geneontology.org. To establish if differentially expressed genes are located in the vicinity of
the IS elements in the genomes of Xoo African strain BAI3 and Xoo Asian strain MAFF311018, we selected a region of 20 kb that flanked the IS elements in both the MAI1 VX-689 and BAI3 genomes. BLAST searches were performed against these flanking sequences, using the Xoo MAI1 non-redundant set of sequences. For the sequences located within the 20-kb sequence flanking the IS elements, the relative distance of each sequence to the IS element was calculated and compared between the two genomes. Southern blot analysis of differentially expressed genes Southern blot analysis was used to confirm that the DNA fragments derived from individual clones were present in the initial tester (Xoo MAI1 strain) and absent in the driver DNA
(Xoo PXO86 or Xoc BLS256 strain). Eight genes (FI978063, FI978069, FI978079, FI978093, FI978109, FI978168, FI978197 and FI978322) were selected according C59 wnt to sequence similarities and library origin. Additionally, the gene FI978197 was selected to screen genomic DNA from different Xoo Asian strains (HN35, PXO339, PXO341, and PXO86), Xoo African strains (MAI1, BAI3, NAI8, and BAI4), Xoc African strains (MAI11 and MAI3), and the Xoc Asian strain BLS256 (Figure 2). Briefly, for each strain, 5 μg of genomic DNA was digested with 10 units of RsaI and run on 0.8% agarose gels. Casein kinase 1 The DNA was transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) by capillary transfer. The insert DNA was amplified by PCR, using the nested primers provided with the PCR-Select™ Bacterial Genome Subtraction Kit (Clontech Laboratories, Inc.). The amplified DNA fragment was gel purified, using the QIAquick Gel Extraction Kit (QIAGEN, Inc.), as recommended by the manufacturer. The DNA fragments were labelled with [α32P] dCTP by random priming (MegaPrime labelling kit, Amersham Biosciences). Conditions of hybridization
and washes were done at 65°C. Filters were washed with three solutions: the first of 2× SSC and 0.1% SDS for 20 min, followed by two washings with 1× SSC and 0.1% SDS for 10 min each, and a final wash with 0.1× SSC and 0.1% SDS for 20 min. Blots were exposed on a PhosphorImager (model Storm 860, Amersham Pharmacia Biotech Inc.-Molecular Dynamics Division, Piscataway, NJ, USA). Validation by quantitative QRT-PCR We selected 14 genes that had been differentially expressed at various time points during infection by Xoo MAI1 for confirmation by QRT-PCR. The primers for quantitative detection were designed, using the Beacon Designer™ software (PREMIER Biosoft International, Palo Alto, CA, USA) (Table 4). All experiments were performed in triplicate. PCR mixtures were prepared, using FullVelocity® SYBR® Green QPCR Master Mix (Stratagene).