Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

Lacerda, A. L. M.; Fonseca, Leonardo N.; Blawid, Rosana; Boiteux, L. S.; Ribeiro, S. G.; Brasileiro, Ana Cristina Miranda

doi:10.1371/journal.pone.0136820

Cited by 47 publications

(34 citation statements)

References 40 publications

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“…Among control genes, the absolute expression levels ( Fig. 3) were in agreement with previously published data in other Solanaceae species (Lu et al 2012;Lacerda et al 2015). Genes involved in cembrene production, NtCBTS2α (78.0 copies/pg),…”

Section: Resultssupporting

confidence: 90%

Identification of two new trichome-specific promoters of Nicotiana tabacum

Pottier

Laterre

Wessem

et al. 2019

Preprint

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Key message3 6 pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were 3 7 identified and their strength and specificity were compared to those of previously described 3 8 promoters in this species. Abstract 4 1 Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and 4 2 derivatives in tall glandular trichomes, which actively synthesize and secrete specialized 4 3 metabolites. However, implementation of an entire biosynthetic pathway in glandular 4 4 trichomes requires the identification of trichome-specific promoters to appropriately drive the 4 5 expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR 4 6 analysis was carried out on wild-type N. tabacum plants to compare the expression pattern 4 7 and gene expression level of NtRbS-T1 and NtMALD1, two newly identified genes expressed 4 8 in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which 4 9 were reported in the literature to be specifically expressed in glandular trichomes. The latter 5 0 were previously investigated separately, preventing any accurate comparison of their 5 1 expression level. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in 5 2 glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 was also 5 3 expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six 5 4promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by 5 5

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Section: Resultssupporting

confidence: 90%

Identification of two new trichome-specific promoters of Nicotiana tabacum

Pottier

Laterre

Wessem

et al. 2019

Preprint

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“…It has been proposed that geNorm, NormFinder and BestKeeper tools tend to generate distinct ranking orders of reference genes because they are based on different algorithms35. Therefore, it is recommended to consider them as complementary statistical methods and analyze results globally.…”

Section: Resultsmentioning

confidence: 99%

“…For example, there are now reference genes available for tomato fruit development31, tomato seeds under different conditions32 and MicroTom- Rg1 genotype fruit33. Similar studies have been conducted in tomatoes under abiotic stresses34 and biotic interactions, such as host responses to viruses353637 and Xanthomonas campestris pv. vesicatoria (Xcv )38.…”

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confidence: 98%

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Pombo

Zheng

Fei

et al. 2017

Sci Rep

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The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.

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“…Recently, taking advantage of a variety of databases comprising data from microarrays, expressed sequence tags, and transcriptomes, other stably expressed genes have been used as novel reference genes for qRT-PCR, such as CUL (Cullin), UBCP (ubiquitin carrier protein) [17], CAP (adenylyl cyclase-associated protein), SKIP1 (ASK-interacting protein 1) [22], F-BOX (F-box/kelch-repeat protein) [23], PP2A (protein phosphatase 2A) [24], SAMDC (s-adenosyl methionine decarboxylase) [12, 25], and SNF (sucrose non fermenting-1 protein kinase) [26]. However, the transcript levels of these reference genes vary among different conditions to some extent [20, 27]. Thus, the selection of ideal reference genes to normalize target gene expression is important to improve the reliability of qRT-PCR results.…”

Section: Introductionmentioning

confidence: 99%

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

Wan

Yang

et al. 2017

PLoS ONE

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Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

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Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

Cited by 47 publications

References 40 publications

Identification of two new trichome-specific promoters of Nicotiana tabacum

Identification of two new trichome-specific promoters of Nicotiana tabacum

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

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