BLAST searches were performed locally, using the MAI1 differentially expressed genes. For the sequences located within the 20-kb sequence flanking the IS elements, the relative distance of each sequence to the IS element in BAI3 was compared with the relative distance of their respective homologues in the Xoo MAFF311018 genome. The designation + indicates upstream location of the sequence relative to the IS element, and the designation – indicates downstream location. For check details IS elements, gene locations within the 20-kb sequence flanking the IS element in
BAI3 and within the genome of Xoo MAFF311018 are presented. Results showed that homologues of the 11 selected Xoo MAI1 differentially expressed genes are located in the vicinity of IS elements in BAI3 genome, within the same 20-kb region (Table 3). In the Xoo MAFF311018 genome, Xoo MAI1 differentially expressed genes are not located in a vicinity of 20 kb of the IS elements. Given that the African Xoo strain BAI3 is more closely related to Xoo MAI1 than Xoo MAFF311018, a similar organization of IS elements and presence of neighbour genes are expected for MAI1. Correlation between differential
expression Selleckchem PF-6463922 of IS elements, genome location, and role played in the control of expression of nearby genes in African Xoo strains need further study. Validation of differentially expressed genes, using QRT-PCR To validate the Xoo MAI1 microarray results, QRT-PCR was performed on a set of 14 genes of different functions and which were up- or down-regulated during infection. Table 4 lists the primers, putative function, and average fold-change expression of genes used for QRT-PCR validation. The genes selected for QRT-PCR correspond to four hypothetical proteins (FI978067, FI978252, FI978305, and FI978328), one gene showing no similarity to known proteins (FI978310), IMP dehydrogenase two putative transposases (FI978288 and FI978099), two genes related to transport and motility (FI978259 and FI978319), one hrpF gene (FI978263), and one avirulence protein from the AvrBs3/pthA family (F1978282), the avr/pth14 gene (M1P3I15),
the xopX gene (ACD57163), and the avrXa7 gene (AF275267). Figure 4 shows five genes out of the 14 tested that were up-regulated by QRT-PCR and having a larger than 4-fold change. Of the 14 genes selected according to the microarray data (Table 4), 13 were up-regulated and 1 (F1978067) was down-regulated. The QRT-PCR results supported these data, and also showed that the gene expression pattern was GF120918 cost identical for all genes tested, except two (FI978259 and FI978319). Gene expression values, however, differed between microarrays and QRT-PCR. As shown in Figure 4, the expression values for the five genes FI978252, FI978263, FI978328, AF275267, and ACD57163 were higher in QRT-PCR than for microarray, indicating that QRT-PCR may be more sensitive than microarray analysis.