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

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. INTRODUCTION Some strains of Xf isolated from host plants other than grape do not sustain viable populations or are not virulent in grape. In particular, many of the almond strains of Xf do not infect grape (Almeida and Purcell 2003). Other studies provide evidence for host specificity among the Xf strains. On a whole genome level, grape strains of Xf were found to cluster together away from oak, plum, mulberry, and periwinkle strains using RFLP data (Chen et al. 1992, Chen et al. 1995). Pooler and Hartung (1995) divided the Xf in 5 groups (citrus, plum, grape-ragweed, almond, and mulberry) based on RAPD-PCR data. Most almond strains are genetically distinct from the grape strains but a few clustered within the grape-strain group whereas oleander, peach, and oak strains were distinct from other strains using RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis (Hendson et al. 2001). Reciprocal inoculation studies in the greenhouse showed that the OLS and PD strains of Xf were not pathogenic to citrus and that the ALS strain was not pathogenic to oleander (Feil et al. unpublished). Based on previous analysis, we estimate that ~4% of the whole genome of the oleander strain is unique to that strain. We hypothesized that the grape strain also possesses ~4% of unique genes in comparison to other strains that do not infect grape. To identify these genes, we will use the grape strain ‘Temecula’ as a reference to perform pairwise comparison experiments via DNA hybridization using each Xf strain that is non-pathogenic to grape. By comparing a large number of strains that both colonize and cause symptoms in grape as well as strains that do not colonize grape we should be able to identify a relatively small number of unique genes that contribute to the virulence of grape by Xf. OBJECTIVES 1. Identify host-specific virulence determinants of the Xf grape strain ‘Temecula1a’. 2. Investigate the role of these specific genes in virulence. RESULTS Strains and Strategy of Screening 70-mer oligodeoxynucleotides were designed using ‘ArrayOligoSelector’ (‘Pick70’) software (http://arrayoligosel.sourceforge.net) based on the coding sequence of 2526 of the 2574 predicted ORFs of Xf ‘Temecula1’. An additional 23 oligos were manually designed from the remaining unrepresented ORFs using the same criteria as ‘Pick70’, except that sequence 5’ or 3’ of ORFs smaller than 70 bases was added to obtain an oligo of the correct size. The remaining 25 ORFs are represented by paralogs with 100% identity found elsewhere in the genome. The designed oligos were generated with a 5’ amino linker that has allowed for covalent binding to aldehyde or epoxy coated slides. The Final number of ORFs represented by gene-specific oligodeoxynucleotides on the arrays is 2551 not including negative and positive