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

Abstract

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.

Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions

“…However, based on our preliminary evaluation and main experimental data, it has been shown that TufA was stably expressed in different tissues and could be considered as a reference gene in H. variegata. Previous studies demonstrated that in different experimental conditions, TufA is one of the most stable reference genes in different species of bacteria (Savard and Roy, 2009;Jiang et al, 2019).…”

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

“…out to evaluate RGs in plant-pathogenic bacteria, demonstrating that it is not possible to define universal RGs that are stably expressed under different experimental conditions (Jiang et al, 2019;Yan et al, 2019;Yang et al, 2018).…”

“…In our study, gyrB, encoding the subunit B of DNA gyrase, was the least stable RG in two experimental conditions, while it has been used as a common RG stably expressed for gram-positive bacteria cultured in nutrient-rich media or under certain stress conditions such as Clavibacter michiganensis (Jiang et al, 2019), Staphylococcus aureus (Sihto et al, 2014), and Corynebacterium pseudotuberculosis (Carvalho et al, 2014). Furthermore, gyrB was selected as the best reference gene, followed by gyrA, rpoB, and gapdh, in X. campestris pv.…”

“…A few studies have been carried out to evaluate suitable candidate RGs for accurate normalization of RT‐qPCR data in plant‐pathogenic bacteria, such as Xanthomonas campestris pv. campestris (Yan et al, 2019), Pectobacterium atrosepticum (Takle et al, 2007), Erwinia amylovora (Kałużna et al, 2017), Ralstonia pseudosolanacearum (Yang et al, 2018), Clavibacter michiganensis (Jiang et al, 2019), Xanthomonas citri subsp. citri (Jacob et al, 2011), and Xanthomonas fragariae (Kałużna et al, 2019).…”

“…In this study, nine RGs belonging to different functional classes were selected. RNA polymerase β subunit ( rpoB ), DNA recombination protein ( recA ), gyrase subunit B ( gyrB ), and gyrase subunit A ( gyrA ) have been validated as RGs in previous studies in other plant‐pathogenic bacteria (Jiang et al, 2019; Kałużna et al, 2017; Takle et al, 2007; Yan et al, 2019; Yang et al, 2018). Other genes such as RNA polymerase γ subunit ( rpoC ), RNA polymerase sigma α factor ( rpoA ), rho termination factor ( rho ), ATP synthase subunit δ ( atpD ), and RNA polymerase σ factor RpoD ( rpoD ) were used as RGs in Gluconacetobacter diazotrophicus (Galisa et al, 2012) and Rahnella aquatilis (Li et al, 2019).…”