Optimizing RT-qPCR normalization for crop plant VIGS: a case study using aphid-infested cotton

Reverse-transcription quantitative PCR (RT-qPCR) is an essential laboratory technique requiring stable reference genes for accurate gene expression normalization. With respect to crop plants, reference gene stability has not been thoroughly investigated under two crucial experimental conditions: 1) virus-induced gene silencing (VIGS) and 2) interactions with insect herbivores. In this study, we comprehensively evaluated six candidate reference genes ( Act7 , Pp2a1 , Ubq7 , Ubq14 , Tmn5 , and Tbl6 ) in wildtype, VIGS-infiltrated, cotton aphid-infested, and uninfested cotton plants, at two time points (14- and 21-days post infestation), in a fully factorial experiment. Combined statistical analyses identified Act7 and Pp2a1 as the most stable reference genes. In contrast, the frequently reported ubiquitin reference genes were the least stable. Expression stability analysis outcomes were validated by evaluating expression of the phytosterol biosynthesis gene GhHydra1 in response to aphid herbivory. Normalization using Act7 / Pp2a1 revealed significant upregulation of GhHydra1 in aphid-infested plants. Conversely, normalization using the unstable reference gene Ubq7 resulted in high variance and reduced sensitivity to detect expression changes. These findings highlight the importance of selecting stable reference genes for accurate expression normalization. Results from the validation study suggest that phytosterols may play a previously unreported role in cotton stress responses to insect herbivory.