Genome-wide analysis of the cell-layer specific expression of pathogen-induced genes in the Arabidopsis root

虽然植物根与周围的微生物（MO）的巨大变化密切接触，转录诱导的根特异性防御病原体的特点很差。由于根是由同心的细胞层构建的，因此可以预期这些层对遗传定义的程序有特异性的响应。应用拟南芥模型，我们建立了一个TRAPseq方法（通过RNA测序翻译核糖体亲和纯化），以研究根对不同MO的细胞层分辨率。由于FLAG标记的核糖体蛋白的细胞类型特异性表达，核糖体/RNA复合物可以被免疫共沉淀以通过随后的RNA测序获得细胞层特异性表达谱。为了比较根的反应，我们分析了三种真菌/卵菌根感染的MO，它们的生活方式不同：（1）寄生疫霉从早期的活体营养阶段切换到坏死营养阶段。(2)长孢轮枝菌穿过根细胞层进入木质部，在那里它作为一种生物营养型维管病原体。在木质部流中运输后，它最终感染叶子。(3)有益真菌梨形孢菌作为内生菌定殖于皮层，支持植物生长并增强病原体防御。对于所有三种MO，获得了细胞层特异性TRAPseq结果，并建立了分析数据的生物信息学工具。重申我们的假设，根细胞层响应特定的表达模式，这取决于MO和他们的生活方式。E. G.我们可以鉴定参与色氨酸衍生的抗微生物化合物的生物合成的基因的细胞层特异性表达，定位激素生物合成/信号传导的差异或观察内胚层屏障功能的强化。在接种P. indica后，观察到营养物质运输的明显差异，这可能解释了有益的特性。相反，有益的真菌，细胞周期基因和根生长的抑制，特别是观察到病原体接种后，在中央圆柱体。在本提案中，我们申请短期支持，通过分析每个MO的第二个感染时间点来完成本研究。挖掘复杂的数据集将使我们能够产生实验验证的假设，这将扩大我们的农学重要植物MO在根际的相互作用的理解。

Although plant roots are in close contact with a tremendous variety of surrounding microorganisms (MOs), transcriptionally induced root-specific defences towards pathogens are poorly characterized. As the root is built by concentric cell-layers, it is anticipated that these layers respond specifically with genetically defined programs. Applying the Arabidopsis model, we established a TRAPseq method (Translating Ribosome Affinity Purification by RNAsequencing) to study root responses towards different MOs on a cell-layer resolution. Due to cell-type specific expression of FLAG-tagged ribosomal proteins, ribosome/RNA complexes can be co-immunoprecipitated to obtain cell-layer specific expression profiles by subsequent RNA sequencing. To compare root responses, we analysed three fungal/oomycete root-infecting MOs, which differ with respect to their lifestyle: (1) Phytophthora parasitica switches from an early biotrophic to a necrotrophic phase. (2) Verticillium longisporum transverses the root cell-layers to enter the xylem, where it performs as a biotrophic vascular pathogen. After transport in the xylem stream, it finally infects the foliage. (3) The beneficial fungus Piriformospora indica colonizes the cortex as an endophyte, supporting plant growth and enhancing pathogen defence. For all three MOs, cell-layer specific TRAPseq results were obtained and bioinformatic tools to analyse the data were established. Confirming our hypothesis, root cell-layers respond with specific expressional patterns, which differ depending on the MOs and their lifestyle. E. g. we could identify cell-layer specific expression of genes involved in biosynthesis of Tryptophan-derived antimicrobial compounds, localize differences in hormone biosynthesis/signalling or observe a fortification of the endodermal barrier function. Distinct differences with respect to altered nutrient transport were observed after inoculation with P. indica, which might explain the beneficial properties. In contrast to the beneficial fungus, repression of cell-cycle genes and root growth was specifically observed in the central cylinder after pathogen inoculation. In this proposal, we apply for a short-term support to complete this study by analysing a second infection time-point for each MO. Mining the complex data-sets will enable us to generate experimentally testable hypotheses which will broaden our understanding of the agronomical important plant MO interactions in the rhizosphere.