Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Albaqami, Mohammed; Reddy, Anireddy S. N.

doi:10.1186/s13007-017-0271-6

Cited by 92 publications

(84 citation statements)

References 80 publications

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“…The specific function of each laccase gene in plants remain to be characterized, therefore we picked one fungal laccase gene to overexpress in Triphysaria. Each gene was driven by the CaMV 35S promoter and transformed into wild type Triphysaria versicolor by Rhizobium rhizogenes mediated root transformation ( Bandaranayake and Yoder, 2018 ). Using this transformation method, Triphysaria plants became chimeric with the shoot remaining wild type while transgenic roots emerge from the shoot-root junction.…”

Section: Resultsmentioning

confidence: 99%

“…Assay of haustorium development in transgenic Triphysaria roots . Transgenic Triphysaria roots were obtained by Rhizobium rhizogenes mediated transformation ( Bandaranayake and Yoder, 2018 ). To assay haustorium development, 1 ml of 1 µM DMBQ was applied to the transgenic root tips and the proportion of roots that formed haustoria 24 h after treatment was counted.…”

Section: Methodsmentioning

confidence: 99%

“…Each amplified gene was inserted into pDS3 vector between PacI and SacI via Gibson Assembly (NEB). The vectors were verified by Sanger sequencing, transformed into Rhizobium rhizogenes stain MSU440 by electroporation ( Sonti et al., 1995 ), and transformed into Triphysaria roots ( Bandaranayake and Yoder, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

“…Transgenic Triphysaria roots were obtained by Rhizobium rhizogenes transformation as described ( Bandaranayake and Yoder, 2018 ) and maintained on square petri plates containing 0.25× Hoagland nutrient media, 0.75% (w/v) sucrose, 0.75% (w/v) Phytagel and 300 mg/L Timentin to control the growth of Rhizobium rhizogenes . For each construct transformed, number of independent plants with transgenic roots analyzed in this study are shown in parenthesis: 35S-AtPrx2 (19), 35S-AtPrx4 (25), 35S-AtPrx21 (19), 35S-AtPrx31 (16), 35S-AtPrx53 (23), 35S-AtPrx71 (27), 35S-AtPrx72 (16), 35S-TvLCC1 (22), empty vector control (19).…”

Section: Methodsmentioning

confidence: 99%

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Kin Recognition in the Parasitic Plant Triphysaria versicolor Is Mediated Through Root Exudates

et al. 2020

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Triphysaria is a facultative parasitic plant in the Orobanchaceae that parasitizes the roots of a wide range of host plants including Arabidopsis , Medicago , rice and maize. The important exception to this broad host range is that Triphysaria rarely parasitize other Triphysaria . We explored self and kin recognition in Triphysaria versicolor and showed that exudates collected from roots of host species, Arabidopsis thaliana and Medicago truncatula , induced haustorium development when applied to the roots of Triphysaria seedlings in vitro while those collected from Triphysaria did not. In mixed exudate experiments, Triphysaria exudates did not inhibit the haustorium-inducing activity of those from host roots. Interestingly, when roots of Triphysari a seedlings were treated with either horseradish peroxidase or fungal laccase, the extracts showed haustorium-inducing factor (HIF) activity, suggesting that Triphysaria roots contain the proper substrates for producing HIFs. Transgenic Triphysaria roots overexpressing a fungal laccase gene TvLCC1 showed an increased responsiveness to a known HIF, 2,6-dimethoxy benzoquinone (DMBQ), in developing haustoria. Our results indicate kin recognition in Triphysaria is associated with the lack of active HIFs in root exudates. Treatment of Triphysaria roots with enzymatic oxidases activates or releases molecules that are HIFs. This study shows that exogenously applied oxidases can activate HIFs in Triphysaria roots that had no previous HIF activity. Further studies are necessary to determine if differential oxidase activities in host and parasite roots account for the kin recognition in haustorium development.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kin Recognition in the Parasitic Plant Triphysaria versicolor Is Mediated Through Root Exudates

et al. 2020

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“…Salad samples were homogenized using an ultra-turax (miccra d-1, Heitersheim, Germany) which was cleaned with 70% IPA and dried between the samples. In order to inhibit lipase activity, all samples were incubated at 75 °C for 30 min under constant shaking [ 27 ]. The warm salad samples were subsequently transferred into glass vials.…”

