Bayogenin 3-O-Cellobioside is a novel anti-blast metabolite produced in rice in response to Pyricularia oryzae infection. Bayogenin 3-O-Cellobioside is a novel anti-blast metabolite produced in rice in response to Pyricularia oryzae infection

Susceptible (CO39 and NPB) and resistant (Pi_gm) rice cultivars were spray inoculated with P. oryzae spore and incubated for 12-hours. Total RNA was extracted from the inoculated rice seedlings (T_Co39, T_NPB, and T_gm) along with their non-inoculated control group C_Co39, C_NPB, and C_gm. The extraction of total RNA from inoculated and non-inoculated control samples was carried-out with RNAprep pure Plant Kit (Tiangen, Beijing) by following processes recommended by the manufacturer. To observe the level of RNA degradation and contamination, the extracted RNA was run on 1% agarose gels. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA concentration was measured with RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, CA, USA). The cDNA library was sequenced on the Illumina sequencing platform (Illumina HiSeq™ 2000) with 150 bp pair-end reads length and 300 bp insert size by Gene Denovo Co. (Guangzhou, China). Novogene in-house Perl script was used to select clean reads by removing adaptor sequences, low quality sequences (reads with more than 50% of bases quality lower than 20) and reads with more than 5% N bases. Overall design: mRNA profile of susceptible and resistant rice seedlings inoculated with P. oryzae for 12-hours and non-inoculated control groups were generated through deep sequencing, data was obtained from one biological experiment with three technical replicates using Illumina HiSeq™ 2000.

将感病水稻品种（CO39与NPB）及抗病水稻品种（Pi_gm）以喷雾法接种稻瘟病菌（P. oryzae）孢子，随后孵育12小时。从接种后的水稻幼苗（T_Co39、T_NPB及T_gm）及其未接种的对照组（C_Co39、C_NPB、C_gm）中提取总RNA。总RNA提取采用天根生化（Tiangen，北京）的RNAprep pure Plant Kit，严格遵循厂商推荐的操作流程完成。为检测提取RNA的降解程度与污染水平，将其在1%琼脂糖凝胶中进行电泳检测。RNA完整性通过美国加利福尼亚州安捷伦科技（Agilent Technologies）Bioanalyzer 2100生物分析仪系统配套的RNA Nano 6000 Assay Kit进行评估；RNA浓度则采用美国加利福尼亚州Life Technologies公司的RNA Assay Kit搭配Qubit 2.0 Fluorometer进行测定。cDNA文库由广州基迪奥生物科技有限公司（Gene Denovo Co., Guangzhou, China）在Illumina测序平台（Illumina HiSeq™ 2000）上完成测序，测序模式为150 bp双端读长，插入片段长度为300 bp。使用诺禾致源（Novogene）自研Perl脚本，通过移除接头序列、低质量序列（质量值低于20的碱基占比超过50%的读段）以及含N碱基占比超过5%的读段，筛选得到clean reads。本研究整体实验设计如下：对接种稻瘟病菌12小时的感病与抗病水稻幼苗，以及未接种的对照组，开展mRNA转录组深度测序；测序数据来自1次生物学重复实验，且设置3次技术重复，采用Illumina HiSeq™ 2000平台完成测序。