Phenotypic Analyses 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.3
DOI:  10.1002/9780471729259.mc03d03s25
Online Posting Date:  May, 2012
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Abstract

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 simple phenotypic characterizations of A. tumefaciens. Curr. Protoc. Microbiol. 25:3D.3.1‐3D.3.14. © 2012 by John Wiley & Sons, Inc.

Keywords: Agrobacterium; taxonomy; opines; plant association; virulence; plasmids; attachment; biofilms

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Potato Tumor Assay
  • Basic Protocol 2: β‐Galactosidase Assay for Agrobacterium tumefaciens
  • Basic Protocol 3: Static Biofilm Coverslip Assay
  • Basic Protocol 4: Short‐Term Binding Assay
  • Basic Protocol 5: Plant Attachment Assay
  • Basic Protocol 6: Swimming Motility
  • Basic Protocol 7: Phenol Flagellar Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Potato Tumor Assay

  Materials
  • Strains to be tested
  • Positive (e.g., A. tumefaciens C58) and negative controls (e.g., A. tumefaciens NTL4)
  • Appropriate medium or buffer (e.g., ATGN)
  • Red‐skinned, organically grown potatoes (purchased no more than 2 days prior to beginning assay)
  • 1.05% sodium hypochlorite (bleach) solution
  • Sterile 1.5% agar water plates (10‐cm diameter)
  • Saturated potassium sulfate solution ( appendix 2A)
  • 28°C incubator
  • Spectrophotometer and cuvettes
  • Metal cork borer, size 6 (must be resistant to ethanol‐dipped flame sterilization)
  • Sterile dissecting tray
  • Scalpels, sterile
  • Parafilm
  • Sealable container (e.g., Tupperware, etc.)

Basic Protocol 2: β‐Galactosidase Assay for Agrobacterium tumefaciens

  Materials
  • Bacterial strain(s) of interest and appropriate medium
  • Z‐buffer (see recipe)
  • 0.05% SDS ( appendix 2A)
  • Chloroform
  • Ortho‐nitrophenyl‐β‐galactoside (ONPG; Sigma, cat. no. N1127)
  • 1 M Na 2CO 3
  • 5‐ml culture tubes
  • 28°C incubator with shaking or rotation
  • Spectrophotometer
  • 2‐ml microcentrifuge tubes
  • Vortex
  • Timer
  • Microcentrifuge
  • Micro‐titer plates or cuvettes

Basic Protocol 3: Static Biofilm Coverslip Assay

  Materials
  • Bacterial strains of interest
  • Medium with appropriate antibiotics (e.g., ATGN or other culture medium)
  • Saturated potassium sulfate solution
  • 0.1% (w/v) crystal violet stain
  • 33% (v/v) acetic acid
  • 5‐ml glass culture tubes
  • 28°C incubator
  • PVC coverslips (Fisher, cat. no. 12‐547)
  • Scissors
  • Aerosol duster (e.g., VWR Whoosh‐Duster, cat. no. 16650‐027)
  • 12‐well tissue culture plates (Fisher, cat. no. 07‐200‐81)
  • UV light source
  • Spectrophotometer
  • Sealable container (e.g., Tupperware, etc.)
  • 200‐ml beaker
  • Forceps
  • Small plastic weigh boats (large enough to hold a coverslip)
  • Microtiter plates (e.g., 96‐well) or cuvettes

Basic Protocol 4: Short‐Term Binding Assay

  Materials
  • A. tumefaciens cells and minimal medium (e.g., ATGN)
  • 1× AT buffer
  • Alexafluor 594‐ and Alexafluor 488‐labeled wheat‐germ agglutinin (Sigma‐Aldrich, cat. no. W11262 and W11261, respectively)
  • 0.05% Tween‐20 in 1× AT buffer
  • 5‐ml glass culture tubes
  • 28°C incubator with shaking
  • Spectrophotometer
  • Glass coverslips
  • 6‐well dish (or 33‐mm petri dishes)
  • Glass microscope slides
  • Fluorescence microscope (with appropriate excitation and emission filter sets; Alexafluor 488, excitation 496, emission 519; Alexafluor 594, excitation 590, emission 617)

