DETERMINATION OF GENES CONFERRING HOST SPECIFICITY IN GRAPE STRAINS OF XYLELLA FASTIDIOSA USING WHOLE-GENOMIC DNA MICROARRAYS

Xylella fastidiosa (Xf) has many plant hosts and causes serious diseases of several crops and ornamentals. Strains of Xf can be classified by the hosts that may be infected. For example, grape strains do not infect oleander and the oleander strains do not infect grape. We are using a DNA Oligo-Microarray based on the genomic sequence of the Xf grape strain ‘Temecula ’ as the reference strain for a genome-wide comparison with DNA from non-virulent strains. Our approach will determine genes unique to grape strains and thus presumably important in growth and virulence of Xf in grape. We hypothesized that the grape strain possesses several unique genes in comparison to other strains that do not infect grape. Initially 2526 of the 2574 predicted ORFs of Xf ‘Temecula ’ were designed using the “pick70 ” software. We manually designed 70-mers oligos for 23 additional ORFs using the same criteria as the program. The remaining ORFs for which oligos were not designed had paralogs elsewhere in the genome with up to 100 % identity. Test arrays have been made to determine optimal concentrations of spotted oligos (probes) using a subset of either four or eight probes. Optimal signal intensity was found for a probe concentration of 15-25 nM/ml. All eight probes tested hybridized with labeled DNA from both the Xf grape strain ‘Temecula ’ and oleander strain ‘Ann’. This indicated that the 8 hypothetical small genes used for the test array were conserved amongst these two genomes. Several quality control tests are underway before we use the full array. The full array includes 2551 70-mer oligos representing the full genome of the Xf grape strain ‘Temecula’. These oligos were generated with a 5 ’ amino linker that allows for covalent binding to aldehyde or epoxy coated slides, therefore minimizing the background