

  • Strains of interest
  • ATGN liquid medium
  • Agarose
  • 0.5× TBE (see recipe)
  • 5× TBE stock (see recipe)
  • 10% SDS in 0.5× TBE (see recipe)
  • 0.3% Sarkosyl in 0.5× TBE (see recipe), cold
  • Eckhardt lysis solution (see recipe; prepared fresh prior to use)
  • Loading dye, optional
  • 0.5 µg/ml ethidium bromide solution: 5 µl ethidium bromide stock and 100 ml sterile water
  • 28°C incubator with shaking
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 5‐ml culture tubes
  • Spectrophotometer
  • Electrophoresis apparatus
  • UV source
  • Gel photography equipment
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  •   FigureFigure 3.D0.1 Schematic of PCR splicing by overlapping extension (SOEing). Light gray bars are representative of DNA sequence to be removed, whereas the black and dark gray bars represent sequence of the upstream and downstream flanking regions, respectively. Lower case letters denote primers, whereas upper case letters represent stretches of DNA.
  •   FigureFigure 3.D0.2 Diagram outlining strategy for plasmid curing by incompatibility. Gray ovals denote cells and black squiggly line represents chromosomal DNA. Plasmids are represented as black circles and associated genes as white rectangles.


Literature Cited

   Hibbing, M.E. and Fuqua, C. 2011 Antiparallel and interlinked control of cellular iron levels by the Irr and RirA regulators of Agrobacterium tumefaciens. J. Bacteriol. 193:3461‐3472.
   Hynes, M.F., Simon, R., and Pühler, A. 1985. The development of plasmid‐free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58. Plasmid 13:99‐105.
   Kaniga, K., Delor, I., and Cornelis, G.R. 1991. A wide‐host‐range suicide vector for improving reverse genetics in Gram‐negative bacteria: Inactivation of the blaA gene of Yersinia enterocolitica. Gene 109:137‐141.
   Lampe, D.J., Akerley, B.J., Rubin, E.J., Mekalanos, J.J., and Robertson, H.M. 1999. Hyperactive transposase mutants of the Himar1 mariner transposon. Proc. Natl. Acad. Sci. U.S.A. 96:11428‐11433.
   Levano‐Garcia, J., Verjovski‐Almeida, S., and da Silva, A.C.R. 2005. Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers. Biotechniques 38:225‐229.
   Luo, Z.Q. and S.K. Farrand, S.K. 1999. Cloning and characterization of a tetracyline resistance determinant present in Agrobacterium tumefaciens C58. J. Bacteriol. 181:618‐626.
   Mersereau, M., Pazour, G.J., and Das, A. 1990. Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90:149‐151.
   Sarker, M. and Cornelis, G.R. 1997. An improved version of suicide vector pKNG101 for gene replacement in Gram negative bacteria. Mol. Microbiol. 23:409‐411.
   Soberón, N., Venkova‐Canova, T., Ramírez‐Romero, M.A., Téllez‐Sosa, J., and Cevallos, M.A. 2004. Incompatibility and the partitioning site of the repABC basic replicon of the symbiotic plasmid from Rhizobium etli. Plasmid 51:203‐216.
   Trevors, J.T. 1986. Plasmid curing in bacteria. FEMS Micr. Rev. 32:149‐157.
Internet Resources
  Genome sequencing of Agrobacterium biovar type strains, Virginia Bioinformatics Institute.
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