Section: Methodsmentioning

confidence: 99%

Chasing the Major Sphingolipids on Earth: Automated Annotation of Plant Glycosyl Inositol Phospho Ceramides by Glycolipidomics

et al. 2020

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Glycosyl inositol phospho ceramides (GIPCs) are the major sphingolipids on earth, as they account for a considerable fraction of the total lipids in plants and fungi, which in turn represent a large portion of the biomass on earth. Despite their obvious importance, GIPC analysis remains challenging due to the lack of commercial standards and automated annotation software. In this work, we introduce a novel GIPC glycolipidomics workflow based on reversed-phase ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry. For the first time, automated GIPC assignment was performed using the open-source software Lipid Data Analyzer (LDA), based on platform-independent decision rules. Four different plant samples (salad, spinach, raspberry, and strawberry) were analyzed and the results revealed 64 GIPCs based on accurate mass, characteristic MS2 fragments and matching retention times. Relative quantification using lactosyl ceramide for internal standardization revealed GIPC t18:1/h24:0 as the most abundant species in all plants. Depending on the plant sample, GIPCs contained mainly amine, N-acetylamine or hydroxyl residues. Most GIPCs revealed a Hex-HexA-IPC core and contained a ceramide part with a trihydroxylated t18:0 or a t18:1 long chain base and hydroxylated fatty acid chains ranging from 16 to 26 carbon atoms in length (h16:0–h26:0). Interestingly, four GIPCs containing t18:2 were observed in the raspberry sample, which was not reported so far. The presented workflow supports the characterization of different plant samples by automatic GIPC assignment, potentially leading to the identification of new GIPCs. For the first time, automated high-throughput profiling of these complex glycolipids is possible by liquid chromatography-high-resolution tandem mass spectrometry and subsequent automated glycolipid annotation based on decision rules.

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Proton‐pumping pyrophosphatase homeolog expression is a dynamic trait in bread wheat (Triticum aestivum)

et al. 2021

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Proton‐pumping pyrophosphatases (H + ‐PPases) have been shown to enhance biomass and yield. However, to date, there has been little work towards identify genes encoding H + ‐PPases in bread wheat ( Triticum aestivum ) ( TaVP s) and limited knowledge on how the expression of these genes varies across different growth stages and tissue types. In this study, the IWGSC database was used to identify two novel TaVP genes, TaVP4 and TaVP5 , and elucidate the complete homeolog sequences of the three known TaVP genes, bringing the total number of bread wheat TaVP s from 9 to 15. Gene expression levels of each TaVP homeolog were assessed using quantitative real‐time PCR (qRT‐PCR) in four diverse wheat varieties in terms of phenotypic traits related to high vacuolar pyrophosphatase expression. Homeolog expression was analyzed across multiple tissue types and developmental stages. Expression levels of the TaVP homeologs were found to vary significantly between varieties, tissues and plant developmental stages. During early development (Z10 and Z13), expressions of TaVP1 and TaVP2 homeologs were higher in shoot tissue than root tissue, with both shoot and root expression increasing in later developmental stages (Z22). TaVP2‐D was expressed in all varieties and tissue types and was the most highly expressed homeolog at all developmental stages. Expression of the TaVP3 homeologs was restricted to developing grain (Z75), while TaVP4 homeolog expression was higher at Z22 than earlier developmental stages. Variation in TaVP4B was detected among varieties at Z22 and Z75, with Buck Atlantico (high biomass) and Scout (elite Australian cultivar) having the highest levels of expression. These findings offer a comprehensive overview of the bread wheat H + ‐PPase family and identify variation in TaVP homeolog expression that will be of use to improve the growth, yield, and abiotic stress tolerance of bread wheat.

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Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Cited by 92 publications

References 80 publications

Kin Recognition in the Parasitic Plant Triphysaria versicolor Is Mediated Through Root Exudates

Kin Recognition in the Parasitic Plant Triphysaria versicolor Is Mediated Through Root Exudates

Chasing the Major Sphingolipids on Earth: Automated Annotation of Plant Glycosyl Inositol Phospho Ceramides by Glycolipidomics

Proton‐pumping pyrophosphatase homeolog expression is a dynamic trait in bread wheat (Triticum aestivum)

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