概要: In rice, a number of resistance (R) genes that counter the blast fungus, Magnaporthe oryzae, have been cloned, but the mechanism by which they trigger disease resistance remains elusive. This is in part due to a lack of comprehensive transcriptome analyses during the resistance or disease progression. Here, we monitored the gene expression profiling in rice during its interactions with Magnaporthe by time-series transcriptome analyses. Distinct from previous studies, we focused on early infection stages within 24 hours post-inoculation. A comparison of those expression changes revealed a general difference in gene expression kinetics between compatible and incompatible interactions, and pointed to that the time when the pathogen just establishes its penetration into rice cells is a key point for the expressional changes. Such conclusions originated from the R gene Pid3-mediated immune responses were validated in the R gene Pi9-mediated responses, suggesting common molecular processes are shared by different R-mediated blast immunity. Our data highlighted the role of jasmonic acid (JA)-triggered signaling pathway (JA pathway) in the hormone signaling network for rice blast resistance. We confirmed that both exogenous and endogenous JA can induce the expression of many defense-related components revealed in above transcriptomic analyses and proved that the knock-down of OsCOI1 which encodes a JA receptor may deprive rice of the blast resistance mediated by R genes. Therefore, it is concluded that JA pathway plays an essential role in the establishment of blast resistance by modulating the expression of other defense-related components.
项目整体设计: We focused on early infection stages within 24 hours post-inoculation, the time when the pathogen just establishes its penetration into rice cells is a key point for the expressional changes. A comparison of kinetics gene expression changes between compatible and incompatible interactions.
Rice seedlings were grown in 32°C day 16 hours and 28°C night 8 hours in greenhouse. Four-week seedlings were used for pathogen inoculation.
处理方案:
Four-week seedlings were subjected to spray inoculation with conidial suspensions (for each strain) of 2.5 × 105 conidia/ml (suspended in 0.25% gelatin solution). For the mock control, the conidial suspensions were replaced with 0.25% gelatin solution. The inoculation was carried out under controlled conditions at 28℃ and 100% relative humidity for 24 h in darkness, then shifted to a 16/8 h light/dark regime.
提取方案:
Total RNAs were isolated using TRIzol reagent (Life Technologies) and treated with RNase-free DNase I (Takara) to remove any contaminating genomic DNA.mRNA extraction was performed using Dynabeads oligo(dT) (Dynal; Invitrogen Corp.). Double-stranded cDNAs were synthesized using reverse transcriptase (Superscript II; Invitrogen Corp.) and random hexamer primers. The cDNAs were then fragmented by nebulization, and the standard Illumina protocol was followed thereafter to create the mRNA-seq libraries.
