NSF 20K Oligo Arrays to Dissect Rice Defense Response Pathways
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ABSTRACT: With the availability of a high quality draft rice genome sequence, large mutant collections, and gene expression oligo arrays for rice, we are now well positioned to dissect rice defense pathways. To do this, we performed global expression analyses to identify genes that are differentially expressed in 10 mutant lines (i.e., Xa21, NH1ox, NRR1ox, GR978, spl11, spl17, NBS2-PI9ox, MPK5ox, OsCoi1ox, and OsNahG1ox), exhibiting altered defense responses. As controls, we used wild typle varieties same with these mutants. Two-condition experiment, 10 mutants vs wild type control with treatment or without treatment. Biological replicates: 2-4 control, independently grown and harvested. Technical replicates: 1-2 control. One replicate per array.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) is a rice pathogen causing bacterial blight, which outbreaks in most rice cultivating countries and reduces yield up to 50% due to no effective pesticide. Urgent responses of Xoo upon the initial contacts with rice at infection site are essential for pathogenesis. We studied the time-resolved gene expression of both transcriptome and proteome in the pathogenicity-activated Xoo cells with an in vitro assay system. Genes related to cell mobility, inorganic ion transport and effectors are early response genes to help Xoo cells invade into damaged rice leaf tissues, obtain rare cofactors, and evade rice immune responses. Although the time-resolved gene expression pattern of Xoo is conserved in both mRNA and protein, there are varied time gaps in genes between the expression peaks of mRNA and protein, which implies there is an additional translational selection step of specific mRNAs for rapid translation. The expression pattern of genes from a polycistronic mRNA in the same gene cluster is strictly conserved. The time-resolved gene expression study of Xoo in both transcriptome and proteome provides a valuable information about the pathogenic responses of Xoo at the initial stage of Xoo-rice interaction.
Project description:Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight disease, is one of the major threats to rice productivity. Yet, the molecular mechanism of rice-Xoo interaction is elusive. Here, we report comparative proteome profiles of Xoo susceptible (Dongjin) and resistant (Hwayeong) cultivars of rice in response to two-time points (3 and 6 days) of Xoo infection. Low-abundance proteins were enriched using a protamine sulfate (PS) precipitation method and isolated proteins were quantified by a label-free quantitative analysis, leading to the identification of 3846 protein groups. Of these, 1128 proteins were significantly changed between mock and Xoo infected plants of Dongjin and Hwayeong cultivars. Based on the abundance pattern and functions of the identified proteins, a total of 23 candidate proteins were shortlisted that potentially participate in plant defense against Xoo in the resistant cultivar. Of these candidate proteins, a mitochondrial arginase-1 showed Hwayeong specific abundance and was significantly accumulated following Xoo inoculation. Overexpression of arginase-1 in susceptible rice cultivar (Dongjin) resulted in enhanced tolerance against Xoo as compared to the wild-type (WT). In addition, expression analysis of defense-related genes encoding PR1, glucanase I, and chitinase II by qRT-PCR showed their enhanced expression in the overexpression lines as compared to WT. Mitochondrial localization of the selected arginase was further confirmed by fluorescent microscopy using GFP-tagged arginase. Taken together, our results uncover the proteome changes in the rice cultivars and highlight the functions of arginase in plant defense against Xoo.
Project description:Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice (Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. Here, we examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some miRNAs and trans-acting siRNAs (ta-siRNAs) were identified, together with a few novel miRNA targets, including a disease resistance gene targeted by osa-miR159a.1. Coordinated expression changes were observed among some miRNA and ta-siRNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or functional correlated signaling pathways, including auxin and GA signaling pathways, nutrition and defense related pathways, etc. Highly abundant small RNAs with pathogen-responsive expression changes were identified from the exonic region of a protein-coding gene, which may present a new class of functional small RNAs. These findings reveal the dynamic and complex roles of small RNAs in rice-pathogen interactions, and identified new targets for regulating plant immune responses. Examination of the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99
Project description:In the present study, we found that Nipponbare and IAC1131 behave equivalently in response to multiple abiotic stress, which included simultaneous application of drought, salt and temperature stress. At the level of molecular analysis by isotopic labelling quantitative proteomics, 6215 proteins were reproducibly identified and quantified across the two genotypes and three time points sampled. Heat shock proteins, late embryogenesis abundant proteins, and photosynthesis-related proteins were all found to be differentially abundant in response to stress treatments in both Nipponbare and IAC1131.
