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Transformation and Transposon Mutagenesis of Leifsonia xyli subsp. xyli, Causal Organism of Ratoon Stunting Disease of Sugarcane

March 2002 , Volume 15 , Number  3
Pages  262 - 268

Stevens M. Brumbley , 1 , 5 Lars A. Petrasovits , 2 Robert G. Birch , 3 and Paul W. J. Taylor 4

1Bureau of Sugar Experiment Stations, 50 Meiers Rd., Indooroopilly, Queensland, Australia 4068; 2Department of Chemical Engineering, University of Queensland, St. Lucia, Australia 4072; 3Department of Botany, University of Queensland, St. Lucia, Australia 4072; 4Joint Center for Crop Improvement, University of Melbourne, Parkville, Victoria, Australia 3052; 5Cooperative Research Centre for Tropical Plant Protection, University of Queensland, Brisbane, Australia 4072

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Accepted 3 December 2001.

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/μg of plas-mid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/μg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/μg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam¯/dcm¯ E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-μm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetra-cycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.

© 2002 The American Phytopathological Society