Abstract
Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.
Highlights
Nicotiana benthamiana has become a very important subject for the study of host–pathogen interactions, those involving plant viruses
To the best of our knowledge, this article describes the first attempt to validate a set of commonly-used candidate reference genes, in N. benthamiana, that can be used for the normalisation of gene expression analysis using Quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR)
We identified 14 reference genes that were suitable for the normalisation of qRT– PCR data, which were obtained from N. benthamiana leaf samples subjected to five different viral infections
Summary
Nicotiana benthamiana has become a very important subject for the study of host–pathogen interactions, those involving plant viruses. It is most likely that N. benthamiana has been adopted as a model plant primarily due to its unparalleled susceptibility to viruses, which is associated with the naturally occurring mutation in the RNA-dependent RNA polymerase gene, NbRdRP1m [3]. Three major technical advances have contributed to increased utilisation of N. benthamiana in the field of plant biology: plant virus-based expression vector systems, virus-induced gene silencing (VIGS) and agro-infiltration. Plant virus-based transient expression systems provide attractive alternatives for the production of antibodies, vaccines, growth factors and many other proteins of pharmaceutical importance, because they offer several potential advantages compared to the traditional transgenic approach, including easy manipulation, high yield and fast manufacturing [1,4,5,6]. N. benthamiana has been used as the experimental host for the development of plant virus-based expression systems, such as Tobacco mosaic virus-based MagnICON vectors, Cowpea mosaic virusbased expression systems, and others [5,7,8]
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