Screening and Validation of Reference Genes for Normalization of qRT-PCR in Rice BLB Pathogen Xanthomonas oryzae pv. oryzae Under Tetramycin Stress

Background: Xanthomonas oryzae pv. oryzae (Xoo) causes rice leaf blight (BLB) and poses a major threat to global rice production. In rice production, tetramycin agents provide good control of rice leaf blight, while the standardization of the reference genes of Xoo under tetramycin stress has not been reported. The aim of this study was to identify the most stable reference genes for quantitative PCR analysis of Xoo under tetramycin stress. Methods: Six candidate reference genes, gyrB (RNA polymerase β gene), GADPH (glyceraldehyde-3-phosphate dehydrogenase gene), recA (recombinase A gene), gyrA (citrate synthase encoding gene), dnaK (molecular chaperone protein gene), and 16S rRNA (16S ribosomal RNA gene) were selected and their expression stability was assessed under tetramycin stress conditions using real-time quantitative PCR (qRT-PCR). GeNorm, NormFinder, BestKeeper and RefFinder were used to assess the expression stability, the relative expression values of the eight genes involved QS (Quorum sensing) pathway under tetramycin stress were used to validate by the rpf (regulation of pathogenic factors) gene family. Results: 16S rRNA expression was most stable under tetracycline stress, whereas GADPH was the least. The rpf gene family showed a highly stable expression level, confirming the reliability of 16S r RNA as a reference gene in the study of Xoo under tetramycin stress. Conclusions: 16S rRNA was identified as the best reference gene for Xoo gene expression analysis under tetramycin stress. It provides a reliable support for the molecular research on the control strategy of rice BLB.