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.
Keywords: food-borne pathogens, lettuce, spinach, rhizosphere, mRNA isolation
Citation: Holmes A, Birse L, Jackson RW and Holden NJ (2014) An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7. Front. Microbiol. 5:286. doi: 10.3389/fmicb.2014.00286
Received: 24 March 2014; Paper pending published: 27 April 2014;
Accepted: 23 May 2014; Published online: 10 June 2014.
Edited by:Adam Schikora, IPAZ, JLU Giessen, Germany
Reviewed by:Saul Burdman, The Hebrew University of Jerusalem, Israel
Copyright © 2014 Holmes, Birse, Jackson and Holden. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Nicola J. Holden, Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK e-mail: email@example.com