A two-genome microarray for the rice pathogens Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola and its use in the discovery of a difference in their regulation of hrp genes

<p>Abstract</p> <p>Background</p> <p><it>Xanthomonas oryzae </it>pv. <it>oryzae </it>(<it>Xoo</it>) and <it>X. oryzae </it>pv. <it>oryzicola </it>(<it>Xoc</it>) are bacterial pathogens of the worldwide staple and grass model, rice. <it>Xoo </it>and <it>Xoc </it>are closely related but <it>Xoo </it>invades rice vascular tissue to cause bacterial leaf blight, a serious disease of rice in many parts of the world, and <it>Xoc </it>colonizes the mesophyll parenchyma to cause bacterial leaf streak, a disease of emerging importance. Both pathogens depend on <it>hrp </it>genes for type III secretion to infect their host. We constructed a 50–70 mer oligonucleotide microarray based on available genome data for <it>Xoo </it>and <it>Xoc </it>and compared gene expression in <it>Xoo </it>strains PXO99^Aand <it>Xoc </it>strain BLS256 grown in the rich medium PSB vs. XOM2, a minimal medium previously reported to induce <it>hrp </it>genes in <it>Xoo </it>strain T7174.</p> <p>Results</p> <p>Three biological replicates of the microarray experiment to compare global gene expression in representative strains of <it>Xoo </it>and <it>Xoc </it>grown in PSB vs. XOM2 were carried out. The non-specific error rate and the correlation coefficients across biological replicates and among duplicate spots revealed that the microarray data were robust. 247 genes of <it>Xoo </it>and 39 genes of <it>Xoc </it>were differentially expressed in the two media with a false discovery rate of 5% and with a minimum fold-change of 1.75. Semi-quantitative-RT-PCR assays confirmed differential expression of each of 16 genes each for <it>Xoo </it>and <it>Xoc </it>selected for validation. The differentially expressed genes represent 17 functional categories.</p> <p>Conclusion</p> <p>We describe here the construction and validation of a two-genome microarray for the two pathovars of <it>X. oryzae</it>. Microarray analysis revealed that using representative strains, a greater number of <it>Xoo </it>genes than <it>Xoc </it>genes are differentially expressed in XOM2 relative to PSB, and that these include <it>hrp </it>genes and other genes important in interactions with rice. An exception was the <it>rax </it>genes, which are required for production of the host resistance elicitor AvrXa21, and which were expressed constitutively in both pathovars.</p

Assessing probe-specific dye and slide biases in two-color microarray data

A primary reason for using two-color microarrays is that the use of two samples labeled with different dyes on the same slide, that bind to probes on the same spot, is supposed to adjust for many factors that introduce noise and errors into the analysis. Most users assume that any differences between the dyes can be adjusted out by standard methods of normalization, so that measures such as log ratios on the same slide are reliable measures of comparative expression. However, even after the normalization, there are still probe specific dye and slide variation among the data. We define a method to quantify the amount of the dye-by-probe and slide-by-probe interaction. This serves as a diagnostic, both visual and numeric, of the existence of probe-specific dye bias. We show how this improved the performance of two-color array analysis for arrays for genomic analysis of biological samples ranging from rice to human tissue.We develop a procedure for quantifying the extent of probe-specific dye and slide bias in two-color microarrays. The primary output is a graphical diagnostic of the extent of the bias which called ECDF (Empirical Cumulative Distribution Function), though numerical results are also obtained.We show that the dye and slide biases were high for human and rice genomic arrays in two gene expression facilities, even after the standard intensity-based normalization, and describe how this diagnostic allowed the problems causing the probe-specific bias to be addressed, and resulted in important improvements in performance. The R package LMGene which contains the method described in this paper has been available to download from Bioconductor

Identification and Functional Analysis of Light-Responsive Unique Genes and Gene Family Members in Rice

Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families

Refinement of Light-Responsive Transcript Lists Using Rice Oligonucleotide Arrays: Evaluation of Gene-Redundancy

Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org). The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics

A Role of Cellulose Binding Module of the Thermophilic Endoglucanase TbCel12A

Endoglucanases are enzymes that play an important role in hydrolysis of lignocellulose by attacking glycosidic linkages in cellulose fibers and other glucans. The cellulose binding module (CBM) is responsible for binding the enzyme to the substrate. However, CBMs in certain enzymes interfere with substrate hydrolysis resulting in moderate or low activity. In a previous study, the processive endoglucanase TbCel12A including its CBM had low activity towards carboxymethyl cellulose (CMC). To assess the effect of the CBM, the catalytic domain of TbCel12A was produced without the CBM. The TbCel12A catalytic domain without the CBM hydrolyzed CMC 23 times more rapidly, while the pH and temperature optima and thermotolerance remained unchanged compared to full-length TbCel12A. Therefore, TbCel12A does not require the CBM for CMC hydrolysis and its application may be improved without it

