Validation of reference genes as an internal control for studying Avena sativa–Puccinia coronata interaction by RT-qPCR

Abstract

In this study we evaluated eleven candidate reference genes in Avena sativa during compatible and incompatible interactions with two different pathotypes of Puccinia coronata f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). The results obtained confirmed that the combination of two genes would be sufficient for reliable normalization of the expression data. In general, the most stable in the tested plant-pathogen system were HNR (heterogeneous nuclear ribonucleoprotein 27C) and EF1A (elongation factor 1-alpha). ARF (ADP-ribosylation factor) and EIF4A (eukaryotic initiation factor 4A-3) could also be considered as exhibiting high expression stability. CYP (cyclophilin) was shown by all assessment methods to be the worst candidate for normalization in this dataset. To date, this is the first report of reference genes selection in A. sativa–P. coronata interaction system. Identified reference genes enable reliable and comprehensive RT-qPCR analysis of oat gene expression in response to crown rust infection. Understanding the molecular mechanisms involved in the host–pathogen interactions may expand knowledge of durable resistance strategies beneficial to modern oat breeding.

Validation of reference gene stability for normalization of RT-qPCR in Phytophthora capsici Leonian during its interaction with Piper nigrum L.

“…The RT-qPCR approach is frequently utilized in studies investigating plant-pathogen interactions to gain insights into the underlying molecular mechanisms 33 – 35 . There is often a concern that despite the widespread use of RT-qPCR, one may not consistently adhere to the recommended guidelines for conducting this experiment, potentially resulting in misleading outcomes 36 .…”