Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

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.

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins

“…Primers used for each gene analyzed were: Nb00051202g0009.1 F: 5′-TGTTGGGTCAAATG ATTCTCACA-3′, R: 5′-GCTCAACCCATTAGAAAC TCTGA-3′; Nb00020954g0005.1 F: 5′-TGTCGGGTC AAATGATTCTC-3′, R: 5′-CAACCCGTTAGAAACTC TCC-3′; Nb00014536g0001.1 F: 5′-AGGCTACCTAATG ATGATGAAA-3′, R: 5′-GGTGGAGTTGAGACAATAG AG-3′; Nb00003176g0019.1 F: 5′-ACCATGAACATT ATGGACTGT-3′, R: 5′-CAACAGAACCTCCACCATT-3′; Nb00029791g0013.1 F: 5′-AGAGGCTCCAAAGTTCGC A-3′, R: 5′-AACCGAACCTCCACCAAT-3′; Nb00042373 g0002.1 F: 5′-ATGATAAGACTACCTGATGATGAC-3′, R: 5′-GGTGGAGTTGAGATAAGAGAATAA-3′. Data were normalized using NbPP2a and NbEF1α [65]. Cycling conditions during qRT-PCR were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s. A pairwise Student's t-test (P <0.01) was used to determine significance differences.…”

Genomic variations in the 3′‐termini of Rice stripe virus in the rotation between vector insect and host plant

“…The fragments from the inner regions of RNA1 and RNA2 were amplified to measure the total RNA levels of RNA1 and RNA2 using primers RNA1‐inner‐F and RNA1‐inner‐R and primers RNA2‐inner‐F and RNA2‐inner‐R, respectively. The transcript levels of the actin genes of the small brown planthopper (Jiao et al ., 2017), rice (AK100267) (Chern et al ., 2013), wheat (TC234027) (Tenea et al ., 2011) and tobacco (AY594294) (Liu et al ., 2012) were quantified with the primers actin‐insect‐F and actin‐insect‐R, actin‐rice‐F and actin‐rice‐R, actin‐wheat‐F and actin‐wheat‐R, and actin‐tobacco‐F and actin‐tobacco‐R, respectively, and were used as internal controls to normalize the levels of the total input RNAs. The primer sequences are listed in Table S1.…”

Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

“…The qPCR primers were BIP_forward (5=-ATCTGACCGGAATTCCTCCAG-3=) and BIP_reverse (5=-TACCAGTGCCCTTATCTTCTGC-3=), ACO3_forward (5=-ATCAGCAGCACCGTTCTTTG-3=) and ACO3_reverse (5=-TGGCA GTAGTTCTGATCTGAGC-3=), and PSBQ2_forward (5=-AACCGTCAT CTCTGCTAAGCC-3=) and PSBQ2_reverse (5=-GCATAGTACTTCTCT GCTTCAGG-3=). L23 primers were based on reference primer sequences previously published (44). Primer annealing temperatures were optimized by RedTaq gradient PCR (Sigma-Aldrich) and determined to be T a ϭ 60°C for all primers.…”