Agricultural Microbes Genome 2 Conference Agricultural Microbes Genome 2 Conference
Poster: Genetic, Mutational, and Biochemical Technology
P06_02.html
Pierce's disease (PD), a lethal disease of grapevine, is caused by the bacterium Xylella fastidiosa, a gram-negative, xylem-limited bacterium, transmitted from plant to plant by xylem sap-feeding insects known as sharpshooters. Strains of Xylella fastidiosa (Xf) have also been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. In order to better understand the complex interactions between the plant, pathogen, and insect vectors it is necessary to identify genes which mediate these processes. The complete genome sequence of Xf has recently been determined (A.D.J. Simpson et al., Nature 406:151-157 (2000)), but a lack of vectors and genetic systems for Xf has hampered progress in understanding its phytopathology. We attempted to use transposon technologies to create mutants that have lost their pathogenicity or their ability to be transmitted by insect vectors. We tested four different suicide plasmid vectors, carrying either Tn5 or Tn10 transposons. The four suicide vectors failed to produce any transposed clones expressing antibiotic resistance. However, the electroporation of a hyperactive Tn5 transposase-transposon complex (Transposome) yielded 6 x 10^3 and 4 x 10^3 colonies per microgram of DNA, respectively, in two grapevine strains of Xf, Fetzer and Temecula. The transposon insertions were single, independent and stable events. DNA sequencing from the ends of inserted transposons in the genomic DNA of individual mutants will be used to directly localize the inserts. The availability of the complete sequence of Xf will facilitate the correlation of mutant phenotypes and specific genes.