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cDNA Libraries

Volume 729 of the series Methods in Molecular Biology pp 3-13

Date:

Isolation of Fungal Infection Structures from Plant Tissue by Flow Cytometry for Cell-Specific Transcriptome Analysis

  • Hiroyuki TakaharaAffiliated withDepartment of Bioproduction Science, Ishikawa Prefectural University
  • , Elmar EndlAffiliated withInstitutes of Molecular Medicine and Experimental Immunology, University of Bonn
  • , Richard O’ConnellAffiliated withDepartment of Plant–Microbe Interactions, Max-Planck-Institute for Plant Breeding Research Email author 

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Abstract

Many plant pathogenic fungi differentiate a series of highly specialized infection structures to invade and colonize host tissues. Especially at early stages of infection, the ratio of fungal to plant biomass is very low. To investigate cell-specific patterns of gene expression, it is necessary to purify the fungal structures of interest from infected plants. We describe here a method to isolate the biotrophic hyphae of Colletotrichum higginsianum from Arabidopsis leaves, based on a combination of pre-enrichment by isopycnic centrifugation followed by further purification by fluorescence-activated cell sorting. This protocol efficiently eliminates contamination by plant components and nontarget fungal cell-types. Moreover, the isolated cells remain alive, providing high-quality RNA for library construction. The method can be readily adapted for cell-specific transcriptome analysis in other plant–microbe interactions.

Key words

Fluorescence-activated cell sorting Flow cytometry Colletotrichum higginsianum Arabidopsis thaliana Biotrophy Transcriptome