title: An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7.
abstract: Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute Invergowrie, Dundee, UK.
authors.forename: Ashleigh
authors.initials: A
authors.lastname: Holmes
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute Invergowrie, Dundee, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Birse
authors.affiliation: School of Biological Sciences, The University of Reading Knight Building, Whiteknights, Reading, UK.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Jackson
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute Invergowrie, Dundee, UK.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Holden
keywords: food-borne pathogens; lettuce; mRNA isolation; rhizosphere; spinach