Characterization of Diluted-acid Pretreatment of Water Hyacinth

Water hyacinth (WH) is an abundant renewable lignocellulosic biomass. The renewable resources are widely studied to produce bioenergy and high value added products. The obstacle of converting the lignocellulosic biomass to products due to its recalcitrant structure, required disruption to enhance the chemical or enzyme accessibility. The aim of this study is to investigate the possibility of WH pretreatment with dilute sulfuric acid (DSA). The addition of sulfuric acid in pretreatment process was varied concentration at 1, 2, 3, and 4% (v/v). The chemical component was analyzed to optimize the pretreatment condition. The results showed that the 2% sulfuric acid had effect on cellulose recovery. The morphological changes of WH due to pretreatment were determined by Scanning Electron Microscopy (SEM). The images showed the destructive surface of all treated samples. The intact surface of native WH was destroyed after pretreatment process while the increment of acid concentration increased the rough surfaces. The Fourier Transform Infrared Spectrometer (FTIR) and X-ray diffraction (XRD) were used for analyzing functional groups and crystallinity, respectively. The FTIR patterns of DSA treated WH were slightly different due to the remained components in samples. The results showed the highest crystallinity index was 55% which was obtained from pretreated WH with 2% sulfuric acid, 80°C, 60 min. In the present study, it was found that DSA pretreatment is possible to modify the chemical structure of WH for developing economical processes

Comparative evaluation of DNA extraction from rice’s root-associated bacterial consortium for population structure study

Understanding in population structure of a plant’s root-associated microbiome is applied in good practices in agricultural activities to improve production yields and enhance plant immune responses. The molecular analysis of bacterial populations inhabited in soil faces difficulties in obtaining high yield and high purity of DNA, and different commercial DNA extraction kits have been developed for this purpose. This study focuses on the comparison of DNA extraction of six different rice root-associated bacterial consortium using three commercial kits with two key technologies, spin-column adsorption and magnetic bead adsorption. The quality and quantity of genomic DNA obtained from these extraction methods were analyzed and compared based on DNA concentration, DNA purity and efficiency to be used as a template for 16sRNA amplification. The results showed that the extraction kit with magnetic bead adsorption technology showed the highest concentration (101.32 ng/μl) compared to other DNA extraction kits (32.67 and 1.89 ng/μl). The purity values (A260/A280) were assessed by using Nano-drop spectrophotometer and resulted in purities of nucleic acids in the range of 1.4-1.7. Thus, it was concluded that the extracted DNA obtained from the extraction kit with magnetic bead adsorption technology can be valuable for molecular analysis of microbial communities present in the soil

Centella asiatica Extract Loaded BSA Nanoparticles Using the Organic and Conventional C. asiatica to Improve Bioavailability Activity and Drug Delivery System

Centella asiatica (CA) extracts have been described for their high phytochemical contents, especially phenolic compounds. Active extracts also showed effectively potential in vitro but not in vivo experiments due to their poor lipid solubility or inappropraietd molecular weight, which resulted in poor bioavailability. This study, the nanoencapsulation process is applied to enhance bioavailability, stability and bioactivity of CA extracts. BSA (Bovine serum albumin) nanoparticles containing phenolic extracts of CA were synthesized by an adapted desolvation method at the ratio between CA extract: BSA at 1:2, 1:3 and 1:4. The entrapment efficiency, loading efficiency, solubility and stability are used to test the efficiency of the nanoparticles. The in vitro released kinetic is monitored for 6-hour period in both artificial gastric buffer at pH 2.0 and intestinal juice buffer at pH 7.4. The result showed that the different ratio of the CA concentrations to BSA nanoparticles had no significant effect to its bioavailability (p &lt; 0.05). On the other hand, the types of extraction solvents including ethanol, chloroform and hexane significantly affected the level of bioavailability (p &lt; 0.05). Especially, the ethanol extracts loaded in BSA at ratio of 1:2 showed the best result, and it was the most economical way due to less consumption of BSA nanoparticle was used. The study of CA extracts loaded in BSA nanoparticles here demonstated the improvement of bioavailability and drug delivery system

A two-genome microarray for the rice pathogens Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola and its use in the discovery of a difference in their regulation of hrp genes.

Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) are bacterial pathogens of the worldwide staple and grass model, rice. Xoo and Xoc are closely related but Xoo invades rice vascular tissue to cause bacterial leaf blight, a serious disease of rice in many parts of the world, and Xoc colonizes the mesophyll parenchyma to cause bacterial leaf streak, a disease of emerging importance. Both pathogens depend on hrp genes for type III secretion to infect their host. We constructed a 50-70 mer oligonucleotide microarray based on available genome data for Xoo and Xoc and compared gene expression in Xoo strains PXO99A and Xoc strain BLS256 grown in the rich medium PSB vs. XOM2, a minimal medium previously reported to induce hrp genes in Xoo strain T7174.Three biological replicates of the microarray experiment to compare global gene expression in representative strains of Xoo and Xoc grown in PSB vs. XOM2 were carried out. The non-specific error rate and the correlation coefficients across biological replicates and among duplicate spots revealed that the microarray data were robust. 247 genes of Xoo and 39 genes of Xoc were differentially expressed in the two media with a false discovery rate of 5% and with a minimum fold-change of 1.75. Semi-quantitative-RT-PCR assays confirmed differential expression of each of 16 genes each for Xoo and Xoc selected for validation. The differentially expressed genes represent 17 functional categories.We describe here the construction and validation of a two-genome microarray for the two pathovars of X. oryzae. Microarray analysis revealed that using representative strains, a greater number of Xoo genes than Xoc genes are differentially expressed in XOM2 relative to PSB, and that these include hrp genes and other genes important in interactions with rice. An exception was the rax genes, which are required for production of the host resistance elicitor AvrXa21, and which were expressed constitutively in both pathovars