an optimised method for the extraction of bacterial mrna from plant roots infected with escherichia coli o157:h7

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ID: 222809
2014
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Ranked #495 of 875 articles by views in journal of magnetic resonance (san diego, calif : 1997)

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Abstract
Analysis of microbial gene expression during host colonisation 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. qPCR, microarray or RNA-seq. The goal of this work was to optimise 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 optimised 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 HMW-PEG and CTAB. 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.
Reference Key
eholmes2014frontiersan Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;Ashleigh eHolmes;Louise eBirse;Robert Wilson Jackson;Nicola eHolden
Journal journal of magnetic resonance (san diego, calif : 1997)
Year 2014
DOI
10.3389/fmicb.2014.00286
URL
http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00286/full
Keywords
lettuce rhizosphere

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