Optimal Reference Gene Selection and Potential Target Gene Identification During Xanthomonas phaseoli pv....

Xanthomonas phaseoli pv. dieffenbachiae (Xpd), the causal agent of bacterial blight in Anthurium within the Araceae family, is listed as an EPPO A2 quarantine organism. Although the whole genome of Xpd has been sequenced, the molecular mechanisms underlying anthurium bacterial blight (ABB) remain unknown. Selecting an optimal reference gene is crucial for obtaining accurate and reliable gene expression profiles during the initial interactions between Xpd and Anthurium. The stability of four reference genes was evaluated by applying three statistical methods-BestKeeper, geNorm, and delta Ct (ΔCt)-using reverse-transcription quantitative PCR (RT-qPCR) data. The rpoD and gyrB genes exhibited the most consistent gene expression profiles, whereas atpD and thyA were less stable at four time points (0, 0.5, 1, and 2 h) during the interactions between Xpd and susceptible A. andreanum cultivar 'Marian Seefurth.' The suitability of these reference gene candidates was validated by normalizing the gene expression levels of four pathogenicity-related genes. The highly upregulated expression of gumD, which encodes xanthan biosynthesis glycosyltransferase, observed after 1 h of interaction, suggests it may be a key virulence determinant in the Xpd-Anthurium pathosystem. The stable reference genes identified here will facilitate more accurate and comprehensive gene expression studies in the Xpd-Anthurium pathosystem going forward.