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Transcriptome profiling of resistant NIL (PB1+Pi9) and susceptible rice line (PB1) 24 hours after M oryzae inoculation

ABSTRACT: Purpose: The aim of this study was to compare transcriptome profiling (RNA-seq) of rice leaf tissue infected with M.oryzae of resistant and suceptible line. The results were validated using real time PCR. Methods: Rice leaves were collected 24 hours after M. Oryza (Mo-nwi-53) infection from resistant and suceptible line, frozen on liquid nitrogen, and RNA was extracted using Spectrum™ Plant Total RNA Kit (Sigma). RNA libraries were prepared for sequencing using standard Illumina protocols. Results: Around 30 million pairs of filtered 101 base paired reads were obtained for each biological replicate. These reads were mapped against RGAP7 using Tophat and approximately 90% aligned to the RGAP 7 while 10% reads were unaligned. Approximately 1-2 million reads returned multiple hits to the RGAP7. After alignment mapped reads were assembled using Cufflink and differentially expressed locus (DEL) were called using Cuffdiff. Statistical test was applied to find significant differentially expressed locus id after multiple corrections (FDR adjusted p value ≤0.05). We obtained 8,418 and 3336 significant (FDR adjusted p value ≤0.05) differentially expressed locus id in the resistant and susceptible rice line, respectively . The number of significant DEL with fold change value greater and les than 2.0 were 1254 (850 upregulated 404 downregulated) and 781 (466 upregulated & 313 downregulated)in in resistance and susceptible lines, respectively. The final data represents the transcriptome of both resistant and susceptible line after mock and M.oryzae infection. The pathway and gene set enrichment analysis was performed to find changes in biological process and molecular function in resistant (PB1+ Pi9) and susceptible (PB1) rice line upon M.oryzae infection. The qRT PCR was performed to validate selected candidate genes playing important role in rice defense upon M.oryzae infection. Conclusions: Our study represents the first detailed transcriptome analysis of resistant near isogenic line (PB1+ Pi9) and its susceptible control (PB1) with biological replicates upon M.oryzae infection, generated by RNA-seq technology. This transcriptome analysis helps to expedite protein network analyses and dissection of complex biological functions. In summary we found that a single functional blast resistant gene Pi9 present in resistant NIL in the background of susceptible line (PB1) activates a cascade of defense response genes, leading to incompatible interaction.

ORGANISM(S): Oryza sativa

PROVIDER: GSE81906 | GEO | 2017/06/12

SECONDARY ACCESSION(S): PRJNA323373

REPOSITORIES: GEO

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Project description:Purpose: To study the differential gene expression by comparing resistant and susceptible rice cultivars against the rice blast fungus M. Oryzae. Methods: Rice leaves were collected after M. Oryzae (MO36) infection from resistant and susceptible cultivars, preserved on deep freezer on -80O C frozen on liquid nitrogen, Total RNA from rice plant tissues was extracted using RNeasy Plant Mini Kit (Qiagen) by following manufacturer protocol. RNA libraries were prepared for sequencing using standard Illumina protocols. Results: Around 35 million pairs of filtered 150 base paired reads were obtained for each biological sample. These reads were mapped against RGAP7 using reference assembly tool of CLC Genomics Workbench. The mapped read data was summarised in to a matrix for each sample. count data was compared between the samples to identify the differentially expressed genes. genes were further filtered based on fold change, p value and FDR p value. The count data was further statistically analysed using Kal's Z test integrated into CGWB. Genes exhibiting 3 fold change and FDR p value <0.05 were filtered as either up regulated or down regulated gene. we obtained 7577 and 4290 significant (FDR adjusted p value ≤0.05) differentially expressed locus id in the resistant and susceptible rice cultivars, respectively . The number of significant DEL with fold change value greater and less than 3 were 3523 upregulated 4054 downregulated and 2190 upregulated & 2100 downregulated in resistance and susceptible cultivars, respectively. 2-way and and 4-way upregulated and downregulated gene comparisions was carried out. To identify the changes in biological process and molecular function the pathway and gene set enrichment analysis was performed in resistant BR2655 and susceptible HR12 rice cultivars. Conclusions: This study represents the first transcriptome analysis of resistant BR2655 and susceptible HR12 upon M.oryzae infection, generated by RNA-seq technology. This transcriptome analysis further helps to understand the differential gene expression analysis response to the M.oryzae. And candidate genes which are involved in triggering defense mechanism in rice plants. Further it helps to understand the mechanism of activating a cascade of defense responses in resistant and susceptible plants.

Project description:Highly pathogenic avian influenza virus (HPAIV) is a permanent threat due to its capacity to cross species barriers and generate severe infections and high mortality in humans. Recent findings have highlighted the potential role of PB1-F2, a small accessory influenza protein, in the pathogenesis process mediated by HPAIV in mammals. In this study, using a recombinant H5N1 HPAIV (wt) and its PB1-F2-deleted mutant (M-NM-^TF2), we studied the effects of PB1-F2 in a chicken model. Unexpectedly, when using low inoculation dose we observed that the wt-infected chickens had a higher survival rate than the M-NM-^TF2-infected chickens, a feature that contrasts with what is usually observed in mammals. High inoculation dose had similar mortality rate for both viruses, and comparison of the bio-distribution of the two viruses indicated that the expression of PB1-F2 allows a better spreading of the virus within chicken embryos. Transcriptomic profiles of lungs and blood cells were characterized at two days post-infection in chickens inoculated with the wild type (wt) or the M-NM-^TF2 mutant viruses. In lungs, the expression of PB1-F2 during the infection induced pathways related to calcium signaling and repressed a large panel of immunological functions. In blood cells, PB1-F2 was associated to a gene signature specific for mitochondrial dysfunction and down-modulated leucocytes activation. Finally we compared the effect of PB1-F2 in lungs of chickens and mice. We identified that gene signature associated to tissue damages is a PB1-F2 feature shared by the two species; by contrast, the early inhibition of immune response mediated by PB1-F2 observed in chickens is not seen in mice. In summary, our data suggest that PB1-F2 expression deeply affect the immune host response in chickens in a way that may attenuate pathogenicity, a feature differing from what was previously observed in mammal species. Three-condition experiment, virus-infected (wt or M-NM-^TF2) vs. Mock-infected chickens. Biological replicates: 2x5 control replicates, 5 wt replicates, 5 M-NM-^TF2 replicates.

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Transcriptome profiling of resistant NIL (PB1+Pi9) and susceptible rice line (PB1) 24 hours after M oryzae inoculation

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