title: Assessment of reference gene stability influenced by extremely divergent disease symptoms in Solanum lycopersicum L.
abstract: Tomato (Solanum lycopersicum L.) is one of the most important vegetables of great worldwide economic value. The scientific importance of the vegetable results from the fact that the genome of S. lycopersicum has been sequenced. This allows researchers to study fundamental mechanisms playing an essential role during tomato development and response to environmental factors contributing significantly to cell metabolism alterations. Parallel with the development of contemporary genetics and the constant increase in sequencing data, progress has to be aligned with improvement of experimental methods used for studying genes functions and gene expression levels, of which the quantitative polymerase chain reaction (qPCR) is still the most reliable. As well as with other nucleic acid-based methods used for comparison of the abundance of specific RNAs, the RT-qPCR data have to be normalised to the levels of RNAs represented stably in a cell. To achieve the goal, the so-called housekeeping genes (i.e., RNAs encoding, for instance, proteins playing an important role in the cell metabolism or structure maintenance), are used for normalisation of the target gene expression data. However, a number of studies have indicated the transcriptional instability of commonly used reference genes analysed in different situations or conditions; for instance, the origin of cells, tissue types, or environmental or other experimental conditions. The expression of ten common housekeeping genes of S. lycopersicum, namely EF1α, TUB, CAC, EXP, RPL8, GAPDH, TBP, ACT, SAND and 18S rRNA were examined during viral infections of tomato. Changes in the expression levels of the genes were estimated by comparison of the non-inoculated tomato plants with those infected with commonly known tomato viral pathogens, Tomato torrado virus, Cucumber mosaic virus, Tobacco mosaic virus and Pepino mosaic virus, inducing a diverse range of disease symptoms on the common host, ranging from mild leaves chlorosis to very severe stem necrosis. It is emphasised that despite the wide range of diverse disease symptoms it is concluded that ACT, CAC and EF1α could be used as the most suitable reference genes in studies of host-virus interactions in tomato.
authors.affiliation: Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection - National Research Institute, Władysława Węgorka 20, 60-318 Poznań, Poland.
authors.forename: Przemysław
authors.initials: P
authors.lastname: Wieczorek
authors.forename: Barbara
authors.initials: B
authors.lastname: Wrzesińska
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Obrępalska-Stęplowska
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D020043:Genes, Essential; D017343:Genes, Plant; D042822:Genomic Instability; D054884:Host-Pathogen Interactions; D018551:Solanum lycopersicum / Q000235:genetics* / Q000821:virology; D010935:Plant Diseases / Q000235:genetics* / Q000821:virology; D012015:Reference Standards
keywords: 18S rRNA; 18S ribosomal RNA; ACT; CAC; CMV; CYP; Cucumber mosaic virus; Cyclophilin; EF1α; EXP; GAPDH; GTP-binding nuclear protein; PepMV; Pepino mosaic virus; Plant viruses; RAN1; RG; RPL8; RPS13; RT-qPCR; Reference genes; Ribosomal protein S13; SAND; SAND family protein; Solanum lycopersicum; TATA-binding protein; TBP; TMV; TUB; ToTV; Tobacco mosaic virus; Tomato torrado virus; actin; clathrin adapter complexes medium subunit; elongation factor 1α; glyceraldehyde-3-phosphate dehydrogenase; quantitative reverse transcription-PCR; reference genes; ribosomal protein L8; α-tubulin; ‘Expressed’ protein