Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to investigate NGS-derived rice leaf transcriptome profiling (RNA-seq) exposed to green rice leafhopper (GRH) and validated RNA-Seq results using quantitative reverse transcription polymerase chain reaction (qRT–PCR). Methods: Rice leaf mRNA profiles of 21-day-old wild-type (WT) and Near Isogenic Line (NIL) rice were generated by deep sequencing, in triplicate, using Illumina’s HiSeq™ 4000 Sequencing System. For assembled genes, longest contigs of the assembled contigs were filtered and clustered into the non-redundant transcripts using CD-HIT-EST (Li et al., 2001; Bolger et al., 2014). qRT–PCR validation was performed using ROX and SYBR Green assays. Results: RNA-Seq results revealed that an average of 1,766,347 (1,240,317 in control and 526,030 GRH treated plants) and 3,676,765 (2,986,748 control and 690,017 GRH) counts per million mapped (CPM) reads were generated from Ilpum and NIL GRH treated plants, respectively. After alignment, more than 75% of all reads were successfully mapped to the reference genome, and 8,859 genes and 15,815,400 transcripts were obtained. GRH infestation caused differential expression of 4265 up-regulated and 4,594 down-regulated genes in Ilpum, while in NIL, 4,479 and 4,380 genes were up- and down-regulated, respectively 48 h post-infestation. A total of 3,424 differentially expressed genes (DEGs, 1,605 up-regulated in Ilpum and down-regulated in NIL; 1,819 genes up-regulated in NIL and down-regulated in Ilpum) were identified. Interestingly, these DEGs were found to be involved in key physiological processes, with interesting molecular functions, including plant defense against diverse biotic stresses, hormone signaling, and other developmental processes. Based on the quantile normalization of the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values followed by student’s t-test (p <0.05) we identified 3,283 DEGs in Ilpum (1,935 upregulated and 1,348 downregulated) and 2,599 DEGs in NIL (1,621 up-regulated and 978 down-regulated) with at least log2 (logarithm base 2) 2-fold change (Log2FC) in expression upon GRH infestation. Conclusions: The current study has provided the first RNA-Seq dataset and comparative transcriptome profile of GRH responsive genes from Ilpum (GRH-susceptible rice cultivar) and a near isogenic line (NIL) carrying Grh1 gene introduced from Shingwang into Ilpum to investigate the possible transcriptional interaction with other defense related genes. In addition, RNA-Seq data and MapMan analysis revealed the activation of multiple regulatory pathways following GRH infestation. Furthermore, the observed differential transcriptome profile between Ilpum and NIL would suggest the existence of a possible interaction network between Grh1 and other defense related genes towards GRH resistance in rice.

Project description:Chilling stress is a major abiotic stress that affects rice growth and development. Rice seedlings are quite sensitive to chilling stress and this harms global rice production. Comprehensive studies of the molecular mechanisms for response to low temperature are of fundamental importance to chilling tolerance improvement. The number of identified cold regulated genes (CORs) in rice is still very small. Circadian clock is an endogenous timer that enables plants to cope with forever changing surroundings including light–dark cycles imposed by the rotation of the planet. Previous studies have demonstrated that the circadian clock regulates stress tolerances in plants show circadian clock regulation of plant stress tolerances. However, little is known about coordination of the circadian clock in rice chilling tolerance. In this study, we investigated rice responses to chilling stress under conditions with natural light-dark cycles. We demonstrated that chilling stress occurring at nighttime significantly decreased chlorophyll content and photosynthesis efficiency in comparison with that occurring at daytime. Transcriptome analysis characterized novel CORs in indica rice, and suggested that circadian clock obviously interferes with cold effects on key genes in chlorophyll (Chl) biosynthesis pathway and photosynthesis-antenna proteins. Expression profiling revealed that chilling stress during different Zeitberger times (ZTs) at nighttime repressed the expression of those genes involved Chl biosynthesis and photosynthesis, whereas stress during ZTs at daytime increases their expression dramatically. Moreover, marker genes OsDREBs for chilling tolerance were regulated differentially by the chilling stress occurring at different ZTs. The phase and amplitude of oscillation curves of core clock component genes such as OsLHY and OsPRR1 are regulated by chilling stress, suggesting the role of chilling stress as an input signal to the rice circadian clock. Our work revealed impacts of circadian clock on chilling responses in rice, and proved that the effects on the fitness costs are varying with the time in a day when the chilling stress occurs.

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Oryza sativa

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