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Reverse transcription-quantitative PCR (RT-qPCR) is an analytical tool for gene expression quantification. Reference genes are not yet available for gene expression analysis during interactions of  Ralstonia solanacearum  with ‘Hawaii 7996’ (the most stable source of resistance in tomato). Here, we carried out a multi-algorithm stability analysis of eight candidate reference genes during interactions of ‘Hawaii 7996’ with one incompatible/avirulent and two compatible/virulent (= resistance-breaking) bacterial isolates. Samples were taken at 24- and 96-h post-inoculation (HPI). Analyses were performed using the ∆∆Ct method and expression stability was estimated using BestKeeper, NormFinder, and geNorm algorithms.  TIP41  and  EF1α  (with geNorm),  TIP41  and  ACT  (with NormFinder), and  UBI3  and  TIP41  (with BestKeeper), were the best combinations for mRNA normalization in incompatible interactions at 24 HPI and 96 HPI. The most stable genes in global compatible and incompatible interactions at 24 HPI and 96 HPI were  PDS  and  TIP41  (with geNorm),  TIP41  and  ACT  (with NormFinder), and  UBI3  and  PDS / EXP  (with BestKeeper). Global analyses on the basis of the three algorithms across 20  R. solanacearum -tomato experimental conditions identified  UBI3, TIP41  and  ACT  as the best choices as reference tomato genes in this important pathosystem.

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Stability analysis of reference genes for RT-qPCR assays involving compatible and incompatible Ralstonia solanacearum-tomato ‘Hawaii 7996’ interactions