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Applied and Environmental Microbiology, June 1999, p. 2558-2564, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis

Pia Linnemannstöns,1 Thorsten Voß,1 Peter Hedden,2 Paul Gaskin,2 and Bettina Tudzynski1,*

Institut für Botanik, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany,1 and IACR---Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS41 9AF, United Kingdom2

Received 16 October 1998/Accepted 1 April 1999

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA12-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.

* Corresponding author. Mailing address: Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, D-48149 Münster, Germany. Phone: (0049) 251-832 4801. Fax: (0049) 251-832 3823. E-mail: Bettina.Tudzynski{at}uni-muenster.de.

Applied and Environmental Microbiology, June 1999, p. 2558-2564, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.