Open AccessExtraction of high‐quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization
Author(s) -
SCHENK ALEXANDER,
WEINGART HELGE,
ULLRICH MATTHIAS S.
Publication year - 2008
Publication title -
molecular plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.945
H-Index - 103
eISSN - 1364-3703
pISSN - 1464-6722
DOI - 10.1111/j.1364-3703.2007.00452.x
Subject(s) - biology , pseudomonas syringae , rna , gene expression , rna extraction , gene , microbiology and biotechnology , virulence , nucleic acid , biochemistry
SUMMARY Plant pathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. In planta gene expression analysis is important to understand the impact of these genes on bacterial virulence. A new mRNA‐based approach using multiplexed Northern hybridization was developed. High‐quality bacterial and plant total RNA was successfully isolated from leaf tissue infiltrated with Pseudomonas syringae . The procedure employs a new extraction buffer formulation containing glycine, sodium dodecylsulphate, cetyltrimethylammonium bromide, high‐molecular‐weight polyethylene glycol and β‐mercaptoethanol. Cell lysis and classical acid–phenol extraction steps followed by LiCl precipitation yielded large amounts of total RNA of high purity and integrity. Multiplexing of DIG and chemically fluorescently labelled RNA probes was developed and expression data were normalized using the 23S rRNA gene as reference. The method was validated by studying in planta expression of the P. syringae genes mucD , cmaA , cfl , corR , corS and corP comprising a selection of highly expressed biosynthetic and low‐expressed regulatory genes. The method was assessed regarding its sensitivity and might by useful for studying a variety of plant–microbe interactions.