Project description:Purpose -Comparison of proteome incubated in different media (rich and minimal media) -Three Xanthomonas species were used (Xanthomonas oryzae pv. oryaze, X. campestris pv. vesicatoria, and X. axonopodis pv. glycines. -Shotgun proteomic was used
Project description:Rice Xa21 resistance gene, which encodes a protein with predicted leucine-rich repeat (LRR), transmembrane, juxtamembrane, and intracellular kinase domains, conferred immunity to diverse strains of Xanthomonas oryzae pv. oryzae (Xoo). We generated Xa21 plant on TP309 background (Oryza Sativa Japonica). Systemic Acquired Resistance (SAR) in plants confers durable broad-spectrum resistance to pathogens and requires a phytohormone, salicylic acid (SA). Arabidopsis NPR1/NIM1 is a key regulator of the SAR response. Recently, we found that rice NPR1 homolog 1 (NH1) mediated enhanced resistance responses for Xoo (Chern et al., 2005b). We further investigated relating pathways in rice by identifying proteins that interact with NH1. One of them, constitutive over-expression of NH1 mediated negative regulator of resistance (NRR) gene caused enhanced susceptibility to Xoo , indicating that this gene product negatively affects to basal resistance response (Chern et al., 2005a). To dissect defense responses for rice bacterial blight pathogen, we planed microarray using two resistant mutant named with Xa21-TP309, NH1ox and one super-susceptible mutant (NRRox) before pathogen inoculation and one day post pathogen inoculation. Keywords: Biotic stress response Two or Three-condition experiment, NH1ox vs wild type control (LG) at two durations of Xoo inoculation (0d and 1d); NRRox vs wild type control (LG) at two durations of Xoo inoculation (0d and 1d); and Xa21vs wild type control (TP309) at three durations of Xoo inoculation (0d,1d and 2d);. Biological replicates: 2 or 4, independently grown and harvested.
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:High-throughput sequencing of small RNAs from rice was used to identify distinct miRNAs that are responsive to elicitors from the fungal pathogen Magnaporthe oryzae. [Expression profiling by array] We used microarrays to determine the expression behaviour of target genes for elicitor-regulated miRNAs. [High throughput sequencing] High-throughput sequencing of rice small RNAs was performed in two different tissues, leaves and roots, and two different time point of elicitor treatment, 30' and 2h Amplicons were prepared by 5M-BM-4and 3M-BM-4adaptor ligation in which the 5'-adaptor contained a 'barcode' consisting of a 4-nucleotide identifier sequence for each sample. The libraries containing unique barcodes were combined and subjected to pyrosequencing (454 Life SciencesTM, Roche) [Expression profiling by array] Leaves from rice plants were harvested at two time points after the onset of treatment (30' and 2h) with elicitors of Magnaporthe oryzae 18.1 and used for RNA extraction and hybridization on Affymetrix microarrays. Mock inoculations were performed with sterile water for control experiments. Three biological replicates were analyzed. Each sample represented a pool of approximately 150 rice plants. [High throughput sequencing] 8 samples examined: leaves and roots, treated or not with elicitors at two different time points, 30' and 2h (2x2x2)
Project description:Host-pathogen interactions, in particular during the early stages of infection, are important for the fate of both the invading pathogen and host. In pathogens, these interactions activate diverse pathogenic signals to cause disease in the host and evade the inevitably activated immune responses of the infected host. In this study, we used in vitro assay system and RNA-seq to analyze the time-resolved genome-wide gene expressions of Xanthomonas oryzae pv. oryzae (Xoo) evoked by the interactions with rice leaf extracts. RNA-seq analysis was carried out with samples of 7 different time points within 1 h. The RNA-seq data were verified by qRT-PCR. The in vitro system successfully initiated the pathogenic signals in Xoo cell culture. Many pathogenicity-related genes were expressed within 15 min. The hrpG gene was transcribed at maximum level within 10 min, and hrpX gene expression reached maximum level in 15 min. Chemotaxis and flagellar biosynthesis-related genes and cyclic-di-GMP synthesizing genes having GGDEF motif, were down-regulated until 10 min and after then up-regulated. Genes related to iron uptake and two component systems of phoB-phoR, phoP-phoQ, and pmrA-pmrB were up-regulated before 5 min. Data of multiple time points enabled non-linear regression fit of the time-resolved expression data, with which we newly developed the continuous time-resolved heat map to help the comparison of gene expression profiles. Essentiality of the transcriptionally up-regulated genes was also analyzed by the pathogenicity assay using the Xoo knockout strains, which showed not necessarily all the up-regulated genes were required for pathogenicity. the time-dependent, 0, 5, 10, 15, 30, 45, and 60 min, transcriptome analysis of genome-wide Xanthomonas oryzae pv. oryzae genes from the pathogenic interactions with RLX via RNA-Seq