Time-resolved genome-wide pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) via RNA-Seq

Host-pathogen interactions, in particular during the early stages of infection, are important for the fate of both the invading pathogen and host. In pathogens, these interactions activate diverse pathogenic signals to cause disease in the host and evade the inevitably activated immune responses of the infected host. In this study, we used in vitro assay system and RNA-seq to analyze the time-resolved genome-wide gene expressions of Xanthomonas oryzae pv. oryzae (Xoo) evoked by the interactions with rice leaf extracts. RNA-seq analysis was carried out with samples of 7 different time points within 1 h. The RNA-seq data were verified by qRT-PCR. The in vitro system successfully initiated the pathogenic signals in Xoo cell culture. Many pathogenicity-related genes were expressed within 15 min. The hrpG gene was transcribed at maximum level within 10 min, and hrpX gene expression reached maximum level in 15 min. Chemotaxis and flagellar biosynthesis-related genes and cyclic-di-GMP synthesizing genes having GGDEF motif, were down-regulated until 10 min and after then up-regulated. Genes related to iron uptake and two component systems of phoB-phoR, phoP-phoQ, and pmrA-pmrB were up-regulated before 5 min. Data of multiple time points enabled non-linear regression fit of the time-resolved expression data, with which we newly developed the continuous time-resolved heat map to help the comparison of gene expression profiles. Essentiality of the transcriptionally up-regulated genes was also analyzed by the pathogenicity assay using the Xoo knockout strains, which showed not necessarily all the up-regulated genes were required for pathogenicity. the time-dependent, 0, 5, 10, 15, 30, 45, and 60 min, transcriptome analysis of genome-wide Xanthomonas oryzae pv. oryzae genes from the pathogenic interactions with RLX via RNA-Seq

宿主与病原体的相互作用，尤其是感染早期阶段，对入侵病原体与宿主的最终命运均至关重要。对于病原体而言，此类相互作用会激活多样化的致病信号，使其能够在宿主体内引发病害，并逃逸受感染宿主必然启动的免疫应答。本研究采用体外测定系统与RNA测序（RNA-seq）技术，分析了稻黄单胞菌稻致病变种（Xanthomonas oryzae pv. oryzae, Xoo）与水稻叶提取物相互作用后，随时间动态变化的全基因组基因表达谱。测序样本覆盖感染后1小时内的7个不同时间点，RNA-seq数据经实时定量PCR（qRT-PCR）验证。该体外系统成功在Xoo细胞培养体系中启动了致病信号通路：诸多致病相关基因在15分钟内即可被检测到表达。hrpG基因的转录水平在10分钟内达到峰值，hrpX基因的表达则在15分钟时达到峰值。趋化作用与鞭毛生物合成相关基因，以及携带GGDEF基序的环二鸟苷酸（cyclic-di-GMP）合成基因，在10分钟前均呈下调趋势，后续则转为上调表达。铁摄取相关基因，以及phoB-phoR、phoP-phoQ与pmrA-pmrB双组分系统相关基因，均在5分钟内即被上调表达。多时间点的转录组数据支持对时间分辨表达谱进行非线性拟合，借此我们全新构建了连续时间分辨率的热图，以辅助不同基因表达谱的对比分析。此外，我们通过构建Xoo基因敲除菌株开展致病力测定，分析了转录上调基因的致病必需性，结果显示并非所有上调基因均为Xoo致病力所必需。本数据集涵盖了0、5、10、15、30、45与60分钟共7个时间点下，Xoo与水稻叶提取物（RLX）发生致病相互作用过程中的全基因组转录组分析数据。