Methods for effective real-time RT-PCR analysis of virus-induced gene silencing

Rotenberg, Dorith; Thompson, Thea S.; German, Thomas L.; Willis, David K.

doi:10.1016/j.jviromet.2006.07.017

Cited by 173 publications

(135 citation statements)

References 26 publications

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“…A possible explanation is that the spread of the silencing signal in tomato needs to be assisted by the virus. Contrary to the situation in N. benthamiana, the severity of TRV-induced silencing symptoms in tomato leaves positively correlates with the amount of virus RNA, indicating that the silencing signal runs parallel with the infective wave and that close proximity of cells to the virus is required for the silencing mechanism to work efficiently (Rotenberg et al, 2006). Since viral movement through the plant is often a stochastic process subjected to complex mechanisms and affected by environmental factors, sinksource relationships, tissue age, clone competition, and others, the patchy penetration of the VIGS phenotype found in tomato and other plant species could be the result of irregular viral distribution throughout the plant.…”

Section: Discussionmentioning

confidence: 66%

“…This mechanism works independently of the presence of the virus, allowing the silencing wave to move ahead of the virus infection wave (Ruiz et al, 1998). In N. benthamiana, silencing extends almost uniformly; consequently, the extent of VIGS symptoms does not correlate with the abundance of the virus (Rotenberg et al, 2006). Since there are no reports showing mutual exclusion of different silencing signals within the same plant tissues, we thought it possible that silencing signals initiated concomitantly at the inoculation site in tomato by simultaneous agrodelivery of pTRV2_DR and pTRV2_GOI could result in cosilencing of all three genes in distal parts of the plant.…”

Section: Discussionmentioning

confidence: 99%

“…With the exception of hypersus-ceptible Nicotiana benthamiana, the penetration of VIGS phenotypes in most plant species is only partial and shows a patchy tissue distribution. Tobacco rattle virus (TRV), for example, which drives a highly efficient VIGS in all newly developed organs of N. benthamiana (Ruiz et al, 1998;Ratcliff et al, 2001;Dong et al, 2007), works less efficiently in potato (Solanum tuberosum) and tomato (Liu et al, 2002;Brigneti et al, 2004;Ryu et al, 2004), probably because parts of the plant remain poorly silenced or not silenced at all (Rotenberg et al, 2006;Senthil-Kumar et al, 2007). Tomato plants inoculated at the cotyledon stages show silenced symptoms in about half of their fruit.…”

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confidence: 99%

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A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

et al. 2009

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Innes Centre, Colney, Norwich NR4 7UH, United Kingdom (E.B., C.M.)Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruitspecific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening.

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Section: Discussionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

et al. 2009

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“…First-strand cDNA was synthesized from 100 to 400 ng of total RNA using SuperScript II reverse transcriptase (Invitrogen; www.invitrogen.com), an oligo(dT)20VN primer (2.5 mM), RT buffer (53 first-strand buffer), deoxyribonucleotide triphosphates (0.5 mM each), and dithiothreitol (5 mM) in a total reaction volume of 20 mL. The occurrence of cDNAs corresponding to TRV2 coat protein was determined by PCR as described previously (Rotenberg et al, 2006).…”

Section: Rt-qpcrmentioning

confidence: 99%

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

Lee

Facchini

2011

Plant Physiology

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Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and a-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5#-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and a-ketoglutarate, with apparent K m values of 1.82 and 0.35 mM, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

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“…Typically, PDS is selected as a marker gene in VIGS system optimization, as a reduction in PDS gene transcripts represents the silencing efficiency of VIGS (Rotenberg et al, 2006). For some plant species in which the PDS gene sequences are unknown, the reduction of their transcript abundances within silenced plants is substituted with chlorophyll (SenthilKumar et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

A novel method for the evaluation of virus-induced gene silencing efficiency

Li¹,

Zhang²,

Ghebremariam³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Virus-induced gene silencing (VIGS) is an important tool for studying gene function. However, a number of factors highly restrict the application of VIGS, such as unstable efficiency and tissue-specific silencing. We developed a novel evaluation method for improving the applicability of VIGS vectors. In this method, 4 indexes were defined and utilized to evaluate VIGS efficiency by silencing the endogenous phytoene desaturase (PDS) gene with a tobacco rattle virus-based VIGS vector. To illustrate the reliability of this evaluation method, we assessed the silencing efficiency of SpPDS and SpMPK1 in Solanum pimpinellifolium. The silencing results of SpPDS showed that an optical density at 600 nm of 2.0 was more suitable than 1.0 for VIGS in S. pimpinellifolium. This suggests that the proposed evaluation method is a valid technique for optimizing the VIGS system of plants. Moreover, the SpMPK1 gene was highly silenced in the 4th-9th leaves with a 50-95% reduction in transcription levels, further demonstrating that this method can be used to select highly silenced candidates for further experiments, particularly when the target gene shows no phenotypic change after being silenced.

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Methods for effective real-time RT-PCR analysis of virus-induced gene silencing

Cited by 173 publications

References 26 publications

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

A novel method for the evaluation of virus-induced gene silencing efficiency

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