Basic Protocol 5: Plant Attachment Assay

  Materials
  • A. thaliana seed stocks
  • 70% ethanol with 0.1% Tween‐20
  • 70% and 95% ethanol
  • 1/2× Murashige Skoog salt plates with 1% agar and 1% sucrose (Sigma, cat. no. M5524) (Murashige and Skoog, )
  • A. tumefaciens derivatives expressing autofluorescent proteins such as GFP
  • ATGN broth with appropriate antibiotics
  • 1 mM CaCl 2/0.4% sucrose buffer (filter sterilized using a 0.22‐µm filter)
  • Nail polish
  • Parafilm
  • 4°C and 28°C incubators
  • Spectrophotometer
  • 35‐ and 100‐mm dishes
  • Sterile, fine‐tipped forceps
  • Sterile scalpel or razor blades
  • Microscope slides
  • Coverslips
  • Fluorescence microscope (with appropriate excitation and emission filter sets)

Basic Protocol 6: Swimming Motility

  Materials
  • Test strains in ATGN medium
  • 0.3% Bacto agar (US Biological, cat. no. A0930) ATGN plates containing 20× AT salts, 20× AT buffer, and 50% glucose
  • Saturated potassium sulfate
  • 5‐ml glass culture tubes
  • 28°C incubator with shaking
  • Sealable container (that can accommodate all plates)
  • Small beaker

Basic Protocol 7: Phenol Flagellar Staining

  Materials
  • Liquid cultures or colonies of test strain(s)
  • Flagellum stain (see recipe)
  • Glass microscope slides
  • Glass coverslips
  • Microscope with 1000× oil immersion lens
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Figures

  •   FigureFigure 3.D0.1 Photograph depicting potato disc tumors induced by Agrobacterium tumefaciens C58. These tumors developed after 14 days of incubation.

Videos

Literature Cited

Literature Cited
   An, D.D., Danhorn, T., Fuqua, C., and Parsek, M.R. 2006. Quorum sensing and motility mediate interactions between Pseudomonas aeruginosa and Agrobacterium tumefaciens in biofilm cocultures. Proc. Natl. Acad. Sci. U.S.A. 103:3828‐3833.
   Anand, V.K. and Heberlein, G.T. 1977. Crown gall tumorigenesis in potato tuber tissue. Am. J. Bot. 64:153‐158.
   Danhorn, T. and Fuqua, C. 2007. Biofilm formation by plant‐associated bacteria. Annu. Rev. Microbiol. 61:401‐422.
   Danhorn, T., Hentzer, M., Givskov, M., Parsek, M.R., and Fuqua, C. 2004. Phosphorous limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR‐PhoB regulatory system. J. Bacteriol. 186:4492‐4501.
   Fuqua, C. and Matthysse, A.G. 2001. Methods for studying bacterial biofilms associated with plants. Meth. Enzymol. 337:3‐18.
   Li, G., Brown, P.J.B., Tang, J.X., Xu, J., Quardokus, E.M., Fuqua, C., and Brun, Y.V. 2012. Surface contact stimulates the just‐in‐time deployment of bacterial adhesins. Mol. Microbiol. 83:41‐51.
   Merritt, P.M., Danhorn, T., and Fuqua, C. 2007. Motility and chemotaxis in Agrobacterium tumefaciens surface attachment and biofilm formation. J. Bacteriol. 189:8005‐8014.
   Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio‐assays with tobacco tissue cultures. Physiol. Plantarum. 15:473‐497.
   O'Toole, G., Kaplan, H.B., and Kolter, R. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54:49‐79.
   Rudrappa, T., Biedrzycki, M.L., and Bais, H.P. 2008. Causes and consequences of plant‐associated biofilms. FEMS Microbiol. Ecol. 64:153‐166.
   Tomlinson, A.D. and Fuqua, C. 2009. Mechanisms and regulation of polar surface attachment in Agrobacterium tumefaciens. Curr. Opin. Microbiol. 12:708‐714.
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