Transcriptome Profiling of Xanthomonas oryzae pv. oryzae knockout mutants at different hybridization conditions and PMTs

Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are important bacterial pathogens of the worldwide staple and grass model, rice. Xoo invades rice vascular tissue to cause bacterial leaf blight, a serious disease of rice throughout the world. Xoc colonizes the parenchyma tissue to cause bacterial leaf steak, a disease of emerging importance. We have designed oligonucleotide probes (50-70-mers) represented 2,858 Xoo genes and 1,816 Xoc genes annotated by The Institute for Genomic Research (TIGR). To validate the Xo arrays, self-hybridization samples and tests of the non-specific hybridization using randomly spotted oligonucleotides corresponding to the hygromycin phosphotransferase gene (hph), and blank spot and of the correlation coefficient between biological replicates as well as between duplicate spots revealed that the data generated from our oligo array were highly reliable and consistent. To optimize the suitable protocol for hybridizing sample onto XOarray slides, we performed hybridization with 4 temperature levels (42 0C, 44 0C, 48 0C, and 52 0C) and 5 numbers of template amounts (10 pMol, 20 pMol, 30 pMol, 40 pMol, and 50 pMol) for hybridization process. Two level of PMT (Power of the scanner photomultiplicator) exposed to hybridized glass slides. Total samples is 36 slides (4 temperatures x 2 technical replicates x 2 PMT level = 16 slides and 5 numbers of template amount x 2 technical replicates x 2 PMT level = 20 slides). Keywords: Condition Optimization Two different Xanthomonas oryzae pv. oryzae knockout mutant strains, phoP and rpfC, were cultured ion PSB media. 2 technical replicates. 4 sets for hybridization temperature optimization. 5 sets for template amount optimization. Every experimental set slides were scanned with two different PMT level, highPMT and lowPMT.