Selection and evaluation of suitable reference genes for quantitative gene expression analysis during infection of Cucumis sativus with Pectobacterium brasiliense

Aims Bacterial soft rot caused by Pectobacterium brasiliense (Pbr) has resulted in severe economic losses of cucumber production in northern China. Quantitative reverse transcription PCR (RT-qPCR) is widely used to determine the fold change in the expression of genes of interest, and an appropriate reference gene played a critical role in the evaluation of genes expression. However, the suitable reference genes for transcript normalization during the interaction between cucumber and Pbr have not yet been systematically validated. In this study, we aimed to identify the suitable reference genes for accurate and reliable normalization of cucumber and Pbr RT-qPCR data. Methods and Results We selected 14 candidate reference genes for cucumber and 10 candidate reference genes for Pbr were analysed by using four algorithms (the deltaCt method, BestKeeper, NormFinder and geNorm). Furthermore, five genes in cucumber involved in plant resistance and five genes in Pbr related to the virulence were selected to confirm the reliability of the reference genes by RT-qPCR. CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) were suggested as the most suitable reference genes for cucumber and Pbr respectively. Conclusion Our results suggested that CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) could be as the reference genes to normalize expression data for cucumber and Pbr during the process of pathogen-host interaction respectively. Significance and Impact of the Study To our knowledge, this is the first systematic study of the optimal reference genes specific to cucumber and Pbr, which could help advance the molecular interactions research in Cucurbitaceae vegetables and Pectobacterium species pathosystems.