ANALYSIS OF XYLELLA FASTIDIOSA TRANSPOSON MUTANTS AND DEVELOPMENT OF PLASMID TRANSFORMATION VECTORS

• Author(s): Kirkpatrick, Bruce; Guilhabert, Magalie;
• Abstract: We screened over 1,000 random Tn5 Xylella fastidiosa (Xf) mutants in Chardonnay grapevines growing in the greenhouse in 2003. Approximately 10 of the mutants exhibited a hypervirulent phenotype, i.e. vines inoculated with these mutants developed symptoms sooner and died sooner than vines inoculated with the wild type Xf parental strain. The identity of the Tn5 insertion sites in these mutants was reported at 2003 PD Symposium. In 2004 we re-inoculated these hypervirulent mutants into another set of Chardonnay, Chenin blanc and Thompson seedless vines and the hypervirulent phenotype was reproduced in all 3 varieties. Movement and populations assays showed that the hypervirulent mutants moved faster and reached higher populations than wild type Xf. In the first Chardonnay screen, we identified an unexpectedly high number of avirulent mutants. Because some of these may have been the result of poor inoculation we sequenced the DNA that flanked the Tn5 insertion in all the mutants. Those mutants with Tn5 insertions in genes other than house keeping genes were reinoculated into a new set of vines and their pathogenic phenotype is being determined. Additional small (1.3kb) native Xf plasmids were engineered as potential Xf/E. coli shuttle vectors. However, like our other similar constructs, these plasmids were not stably maintained without antibiotic selection, and not useful tools for in planta gene complementation studies.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium