Evaluation of suitable reference genes for normalization of quantitative reverse transcription PCR analyses in Clavibacter michiganensis

Clavibacter michiganensis, the causal agent of bacterial canker of tomato, is a Gram-positive bacterium and a model for studying plant diseases. The real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay is widely used to quantify gene expression in plant pathogenic bacteria. However, accurate quantification of gene expression requires stably expressed reference genes that are consistently expressed during the experimental conditions of interest. The use of inappropriate reference genes leads to a misinterpretation of gene expression data and false conclusions. In current study, we empirically assessed the expression stability of six housekeeping genes (gyrB, rpoB, tufA, bipA, gapA, and pbpA) of C. michiganensis under five experimental conditions using two algorithms, geNorm and NormFinder. C. michiganensis expressed gyrB, bipA, and gapA stably when growing in nutrient-rich broth (TBY broth and modified M9 broth). We concluded that pbpA, tufA, and gyrB were suitable reference genes in C. michiganensis-tomato interaction studies. We also recommended bipA and rpoB to be used to study bacterial gene expression under nutrient-poor conditions. Finally, gyrB, pbpA, and rpoB can be used to normalize the quantification of C. michiganensis gene expression while the bacterium is in the viable but nonculturable (VBNC) state. This study identified the most suitable reference genes depending on the experimental conditions for calibrating real-time qRT-PCR analyses of C. michiganensis and will be useful in studies that seek to understand the molecular interactions between C. michiganensis and tomato.