Genome-Wide Identification and Validation of Reference Genes in Infected Tomato Leaves for Quantitative RT-PCR Analyses

• Published in: PloS one Vol. 10; no. 8; p. e0136499
• Main Authors: Müller, Oliver A, Grau, Jan, Thieme, Sabine, Prochaska, Heike, Adlung, Norman, Sorgatz, Anika, Bonas, Ulla
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27-08-2015; Public Library of Science (PLoS)
• Subjects: Algorithms; Apoptosis; Biology; Capsicum annuum; Case studies; Cytosol; Dehydrogenases; DNA microarrays; Flowers & plants; Gene expression; Genes; Genes, Plant; Genetic aspects; Genetics; Genome-Wide Association Study; Genomes; Genomics; Homology; Host-Pathogen Interactions - physiology; Infections; Kinases; Leaves; Lycopersicon esculentum - genetics; Lycopersicon esculentum - metabolism; Lycopersicon esculentum - microbiology; Pathogens; Plant Diseases - genetics; Plant Diseases - microbiology; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Leaves - microbiology; Plant Proteins - biosynthesis; Plant Proteins - genetics; Polymerase chain reaction; Proteins; Reliability analysis; Reverse Transcriptase Polymerase Chain Reaction; snRNA; Solanum lycopersicum; Spot; Tomatoes; Transcription; Transcription (Genetics); Translocation; Xanthomonas campestris; Xanthomonas vesicatoria - physiology; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0136499

The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot disease of pepper and tomato by direct translocation of type III effector proteins into the plant cell cytosol. Once in the plant cell the effectors interfere with host cell processes and manipulate the plant transcriptome. Quantitative RT-PCR (qRT-PCR) is usually the method of choice to analyze transcriptional changes of selected plant genes. Reliable results depend, however, on measuring stably expressed reference genes that serve as internal normalization controls. We identified the most stably expressed tomato genes based on microarray analyses of Xcv-infected tomato leaves and evaluated the reliability of 11 genes for qRT-PCR studies in comparison to four traditionally employed reference genes. Three different statistical algorithms, geNorm, NormFinder and BestKeeper, concordantly determined the superiority of the newly identified reference genes. The most suitable reference genes encode proteins with homology to PHD finger family proteins and the U6 snRNA-associated protein LSm7. In addition, we identified pepper orthologs and validated several genes as reliable normalization controls for qRT-PCR analysis of Xcv-infected pepper plants. The newly identified reference genes will be beneficial for future qRT-PCR studies of the Xcv-tomato and Xcv-pepper pathosystems, as well as for the identification of suitable normalization controls for qRT-PCR studies of other plant-pathogen interactions, especially, if related plant species are used in combination with bacterial pathogens.