Summary: We report about the effects of temperature increase on rice response to Xanthomonas oryzae pv. oryzae using high throughput sequencing (RNA-Seq). The time course transcriptomic analysis revealed that temperature enhanced IRBB67 resistance to combined heat and Xoo. Our findings highlight altered cellular compartment by Xoo and heat stress in both susceptible (IR24) and the resistant (IRBB67). Interestingly, up-regulation of trehalose-6-phosphatase gene and low affinity cation transporter in IRBB67 suggest that IRBB67 maintained a certain homeostasis under high temperature to have its resistance enhanced. The interplay of both heat stress and Xoo responses as determined by up-regulated and down-regulated genes demonstrates how resistant plant cope with combined biotic and abiotic stresses. This study provides an understanding of how IRBB67 mediated resistance to Xoo under temperature get insight to cross talk in abiotic and biotic stress regulatory pathway.
Overall Design: Leaf mRNA profile of 21 days old plants of IR24 (susceptible) and IRBB67 (resistant) inoculated with Xanthomonas oryzae pv. oryzae strain PXO145 under two temperature regimes. Samples were collected at three time points (3hours post inoculation, hpi; 72 hpi and 120 hpi).
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Growth Protocol: | Rice genotypes IR24 (susceptible), and IRBB67 (Xa4+Xa7) seeds were pre-germinated for 4 days at 37°C and transferred in pot for further growth under greenhouse conditions (12h light and 12h dark). Two weeks old healthy plants were then transferred into indoor growth chambers under two temperature regimes (29/21 °C and 35/31 °C; day/night temperatures) and 70% of relative humidity and inoculated at 21 days old. Philippines Xoo race 7, strain PXO145 (avrXa4+avrXa7) inoculums was prepared from 3 days old culture. Seedling of IR24 and IRBB67 at 21 days old were syringe needless inoculated with PXO145 and sterilized demineralized water (Mock) for RNA-Seq gene expression analysis. Leaf at the second position of the main tiller was inoculated and leaves were sampled at 3, 72 and 120 hours post inoculation (hpi). A pulled leaves from 5 plants for each rice genotype treatment at each time point and immediately frozen in liquid nitrogen, and stored at -80°C until total RNA extraction. |
Treatment Protocol: | Twenty-one day-old seedlings of IR24, IRBB4, IRBB7 and IRBB67 were inoculated by the leaf clipping method78 while IR24 and IRBB67 plants were inoculated at 3 points of infiltration using syringe needless with PXO145 and with sterilized demineralized water (mock). |
Extract Protocol: | Total RNA was extracted from syringe needless inoculated leaves using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer protocol. Total RNA was then treated with DNase (Promega) and quantified using NanoDrop. RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). For each time point and condition, two biological replicates and cleaned total RNA. |
Library Construction Protocol: | Libraries were prepared according to Illumina's instructions. Briefly, Single-end fragment library of 100bp length was generated from cleaned total RNA following the TruSeq RNA Sample Preparation kit. Cluster generation of the produced libraries was performed using Illumina_ TruSeq SR Cluster Kit v3 - cBot - HS, and sequenced on a HiSeq 2000 platform (Illumina) with single-end 100-bp reads. |
Molecule Type: | poly(A)+ RNA |
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Library Layout: | SINGLE |
Library Strand: | - |
Platform: | ILLUMINA |
Instrument Model: | Illumina HiSeq 2000 |
Strand-Specific: | Unspecific |
Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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