A small-scale RNA isolation protocol useful for high-throughput extractions from recalcitrant plants

Smart, Mariette; Roden, Laura C.

doi:10.1016/j.sajb.2010.01.002

Cited by 21 publications

(15 citation statements)

References 10 publications

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“…Tissue was harvested at 4 hpi and total RNA extracted as previously described [45]. cDNA synthesis was carried out using ImpromII reverse transcription system (Promega), following the manufacturer's protocol except that half reaction-volumes and random hexamer primers were used.…”

Section: Methodsmentioning

confidence: 99%

Defence Responses of Arabidopsis thaliana to Infection by Pseudomonas syringae Are Regulated by the Circadian Clock

et al. 2011

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The circadian clock allows plants to anticipate predictable daily changes in abiotic stimuli, such as light; however, whether the clock similarly allows plants to anticipate interactions with other organisms is unknown. Here we show that Arabidopsis thaliana (Arabidopsis) has circadian clock-mediated variation in resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), with plants being least susceptible to infection in the subjective morning. We suggest that the increased resistance to Pst DC3000 observed in the morning in Col-0 plants results from clock-mediated modulation of pathogen associated molecular pattern (PAMP)-triggered immunity. Analysis of publicly available microarray data revealed that a large number of Arabidopsis defence-related genes showed both diurnal- and circadian-regulation, including genes involved in the perception of the PAMP flagellin which exhibit a peak in expression in the morning. Accordingly, we observed that PAMP-triggered callose deposition was significantly higher in wild-type plants inoculated with Pst DC3000 hrpA in the subjective morning than in the evening, while no such temporal difference was evident in arrhythmic plants. Our results suggest that PAMP-triggered immune responses are modulated by the circadian clock and that temporal regulation allows plants to anticipate and respond more effectively to pathogen challenges in the daytime.

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Section: Methodsmentioning

confidence: 99%

Defence Responses of Arabidopsis thaliana to Infection by Pseudomonas syringae Are Regulated by the Circadian Clock

et al. 2011

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“…Other conventional methods employing the hash lysate buffer such as sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), N-lauroyl sarcosine, and urea have also been widely used for RNA isolation in plant species (Liao et al, 2004;Almarza et al, 2006;Fort et al, 2008;Rio et al, 2010;Wu et al, 2011). Special measures could be adopted to address the problems caused by inferences of polysaccharides, phenolic compounds, starches, and pigments (Rodrigues et al, 2007;Kansal et al, 2008;Smart and Roden, 2010).…”

Section: Introductionmentioning

confidence: 99%

Methodology An alternative cetyltrimethylammonium bromide-based protocol for RNA isolation from blackberry (Rubus L.)

Chen

Yu²,

Wang³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. Isolation of high-quality RNA free of contaminants, such as polyphenols, proteins, plant secondary metabolites, and genomic DNA from plant tissues, is usually a challenging but crucial step for molecular analysis. We developed a novel protocol based on the cetyltrimethylammonium bromide method to isolate high-quality RNA from blackberry plant tissues, especially fruits. Most DNA was removed when acetic acid was utilized, before RNA precipitation. Thus, lithium chloride, a reagent widely used for RNA purification, was not needed. The isolation time was shortened to less than 3 h. The RNA was quite pure, with little DNA contamination. The quality of the RNA was assessed by spectrophotometric readings and electrophoresis on agarose gels. It was good enough for downstream enzymatic reactions, such as reverse transcription-PCR, cloning and real-time PCR assay. The method yielded an amount of total RNA comparable to previously described protocols.

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“…Total RNA was extracted from 50 mg from each of twelve individual spikes using the Cetyl trimethylammonium bromide (CTAB) method [ 79 ] with slight modifications. Briefly, spikes were grounded separately using Geno/Grinder (SPEX Sample prep.…”

Section: Materials Methodsmentioning

confidence: 99%

A Comparative Transcriptome Analysis, Conserved Regulatory Elements and Associated Transcription Factors Related to Accumulation of Fusariotoxins in Grain of Rye (Secale cereale L.) Hybrids

Mahmood

Orabi²,

Kristensen³

et al. 2020

IJMS

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Detoxification of fusariotoxin is a type V Fusarium head blight (FHB) resistance and is considered a component of type II resistance, which is related to the spread of infection within spikes. Understanding this type of resistance is vital for FHB resistance, but to date, nothing is known about candidate genes that confer this resistance in rye due to scarce genomic resources. In this study, we generated a transcriptomic resource. The molecular response was mined through a comprehensive transcriptomic analysis of two rye hybrids differing in the build-up of fusariotoxin contents in grain upon pathogen infection. Gene mining identified candidate genes and pathways contributing to the detoxification of fusariotoxins in rye. Moreover, we found cis regulatory elements in the promoters of identified genes and linked them to transcription factors. In the fusariotoxin analysis, we found that grain from the Nordic seed rye hybrid “Helltop” accumulated 4 times higher concentrations of deoxynivalenol (DON), 9 times higher nivalenol (NIV), and 28 times higher of zearalenone (ZEN) than that of the hybrid “DH372” after artificial inoculation under field conditions. In the transcriptome analysis, we identified 6675 and 5151 differentially expressed genes (DEGs) in DH372 and Helltop, respectively, compared to non-inoculated control plants. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs were associated with glycolysis and the mechanistic target of rapamycin (mTOR) signaling pathway in Helltop, whereas carbon fixation in photosynthesis organisms were represented in DH372. The gene ontology (GO) enrichment and gene set enrichment analysis (GSEA) of DEGs lead to identification of the metabolic and biosynthetic processes of peptides and amides in DH372, whereas photosynthesis, negative regulation of catalytic activity, and protein-chromophore linkage were the significant pathways in Helltop. In the process of gene mining, we found four genes that were known to be involved in FHB resistance in wheat and that were differentially expressed after infection only in DH372 but not in Helltop. Based on our results, we assume that DH372 employed a specific response to pathogen infection that led to detoxification of fusariotoxin and prevented their accumulation in grain. Our results indicate that DH372 might resist the accumulation of fusariotoxin through activation of the glycolysis and drug metabolism via cytochrome P450. The identified genes in DH372 might be regulated by the WRKY family transcription factors as associated cis regulatory elements found in the in silico analysis. The results of this study will help rye breeders to develop strategies against type V FHB.

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A small-scale RNA isolation protocol useful for high-throughput extractions from recalcitrant plants

Cited by 21 publications

References 10 publications

Defence Responses of Arabidopsis thaliana to Infection by Pseudomonas syringae Are Regulated by the Circadian Clock

Defence Responses of Arabidopsis thaliana to Infection by Pseudomonas syringae Are Regulated by the Circadian Clock

Methodology An alternative cetyltrimethylammonium bromide-based protocol for RNA isolation from blackberry (Rubus L.)

A Comparative Transcriptome Analysis, Conserved Regulatory Elements and Associated Transcription Factors Related to Accumulation of Fusariotoxins in Grain of Rye (Secale cereale L.) Hybrids

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