NSF 20K Oligo Arrays to Dissect Rice Defense Response Pathways
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: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:affy_riz_2011_7 - affy_riz_2011_7 - 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 (TTSS) that is devoted to the injection of type 3 effectors (T3Es) 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. During an incompatible interaction, T3Es may act as Avr proteins and stimulate Effector-Triggered-Immunity. We aim at evaluating the transcriptomic response of rice leaves challenged with avirulent strains of Xoo BAI3 and MAI1 on resistant lines IR64 and IRBB4 versus the reference susceptible rice line Nipponbare. In addition, we investigated the transcriptomic response of rice leaves upon inoculation of an XoohrcC mutant strain affected in the production of a functional TTSS.-The goal of the experiment is to characterize the rice leaf transcriptome response, upon the inoculation of susceptible and resistant rice leaves 24 hours post-infection. To that end, the experimental design includes the inoculation of susceptible Nipponbare rice leaves with Xoo strains BAI3 (race A1) and MAI1 (race A3), that will be compared to the response of resistant lines IRBB4 and IR64 rice lines. In addition, Nipponbare rice leaves will also be challenged with the BAI3hrcC mutant that is affected in the production of a functional TTSS. 18 arrays - rice; avirulent vs virulent
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: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 5´and 3´adaptor 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: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: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
Project description:Analysis of transgenic rice overexpressing OsWRKY28, a WRKY type transcription factor. Results provide insight into the role of OsWRKY28 in the defense signaling against rice blast fungus. Expression profiling in wild-type and OsWRKY28 overexpressing rice leaves infected with or without Magnaporthe Oryzae was analyzed using one-color method with three biological replicates.
Project description:Three week old seedlings of rice cultivar Jumli Marshi (spp. Japonica) was exposed to cold (+4C) temperatures for varying time periods. Tissue samples were collected in replicates. Microarray data analysis was then performed on the extracted totalRNA from the samples.
Project description:Analysis of transgenic rice plants overexpressing the rice WRKY transcription factor OsWRKY53 or its phospho-mimicking mutant (OsWRKY53SD). Results provide insight into the roles of OsWRKY53 and its phosphorylation in the basal defense signaling against the rice blast fungus. Expression profiling in wild-type, OsWRKY53- or its phospho-mimicking mutant-overexpressing rice leaves infected with or without Magnaporthe Oryzae was analyzed using one-color method with four biological replicates.
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