Transcriptome-based identification of N. benthamiana-bacteria interaction RT-qPCR reference genes

ZHEN, YI; ROSLI, HERNÁN GUILLERMO; POMBO, MARINA ALEJANDRA; GOMEZ-LOBATO, MARÍA EUGENIA; RAMOS, ROMINA NAHIR; MARTIN, GREGORY

Paraná, Entre Ríos, Argentina

Congreso; LIV Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on anormalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from thoseused in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genesfor the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stablyexpressed genes. Nicotianabenthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants,combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpAexhibit less variable gene expressionin our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis.We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.