Next Generation Sequencing Facilitates Small RNA Mediated Rice Resistance to Xoo
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ABSTRACT: Purpose: The goal of this study is to identify small non-conding RNAs which are involved in rice resistance to Xoo. Methods: Rice leaves were inoculated with the Xoo strain PXO61 at the four-leaf to five-leaf stage by the leaf-clipping method. Control rice plants were inoculated with water (mock inoculation). And then, total RNA was extracted to be sequenced using Illumina GAIIx. Results: Using an optimized data analysis workflow to count the expression level of small ncRNA, we found several differentially expressed small ncRNA which may be participated in the interaction between rice and Xoo. Conclusions: Small ncRNA have be found to function in a variety of biological processes. Our study here has showed that several candidate miRNA or siRNA may play a significant role in rice immunity.
Project description:Purpose: The goal of this study is to identify small non-conding RNAs which are involved in rice resistance to Xoo. Methods: Rice leaves were inoculated with the Xoo strain PXO61 at the four-leaf to five-leaf stage by the leaf-clipping method. Control rice plants were inoculated with water (mock inoculation). And then, total RNA was extracted to be sequenced using Illumina GAIIx. Results: Using an optimized data analysis workflow to count the expression level of small ncRNA, we found several differentially expressed small ncRNA which may be participated in the interaction between rice and Xoo. Conclusions: Small ncRNA have be found to function in a variety of biological processes. Our study here has showed that several candidate miRNA or siRNA may play a significant role in rice immunity. Plants were inoculated with the Xoo strain PXO61 at the four-leaf to five-leaf stage by the leaf-clipping method. Control rice plants were inoculated with water (mock inoculation). Samples were collected before inoculation (ck) and at 2, 4, and 24 hours after PXO61 or mock inoculation from Rb49 and MDJ8. Leaf fragments approximately 2 cm in length that were immediately next to the inoculation site were collected.
Project description:Japonica rice (Oryza sativa ssp. japonica) variety Mudanjiang 8 (MDJ8) is the wild-type and is susceptible to Xoo. Transgenic rice line Rb49 carries the MR gene Xa3/Xa26, which is driven by its native promoter with the genetic background of MDJ8, and this line is resistant to certain strains (including strain PXO61) of Xoo. Although many studies on Xa3/Xa26-mediated resistace to rice Xoo have been published, the molecular mechanism of this major resistance gene remains poorly understood. Here, we use affymetrix microarray technology to analyze the regulated network mediated by Xa3/Xa26 We used microarrays to study the gene expression network mediated by Xa3/Xa26. Plants were inoculated with the Xoo strain PXO61 at the four-leaf to five-leaf stage by the leaf-clipping method. Control rice plants were inoculated with water (mock inoculation). Samples were collected before inoculation (ck) and at 2, 4, and 24 hours after PXO61 or mock inoculation from Rb49 and MDJ8. Leaf fragments approximately 2 cm in length that were immediately next to the inoculation site were collected.
Project description:affy_xoo_rice - affy_xoo_rice - The Bacterial Leaf Blight disease of rice is due to Xanthomonas oryzae pv. oryzae. As for many pathogenic bacteria, it relies on a type 3 secretion system that is devoted to the injection of type 3 effectors into the eukaryotic host cell. These proteins are meant to suppress host basal defense responses and/or mimic some host regulatory function promoting bacterial survey in the plant. We are interested in the functional analysis of a subgroup of Xoo T3Es, that are specialized in host cell transcriptome remodelling. These effectors, therefore called TAL for Transcription Activator-Like proteins (also named AvrBs3/PthA-like), are often key virulence factors essential to Xoo pathogenicity such as the effector protein Talc of african Xoo strain BAI3. Our goal is to understand its function during disease development, by identifying rice host genes that are being directly up- or down-regulated by Talc. To that end, we aim at performing Affymetrix transcriptomic analysis, comparing leaf samples of a susceptible rice line inoculated with Xoo to leaves challenged with a Talc-deficient mutant and water-treated leaves. Highly induced genes are likely to be Talc primary targets and therefore potentially good susceptibility gene candidates.-The goal of the experiment is to identify the rice genes up- or down-regulated by the type III effector Talc from Xoo African strain BAI3, upon the inoculation of susceptible rice leaves 24 hours post-infection. To that end, the experimental design includes the inoculation of Nipponbare rice leaves with the virulent Xoo strain BAI3, that will be compared to Nipponbare rice leaves inoculated with a talc K.O. mutant strain and water. Keywords: wt vs virulence mutant
Project description:An indica rice cultivar IET8585 (Ajaya), resists diverse races of the Xanthomonas oryzae pv oryzae (Xoo) pathogen attack, and is often cultivated as bacterial leaf blight (blb) resistant check in India. Earlier we reported a recessive blb resistance gene mapped to the long arm of chromosome 5 in IET8585. To further understand the mechanism of recessive and durable resistance response, two indica rice genotypes namely, i) IET8585 (Ajaya), a disease resistant indica veriety from India and ii) IR24, a bacterial leaf blight disease susceptible genotype were selected for this study. We used the 22K rice Oligoarray from Agilent technologies to study the transcript profile in the leaves of the two contrasting rice genotypes under inoculated and un-inoculated conditions during seedling stage. Keywords: Bacterial leaf blight disease resistance mechanism
