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Related Experiment Videos

Small, stable shuttle vectors for use in Xanthomonas.

R DeFeyter1, C I Kado, D W Gabriel

  • 1Plant Pathology Department, University of Florida, Gainesville 32611.

Gene
|March 30, 1990
PubMed
Summary

New plasmids, pUFR027 and pUFR034, offer stable cloning vector options for Xanthomonas campestris pv. malvacearum (Xcm) and related bacteria. These vectors overcome limitations of previous broad-host-range plasmids, enabling genetic research in Xcm.

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Area of Science:

  • Molecular Biology
  • Bacteriology
  • Plant Pathology

Background:

  • Broad-host-range (bhr) plasmids are crucial tools for genetic manipulation in bacteria.
  • Previous cloning vectors showed instability and low transformation efficiency in Xanthomonas campestris pv. malvacearum (Xcm).
  • Effective cloning vectors are needed for Xcm, the causative agent of bacterial blight of cotton.

Purpose of the Study:

  • To evaluate existing bhr plasmids for cloning in Xcm.
  • To construct and characterize novel plasmids suitable for Xcm genetic studies.
  • To assess plasmid stability and maintenance in Xcm and other Xanthomonas species.

Main Methods:

  • Testing IncP, IncQ, and IncW plasmids for transformation frequency and stability in Xcm.
  • Constructing new plasmids (pUFR027, pUFR034) with specific replication origins, partition loci, selection markers, and cloning sites.

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  • Mobilizing plasmids into Xcm via conjugation and assessing their stability in vitro and in planta.
  • Evaluating plasmid stability and maintenance in various Xanthomonas pathovars.

    • Main Results:

    • IncP, IncQ, and IncW plasmids exhibited poor transformation efficiency or instability in Xcm.
    • Constructed plasmids pUFR027 (9.3 kb) and pUFR034 (8.7 kb) demonstrated high conjugational transfer frequency (approx. 1 x 10(-6)) into Xcm.
    • These novel plasmids showed excellent stability in Xcm ( >95% retention over 36 generations) under non-selective conditions, both in vitro and in planta.
    • Plasmids were stably maintained in X. citri and several X. campestris pathovars, with moderate stability in X. phaseoli.

    Conclusions:

    • pUFR027 and pUFR034 are effective and stable cloning vectors for Xcm and related xanthomonads.
    • These plasmids overcome limitations of previously used vectors, facilitating genetic research in Xanthomonas.
    • The developed vectors show no adverse effects on pathogenicity, making them suitable for virulence studies.