Rapid identification of insertion sites of green florescent protein: Labeled mutants of Xylella fastidiosa

• Author(s): Hartung, J.; Li, W.; Qin, X.;
• Abstract: Xylella fastidiosa (Xf), an emerging plant pathogenic bacterium, causes several of the most significant plant diseases in the Americas. Little is known about the basis of pathogenicity, and what is known suggests that the interactions of this bacterium with its hosts are unique. Four strains of the bacterium, isolated from different plant hosts, have had their genomes totally sequenced. Approximately 3,000 open reading frames (ORFs) have been identified in each genome, and approximately half of the ORFs have no predicted function. Some ORFs have been identified as possibly encoding factors that the pathogen uses to cause disease in plants, but little work has been done on its virulence or pathogenicity. Systems for genetic analysis of the bacterium have been recently developed. However, there has not been a good method to precisely identify mutations in a large number of strains. We report the successful use, of a semi-random, two-step PCR (ST-PCR) technique to localize a series of GFP-labeled insertions in X. fastidiosa. We determined rapidly and easily the insertion sites of mini-Tn5 GFP transposons in our collection of X. fastidiosa mutants obtained from a wild strain isolated from citrus. This shows that the ST-PCR method will be a reliable, rapid and simple method to identify insertion sites in X. fastidiosa mutants on a large scale. The transposons were randomly inserted into the whole genome and were stable in vitro and in planta. The mutants were found to have a single transposon insertion, most often in a gene coding a hypothetical protein. One had an insertion in the gene encoding ribonuclease G. None of the genes mutated to date are essential to virulence or pathogenicity of the bacterium. This was verified by inoculating the mutants into Chardonnay grapevines, a very susceptible experimental host. Our results indicate that it will be possible to make a random library of GFP-labeled mutants and define their insertion sites prior to in planta studies. This will be very useful to functional genomic studies on X. fastidiosa.
• Publication Date: Jan 2003
• Journal: Abstracts Of The General Meeting Of The American Society For Microbiology