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

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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.