Project description:affy_xoo_rice - affy_xoo_rice - The Bacterial Leaf Blight disease of rice is due to Xanthomonas oryzae pv. oryzae. As for many pathogenic bacteria, it relies on a type 3 secretion system that is devoted to the injection of type 3 effectors into the eukaryotic host cell. These proteins are meant to suppress host basal defense responses and/or mimic some host regulatory function promoting bacterial survey in the plant. We are interested in the functional analysis of a subgroup of Xoo T3Es, that are specialized in host cell transcriptome remodelling. These effectors, therefore called TAL for Transcription Activator-Like proteins (also named AvrBs3/PthA-like), are often key virulence factors essential to Xoo pathogenicity such as the effector protein Talc of african Xoo strain BAI3. Our goal is to understand its function during disease development, by identifying rice host genes that are being directly up- or down-regulated by Talc. To that end, we aim at performing Affymetrix transcriptomic analysis, comparing leaf samples of a susceptible rice line inoculated with Xoo to leaves challenged with a Talc-deficient mutant and water-treated leaves. Highly induced genes are likely to be Talc primary targets and therefore potentially good susceptibility gene candidates.-The goal of the experiment is to identify the rice genes up- or down-regulated by the type III effector Talc from Xoo African strain BAI3, upon the inoculation of susceptible rice leaves 24 hours post-infection. To that end, the experimental design includes the inoculation of Nipponbare rice leaves with the virulent Xoo strain BAI3, that will be compared to Nipponbare rice leaves inoculated with a talc K.O. mutant strain and water. Keywords: wt vs virulence mutant 9 arrays - rice
Project description:ABA deficient mutant Osaba1-1 exhibits great resistance to Xanthomonas oryzae pv. oryzae (Xoo) infection. To investigate gene expression profile changes at whole genome level between Osaba1-1 and wild-type (Nipponbare) rice during Xoo infection, we employed microarray expression profiling as a discovery platform. Osaba1-1 and wild-type rice plants about 6.5 leaf stage were used in this experiment. For Xoo inoculation, tips (about 3cm) of the fifth and sixth fully expanded leaves were cut off, and then immersed into Xoo (PXO99 strain) inoculum (suspended in sterile distilled water containing 10mM MgCl2, OD≈0.5) immediately for about fifteen seconds. Inoculated rice leaves were collected (approximately 2 cm leaf fragment from the inoculation site) at 0h and 72h post inoculation. Three independent replicate samples were collected at each time point for microarray.
Project description:Lipopolysaccharide is a Microbe Associated Molecular Pattern (MAMP) that is known to induce defense responses in plants. In rice we have shown that Xoo LPS induce callose deposition, reactive oxygen production and induced resistance response. The exopolysaccaride (EPS) secreted by Xoo might be involved in supressing these defense responses. We have performed transcriptional profiling of rice leaf gene expression changes after treatment with Xoo strains BXO1003 (LPS-, EPS-), BXO1002 (LPS+ EPS-) and BXO43 (wild type) along with milliQ treated leaves to identify the genes that are differentially expressed.
Project description:An indica rice cultivar IET8585 (Ajaya), resists diverse races of the Xanthomonas oryzae pv oryzae (Xoo) pathogen attack, and is often cultivated as bacterial leaf blight (blb) resistant check in India. Earlier we reported a recessive blb resistance gene mapped to the long arm of chromosome 5 in IET8585. To further understand the mechanism of recessive and durable resistance response, two indica rice genotypes namely, i) IET8585 (Ajaya), a disease resistant indica veriety from India and ii) IR24, a bacterial leaf blight disease susceptible genotype were selected for this study. We used the 22K rice Oligoarray from Agilent technologies to study the transcript profile in the leaves of the two contrasting rice genotypes under inoculated and un-inoculated conditions during seedling stage. Experiment Overall Design: We used Agilent rice gene chips (G4138A) to investigate the transcript level changes in rice leaf tissues during bacterial pathogen infection. We used two contrasting rice genotypes (IET8585 (Ajaya) blb resistant IR24 blb susceptible) differing in bacterial disease response. Plants were grown growth chambers and inoculated with bacterial pathogen on 18th DAS. Leaf sampling was done in both un-inoculated and inoculated plants at 3 time points. Two replications of microarray experiments were carried out by hybridizing the resistant samples against the susceptible samples.
Project description:The plant cell wall degrading enzyme LipA (Lipase/Esterase A) is a Type II secretion system secreted protein of Xanthomonas oryzae pv. oryzae (Xoo; the casual of bacterial leaf blight of rice). LipA is an Xoo virulence factor. However, LipA is a double edged sword for Xoo as it induces rice defense responses such as programmed cell death/hypersensitive response like reaction (HR) and callose deposition. Prior treatment with LipA enhances resistance against subseqent Xoo infection. In order to understand the molecular events associated with Esterase (LipA) induced innate immune responsein rice , whole genome transcriptional profiling was performed using Affymetrix Rice GeneChips
Project description:Lipopolysaccharide is a Microbe Associated Molecular Pattern (MAMP) that is known to induce defense responses in plants. In rice we have shown that Xoo LPS induce callose deposition, reactive oxygen production and induced resistance response. The exopolysaccaride (EPS) secreted by Xoo might be involved in supressing these defense responses. We have performed transcriptional profiling of rice leaf gene expression changes after treatment with Xoo strains BXO1003 (LPS-, EPS-), BXO1002 (LPS+ EPS-) and BXO43 (wild type) along with milliQ treated leaves to identify the genes that are differentially expressed. RNA was isolated from mid veins of rice leaves 15 hours after injecting them with Xoo strains BXO1003 (LPS-, EPS-), BXO1002 (EPS-), BXO43 (wild type) or milli-Q water. The rice gene expression in each of the treatment was normalized based on the gene expression in the milli-Q treatment.