Labeled and defined mutants of Xylella fastidiosa by triparental matings

• Author(s): Hartung, J.; Li, W.;
• Abstract: Although the complete genome sequence of four strains of Xylella fastidiosa (Xf) has been determined, post-genomic research is just beginning. Defined mutants are required to exploit the genomic sequence data. A triparental mating system was employed to introduce a green fluorescent protein (GFP) labeled Mini-Tn5 transposon into the chromosomal DNA of a rifampicin resistant mutant of X. f. isolated from coffee. More than 200 putative transconjugants were obtained and confirmed by PCR using simultaneously two pairs of primers, one specific for X. f. and the other for GFP. The insertion sites of the GFP labeled transconjugants were identified using a semi-random, two-step PCR (ST-PCR) technique. Transconjugants with mutations in defined genes are being evaluated in vitro and also in planta. Conjugational matings with E. coli donors combined with our ST-PCR method to define transposon insertions in the recipient, will provide an efficient method to create defined mutants needed for the functional genomic analysis of X. f. GFP labeled mutants will also be useful for localizing the bacterium in infected plants.
• Publication Date: Jun 2003
• Journal: Phytopathology