ROLE OF UNIQUE GENES OF XYLELLA FASTIDIOSA GRAPE STRAIN IN HOST SPECIFICITY AND VIRULENCE TO GRAPE AND TO INSECT USING MICROARRAY Project Leader:

Xylella fastidiosa (Xf) is a group of genetically similar strains that infect a wide range of plants. We hypothesized that differing genetic factors among the strains determine the ability of a strain to infect a particular host plant. To better understand what makes grape a good host for all grape strains but not for strains such as oleander and almond that cannot colonize grape, we conducted experiments to look for host specific genes of the grape strain. Through our microarray and in silico genomic studies, we have so far identified 52 potential Xf grape strain virulence genes. We have constructs for knocking out 12 of the 52 identified genes. The genes we chose from our list were greater than 300 bp and were not part of a remnant phage. Our constructs have a Kanamycin gene inserted near the 5’ end of the gene for optimum efficiency in knocking out our gene and preventing Xf from making partial transcripts. We plan to inoculate plants with our knock-out mutants once they are confirmed. We noticed that the microarray studies have produced fewer genes than expected, indicating that the similarity between Xf ‘Temecula’ and other non-grape strains must be greater than expected. Our in silico comparisons revealed a high level of similarity as well. Because of this, we are now using dual labeling with our microarray studies. This is a more sensitive way to identify differences in gene sequence between the strains. INTRODUCTION Xf is a group of genetically similar strains that infect a wide range of plants. A particular strain often has a relatively reduced and distinct host range when compared to other strains. Some strains of Xf originating 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). This strongly suggests that differing genetic factors among the strains determine the ability of a strain to infect a particular host plant. Other studies provide evidence for host specificity among the Xf strains (Chen et al. 1992; Chen et al. 1995; Pooler and Hartung1995; Hendson et al. 2001; Bhattacharyya et al. 2002a, 2002b). For example, cross inoculations in greenhouse studies showed that the oleander and grape strains of Xf were not pathogenic to citrus and that the almond strain was not pathogenic to oleander (Feil et al. unpublished). In California, we have three identified groups of strains of Xf as designated by their host range; the grape strains, the almond strains, and the oleander strains. To better understand the underlying genetics of Xf as it relates to pathogenesis, several strains have been sequenced. Strain Xf ‘9a5c’, a citrus pathogen, was fully sequenced in Brazil (Simpson, 2000). The draft-genome sequences of the almond and oleander strains of Xf, ‘Dixon’ and ‘Ann1’, respectively, are also publicly available. We used this information to identify a list of genes present in the grape strain genome but missing in other strains that do not sustain viable colonies in grape. We used target DNA from non-grape strains for hybridizing to probes designed from the sequenced reference strain, Xf ‘Temecula’, which are affixed to epoxy slides. During this process, we determined that most strains are highly similar to each other and require a much more sensitive approach to identify genetic distinctions with the grape strain. That is, few genes are completely missing in non-grape strains compared to grape strains and vice versa. We are thus using a duel labeling approach where the reference strain and the target strain are labeled differentially and co-hybridized on the same array, and sequence differences are reveled by competitive hybridization. OBJECTIVES 1. Complete our initial work on host-specific gene identification using DNA/DNA microarray studies and to better define the role of 52 genes thus far identified as unique to the grape strain. 2. Produce knock-outs of the unique genes to the grape strain and to test for virulence of these knock-outs in grape. RESULTS Objective 1 To arrive at a list of 52 genes unique to the grape strain, we have hybridized DNA from six different strains to the DNA on the grape strain array. These strains were isolated from almond (two different isolates), oleander (two different isolates), oak, plum, olive, and maple. We also used two other grape isolates (‘STL’ and ‘Fetzer’) in our studies. Eighteen of these 52 genes are presented here (Table 1). There are 34 additional genes not listed that are either less than 300 bp in size or are