Selection and validation of reference genes for gene expression studies in Xanthomonas arboricola pv. juglandis subjected to abiotic stress

The selection and validation of reference genes for reverse-transcription quantitative PCR (RT-qPCR) is key in the normalization of data to evaluate the expression levels of target genes. In this study, the expression levels of nine candidate reference genes in Xanthomonas arboricola pv. juglandis (Xaj) undergoing three abiotic stresses, including thermal stress at 15 +/- 1 degrees C and 35 +/- 1 degrees C and UV light, were evaluated by RT-qPCR. A ranked list of reference genes based on expression stability was assessed using BestKeeper, NormFinder, GeNorm, and Delta C-t methods. In Xaj undergoing all tested abiotic stress conditions, two reference genes (RGs) were sufficient for effective normalization of RT-qPCR data. ATP synthase subunit delta (atpD) and RNA polymerase delta factor (rpoD) were the most stably expressed RGs, while gyrase subunit A (gyrA) and gyrase subunit B (gyrB) were the least stable. A combination of the most and least stable RGs was used for the normalization of two isoforms of three target genes, catalase (cat1 and cat2), superoxide dismutase (sod1 and sod2), and glutathione peroxidase (gpx1 and gpx2), showing that the validation of RGs was needed in each experimental condition to avoid misinterpretation of the expression levels of target genes. These results can be useful in studies that seek to understand the biochemical and molecular complex pathways involved in stress tolerance mechanisms in Xaj and to evaluate the effects of stress conditions on plant-pathogen interactions.